Gene editing for improved joint function

Abstract

The present disclosure provides compositions and methods for treating joint conditions characterized by inflammatory components. In some aspects, the compositions and methods are useful for preventing the progression of osteoarthritis and other arthritis in mammalian joints, as well as for treating osteoarthritis and other arthritis.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 222,972 filed on July 17, 2021, U.S. Provisional Patent Application No. 63 / 300,822 filed on January 19, 2022, U.S. Provisional Patent Application No. 63 / 326,571 filed on April 1, 2022, and International Application No. WO 2022 / 016121 filed on July 16, 2021, the contents of which are hereby incorporated by reference in their entirety for all purposes. Technical Field

[0003] Compositions and methods for treating synovial joint dysfunction are described herein. In addition, methods for gene editing synovial cells and / or synoviocytes, chondrocytes, synovial macrophages and synovial fibroblasts, and uses of gene-edited synovial cells, chondrocytes, synovial macrophages and synovial fibroblasts in treating diseases such as osteoarthritis are disclosed herein. Background Art

[0004] Treatment of osteoarthritis, degenerative joint disease and other joint dysfunctions is complex and there are few long-term options for symptom relief or restoration of joint function. Osteoarthritis (OA) is the leading cause of disability due to pain. Neogi, Osteoarthritis Cartilage, 2013;21:1145-53. All mammalian species are affected: working animals, livestock and their owners all experience discomfort, pain and disability associated with OA, depending on the extent of disease progression.

[0005] OA is a complex disease characterized by a progressive course of disability. Systemic inflammation is associated with OA and OA disease progression. Inflammation is driven by increased levels of proinflammatory cytokines. New methods and compositions for treating this disease are urgently needed. Disclosed herein are compositions and methods that can be used to treat OA and other inflammatory joint disorders. Summary of the invention

[0006] The present disclosure provides compositions and methods for treating joint disorders characterized by inflammatory components. In some aspects, the compositions and methods are used to prevent the progression of osteoarthritis and other arthritis in mammalian joints, and to treat osteoarthritis and other arthritis. According to an exemplary embodiment, at least a portion of joint synovial cells and / or synovial cells, chondrocytes, synovial macrophages or synovial fibroblasts are gene-edited to reduce the expression of inflammatory cytokines. In some aspects, at least a portion of joint synovial cells and / or synovial cells, chondrocytes, synovial macrophages or synovial fibroblasts are gene-edited to reduce the expression of IL-1α, IL-1β or IL-1α, IL-1β.

[0007] In some embodiments, gene editing silences or reduces the expression of one or more cytokines and / or growth factor genes in at least a portion of cells comprising a mammalian joint. In some aspects, the cell is a synovial cell. In some aspects, the cell is a synovial fibroblast. In some aspects, the cell is a synovial cell. In some aspects, the cell is a chondrocyte. In some aspects, the cell is a synovial macrophage.

[0008] In some embodiments, the one or more cytokine and / or growth factor genes are selected from the group consisting of IL-1a and IL-1Bb.

[0009] In some embodiments, gene editing comprises the use of programmable nucleases that mediate the generation of double-stranded or single-stranded breaks at the one or more cytokine and / or growth factor genes.

[0010] In some embodiments, gene editing comprises one or more methods selected from the group consisting of CRISPR methods, TALE methods, zinc finger methods, and combinations thereof.

[0011] In some embodiments, gene editing comprises CRISPR methods.

[0012] In some embodiments, the CRISPR method is a CRISPR-Cas9 method.

[0013] In some embodiments, gene editing comprises a TALE approach.

[0014] In some embodiments, gene editing comprises a zinc finger approach.

[0015] In some embodiments, gene editing silences or reduces the expression of one or more cytokine and / or growth factor genes in at least a portion of cells comprising a joint. In some embodiments, the edited cell portion is a synovial cell. In one aspect, the edited cell portion is a synovial fibroblast. In some embodiments, the edited cell portion is a synovial cell. In some embodiments, the edited cell portion is a chondrocyte. In some embodiments, the edited cell portion is a synovial macrophage.

[0016] In some embodiments, an adeno-associated virus (AAV) delivery system is used to deliver the gene editing system. In some embodiments, the AAV delivery system is injected into a joint.

[0017] Some aspects of the present disclosure provide a pharmaceutical composition for treating or preventing a joint disease or condition, comprising a gene editing system and a pharmaceutically acceptable carrier. In one aspect, the gene editing system comprises one or more nucleic acids targeting one or more gene loci selected from the group consisting of IL-1α, IL-1β, TNF-α, IL-6, IL-8, and IL-18.

[0018] In some embodiments, the gene editing system comprises a composition for treating or preventing a joint disease or condition, the composition comprising an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease and at least one guide RNA or a nucleic acid encoding at least one guide RNA, the guide RNA targeting an IL-1α or IL-1β gene, wherein the guide RNA specifically binds to a target sequence adjacent to a protospacer adjacent motif (PAM) sequence of a Cas9 protein.

[0019] In some embodiments, the at least one guide RNA targets human IL-1α and comprises a crRNA sequence that is at least 85%, 90%, 95%, or 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 168-187, 298-387, and 681-710.

[0020] In some embodiments, the at least one guide RNA targets a human IL-1β gene and comprises a crRNA sequence that is at least 85%, 90%, 95%, or 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 188-201, 388-496, and 711-740.

[0021] In some embodiments, the at least one guide RNA targets a canine IL-1 alpha gene and comprises a crRNA sequence that is at least 85%, 90%, 95%, or 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 202-216, 552-590, and 741-770.

[0022] In some embodiments, the at least one guide RNA targets a canine IL-1 β gene and comprises a crRNA sequence that is at least 85%, 90%, 95%, or 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 217-235, 497-551, and 771-800.

[0023] In some embodiments, the gene editing system comprises one or more lipid nanoparticles (LNPs) that collectively comprise an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease and at least one guide RNA or a nucleic acid encoding at least one guide RNA.

[0024] In some embodiments, the LNPs comprise a first plurality of LNPs comprising a first nucleic acid and a second plurality of LNPs comprising a second nucleic acid, wherein the first plurality of LNPs encapsulates a nucleic acid encoding an RNA-guided nuclease and the second plurality of LNPs encapsulates a nucleic acid encoding the at least one guide RNA.

[0025] In some embodiments, the LNPs comprise a first plurality of LNPs encapsulating an RNA-guided nuclease and a second plurality of LNPs comprising a second nucleic acid, the second plurality of LNPs encapsulating a nucleic acid encoding at least one guide RNA.

[0026] In some embodiments, the LNP comprises a single nucleic acid, wherein the single nucleic acid encodes an RNA-guided nuclease and at least one guide RNA.

[0027] In some embodiments, the gene editing system comprises one or more liposomes that collectively comprise an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease and at least one guide RNA or a nucleic acid encoding at least one guide RNA.

[0028] In some embodiments, the nucleic acid encoding the RNA-guided nuclease and / or the nucleic acid encoding at least one guide RNA is present in a naked state.

[0029] In some embodiments, the RNA-guided nuclease in the gene editing system is a Cas9 protein. In some embodiments, the Cas9 protein is spCas9. In some embodiments, the Cas9 protein is espCas9. In other embodiments, the Cas9 protein is saCas9.

[0030] One embodiment provides a method for treating canine lameness, the method comprising administering a gene editing composition, wherein the composition silences or reduces the expression of IL-1α and IL-1β in a portion of synovial cells, chondrocytes, synovial macrophages, or synovial fibroblasts in a lame joint.

[0031] One embodiment provides a method for treating a joint disease or condition in a subject in need thereof. In some embodiments, the joint disease or condition is arthritis. In some embodiments, the joint disease or condition is osteoarthritis.

[0032] In some embodiments, the gene editing composition is formulated for parenteral administration. In some embodiments, the gene editing composition is formulated for intra-articular injection into a joint of a subject.

[0033] In some embodiments, the above methods further comprise one or more features listed in any of the methods and compositions described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The embodiments of the present disclosure will be further explained with reference to the accompanying drawings. The drawings shown are not necessarily drawn to scale, with emphasis instead generally being placed upon illustrating the principles of the embodiments of the present disclosure.

[0035] Figure 1A Agarose gel electrophoresis analysis of 100 ng mouse DNA (gBlocks, Integrated DNA Technologies) designed for the Mus musculus Il1a and Il1b genes, which was in vitro activated by 0.5 μg SpyCas9 (TrueCut TM Cas9 protein v2, ThermoFisher Scientific) and 200 ng of phosphorothioate-modified single-stranded guide (sg) RNA targeting Il1a gene (#43-46) and Il1b gene (#47-50) for cleavage.

[0036] Figure 1B Agarose gel electrophoresis analysis of 100 ng mouse DNA (gBlocks, Integrated DNA Technologies) designed for the Il1a and Il1b genes of Mus musculus, which was stimulated by 0.5 μg of SauCas9 (GeneSnipper TM Cas9, BioVision) and 200ng of phosphorothioate-modified guide sgRNAs targeting Il1a (#51-53) and IL1b (#54-56) genes for cleavage.

[0037] Figure 2A , 2B , 2C and 2D collectively show graphs showing the editing efficiency of SpyCas9 and SauCas9 used with a range of guide RNAs in J774.2 (“J”) and NIH3T3 (“N”) cells; Figure 2A : In vivo cleavage of Il1a edited with 4x sgRNAs (Spy Cas9) in two separate pools (Pool 1 and Pool 2) between two cell lines, NIH 3T3 (“N”) and J774.2 (“J”); Figure 2B : In vivo cleavage of Il1b edited with 4x sgRNAs (Spy Cas9) in two separate pools (Pools 1 and 2) between two cell lines, NIH 3T3 (“N”) and J774.2 (“J”); Figure 2C : In vivo cleavage of Il1a edited with 3x sgRNAs (Sau Cas9) in two separate pools (Pools 1 and 2) between two cell lines, NIH 3T3 (“N”) and J774.2 (“J”); Figure 2D : In vivo cleavage of Il1b edited with 3x sgRNAs (saCas9) in two separate pools (Pool 1 and Pool 2) between two cell lines, NIH 3T3 (“N”) and J774.2 (“J”); editing efficiency determined by deconvolution using Sanger sequencing traces (ICE tool, Synthego) of each pool.

[0038] Figure 3 GFP expression measured using the IVIS system is shown. Flux values ​​are based on a region of interest centered on the animal's injected knee joint. Data are presented as the mean (SD) of four samples per group.

[0039] Figure 4 The design of the study as described in Example 5 of the present disclosure is shown.

[0040] Figure 5 Shown are survival outcome measures obtained in the study as described in Example 5 of the present disclosure.

[0041] Figure 6 Shown are changes in body weight of mice treated with intra-articular (IA) injections of PBS, AAV-6 with a scrambled vector, AAV-6 with CRISPR-Cas guides 1 and 2, AAV-5 with a scrambled vector, or AAV-5 with CRISPR-Cas guides 1 and 2 in a study as described in Example 5 of the present disclosure.

[0042] Fig. 7A and 7BShown together are (A) knee caliper measurements relative to baseline in mouse joints over time, and (B) mean differences in ankle caliper measurements from AUC in mice treated with intra-articular (IA) injections of PBS, AAV-6 with a scrambled vector, AAV-6 with CRISPR-Cas guides 1 and 2, AAV-5 with a scrambled vector, or AAV-5 with CRISPR-Cas guides 1 and 2 in a study as described in Example 5 of the present disclosure.

[0043] Fig. 8A and 8B Shown together are (A) changes in von Frey measurements, and (B) average absolute thresholds of von Frey measurements, obtained from mice treated with intra-articular (IA) injections of PBS, AAV-6 with a scrambled vector, AAV-6 with CRISPR-Cas guides 1 and 2, AAV-5 with a scrambled vector, or AAV-5 with CRISPR-Cas guides 1 and 2 in a study as described in Example 5 of the present disclosure.

[0044] Fig. 9 Shown are the results of qPCR assays for IL-1β expression in synovial fluid obtained from mice treated with intra-articular (IA) injections of PBS, AAV-6 with a scrambled vector, AAV-6 with CRISPR-Cas guides 1 and 2, AAV-5 with a scrambled vector, or AAV-5 with CRISPR-Cas guides 1 and 2 in a study as described in Example 5 of the present disclosure.

[0045] Fig. 10A , 10B , 10C, and 10D collectively show the immunohistochemistry of murine IL-1β in synovial tissues of MSU-injected animals pretreated with PBS (A, B) and treated with CRISPR (C, D). Fig. 10B and 10D They are shown respectively Fig. 10A and 10C Isotype controls for each of the .

[0046] Fig.11A , 11B and 11C together show the alignment between mouse, human, equine, feline and canine IL-1α genes.

[0047] Fig. 12A , 12B , 12C and 12D collectively show the alignment between mouse, human, equine, feline and canine IL-1β genes.

[0048] Fig.13A, 13B , 13C and 13D collectively show exemplary CRISPR / Cas9 crRNA sequences designed for editing the human IL-1α gene.

[0049] Fig.14A , 14B , 14C, 14D, and 14E collectively show exemplary CRISPR / Cas9 crRNA sequences designed for editing the human IL-1β gene.

[0050] Fig.15A , 15B 15C and 15C together show an exemplary CRISPR / Cas9 crRNA sequence designed to edit the canine IL-1α gene.

[0051] Fig.16A and 16B Shown together are exemplary CRISPR / Cas9 crRNA sequences designed to edit the canine IL-1β gene.

[0052] Fig.17A , 17B , 17C and 17D collectively show targeting of the human IL-1α gene ( Fig. 7A )、human IL-1β gene ( Figure 7B ), canine IL-1α gene (Figure 7C), and canine IL-1β gene (Figure 7D) results of cell-based and in silico gene editing analysis of crRNA sequences, as described in Example 8. ° CRISPR cleavage position within the amino acid (AA) translation frame. * Optimization score from Doench, Fusi et al. (2016). The score is optimized for a 20 bp guide with NGG. The score ranges from 0 to 100. Higher is better. ** Specificity score from Hsu et al. (2013). The score ranges from 0 to 100. Higher is better. *** The score is based on experiments in U2OS. High accuracy scores (>0.4) mean that DNA repair results are consistent and only a few unique genotypes are enriched. **** The score is based on experiments in U2OS. Higher (>80%) frameshift frequencies will tend to knock protein-coding genes out of frame. Typical genomic frameshift frequencies are above 66%, as 1-bp insertions and 1-2bp deletions are particularly common repair outcomes. ^Comprehensive score = (off-target score + accuracy score*100 + frameshift) / 3. The pipe symbol '|' indicates a CRISPR cut site. Curly brackets '{}' indicate insertions. Hyphens '-' indicate deletions. $ Potential off-target sites. Scoring according to Hsu et al. (2013). On-target sites have a score of 100.

[0053] Fig.18A , 18B , 18C and 18D collectively show that 6 hours after exposure to PBS or LPS ( Fig.18A and 18C ) and 24 hours ( Fig.18B and 18D )Canine IL-1α( Fig.18A and 18B ) and canine IL-1β release from unedited (control) and double IL-1α / IL-1β KO (edited) canine chondrocytes ( Fig. 18C and 18D ), as described in Example 9.

[0054] Fig.19A , 19B , 19C and 19D collectively show that 6 hours after exposure to PBS or LPS ( Fig.19A and 19C ) and 24 hours ( Fig.19B and 19D ) Human IL-1α( Fig.19A and 19B ) and canine IL-1β ( Fig.19C and 19D ) from unedited (control) and double IL-1α / IL-1β KO (edited) canine chondrocytes, as described in Example 9.

[0055] Fig. 20 Shown are the results of tissue-specific splicing and expression analysis of the human IL1A (IL-1a) gene.

[0056] Fig.21 Shown are the results of tissue-specific splicing and expression analysis of the human IL1B (IL-1b) gene.

[0057] Fig. 22The results of computer analysis of human IL-1a-targeted and IL-1b-targeted gRNA targeting domains are shown. The on-target score is optimized for a 20-bp gRNA with an NGG protospacer sequence adjacent motif (PAM) (see Doench et al.). The score range is 0 to 1. The accuracy score is based on experiments in U2OS cells. High accuracy scores (>0.4) mean that the DNA repair results are consistent and only a few unique genotypes are enriched. The frameshift percentage is based on experiments in U2OS cells. A higher (>80%) frameshift frequency will tend to knock protein-coding genes out of frame. Typical genomic frameshift frequencies are higher than 66%, because 1-bp insertions and 1-2bp deletions are particularly common repair results. The off-target score from CRISPOR evaluates the number of matches with a given number of mismatches in the genome. Mismatches in the seed sequence have a more harmful effect.

[0058] Fig.23A , 23B , 23C and 23D collectively show the results of splicing and functional analysis of canine IL1A (IL-1a) and IL1B (IL-1b) genes. The reference canine genome assembly (CanFam3.1) was used for these analyses. gRNA design can be customized for individual breeds as needed.

[0059] Fig.24 Shown are the results of in silico analysis of canine IL-1a-targeted and IL-1b-targeted gRNA targeting domains.

[0060] Fig.25A , 25B and 25C together show human chondrocytes ( Fig.25A ), canine chondrocytes ( Fig.25B ) and synaptic cells ( Fig.25C ) in the knockout efficacy of the selected gRNA targeting domain. Genomic DNA was collected from the pooled cells 8-10 days after administration prior to sequencing analysis.

[0061] Fig.26 Shown are the results of in silico analysis of off-target effects of lead candidate gRNA targeting domains in canine cells.

[0062] Fig.27A and 27B Together, we show the efficacy of enhanced-specificity Cas9 (espCas9) in eliminating off-target editing of sgRNA242 in canine cells. Fig.27A The effects of a typical spCas9 are shown, resulting in strong on-target editing but also high off-target activity. Fig.27B It was shown that espCas9 maintained high on-target efficiency without off-target effects.

[0063] Fig.28A and 28B Editing activity of sgRNAs targeting IL-1a and IL-1b in human (A) and canine (B) chondrocytes is shown together.

[0064] Fig.29 Shown are the results of simultaneous or sequential co-administration of lead candidate sgRNAs in canine cells.

[0065] Fig. 30A and 30B Shown are sequence alignments of IL1A (A) and IL1B (B) genomic sequences from human, equine, murine, and canine with potential cross-species targets for individual sgRNAs.

[0066] Fig.31A , 31B and 31C together show CRISPR-mediated editing of canine IL-1A. Specifically, Fig.31A Shown are the average KO scores determined by inferring CRISPR editing from Sanger traces in canine chondrocytes (n=3+), fibroblast-like synaptic cells (n=3), and monocytic DH82 cells (n=2). The stacked bar graphs show the top and all remaining KO genotypes of sgRNAs assembled with wild-type Cas9 in light and dark colors, respectively. Fig.31B The amino acid sequences of wild-type and truncated canine IL1A before (top) and after (bottom) CRISPR-mediated genome editing are shown. Wild-type IL1A includes a propeptide that is cleaved to make a functional (mature) protein (highlighted). Fig.31C The predicted three-dimensional structures of wild-type and truncated IL1A as predicted by AlphaFold2 are shown. N indicates the N-terminus, ie, the typical translation start point.

[0067] Fig.32A , 32B and 32C collectively show the design and testing of eight canine IL1A-targeting sgRNAs: sg239 / OCA01, sg360 / OCA02, sg240 / OCA03, sg359 / OCA04, sg358 / OCA05, sg251 / OCA07, sg361 / OCA06, and sg252 / OCA08. Fig.32A The target sequence edited by the guide is shown (equivalent to the crRNA sequence in the guide, except that the guide sequence includes uracil instead of thymidine). Fig.32B Scores for predicted on-target, off-target, and frameshift effects are shown for each gRNA, as described in Example 16. Fig.32CShown are the results of an in vitro editing assay with sequencing analysis of the edited targets.

[0068] Fig.33A , 33B , 33C and 33D collectively show the design and testing of eight canine IL1B-targeting sgRNAs: sg241 / OCB01 (SEQ ID NO: XX), sg242 / OCB02 (SEQ ID NO: XX), sg352 / OCB06 (SEQ ID NO: XX), sg353 / OCB04 (SEQ ID NO: XX), sg354 / OCB08 (SEQ ID NO: XX), sg355 / OCB05 (SEQ ID NO: XX), sg356 / OCB07 (SEQ ID NO: XX) and sg357 / OCB03 (SEQ ID NO: XX). Fig.33A The target sequence edited by the guide is shown (equivalent to the crRNA sequence in the guide, except that the guide sequence includes uracil instead of thymidine). Fig.32B Scores for predicted on-target, off-target, and frameshift effects are shown for each gRNA, as described in Example 17. Fig.32C Shown are the results of an in vitro editing assay with sequencing analysis of the edited targets. Fig.33D Shown are the results of an anti-IL-1B ELISA assay on supernatants of canine monocytes targeted by the editing construct.

[0069] Fig.34A and 34B The performance of IL1B-targeting sgRNA #241 (OCB01) and #242 (OCB02) is shown together, as inferred from the average IL1B KO score of Sanger sequencing CRISPR-mediated genome editing in chondrocytes, synaptic cells, and monocytes DH82 ( Fig.34A ) and ELISA results of IL1B in control or two IL1A / B double KO monocytes generated by different combinations of sgRNAs ( Fig.34B ; KO score inferred by Sanger sequence for each combination on the left).

[0070] Fig.35A and 35B Shown together are the effects of electroporation on canine monocytes harboring OCB02 / sg242(KO1) or OCB01 / sg241(KO2), as measured by supernatant ELISA at (A) 6 hours and (B) 24 hours after challenge with lipopolysaccharide (LPS).

[0071] Fig.36Shown is a comparison of the editing efficiency of five potential gRNAs targeting canine IL-1β in monocytes as measured by the Abcam IL-1β ELISA kit.

[0072] Fig.37A and 37B The effects of different Cas9 mutants with enhanced specificity are shown together, including (A) comparison of on-target editing efficiency with OCB02 gRNA plus standard Cas9 or enhanced specificity / fidelity Cas9 variants, and (B) comparison of on-target editing efficiency of OCB01 with ARCas9 variants or wild-type Cas9.

[0073] Fig.38A and 38B Together, the in vitro performance of Cas9 mutants with enhanced specificity is shown. Fig.38A On-target and off-target editing efficiencies are shown, with percentages representing KO score and editing efficiency, respectively. Fig.38B Predicted off-target editing sites of OCB01 are shown.

[0074] Fig.39A and 39B Shown together are exemplary CRISPR / Cas9 crRNA sequences designed for editing the human IL-1α gene.

[0075] Fig.40A and 40B Shown together are exemplary CRISPR / Cas9 crRNA sequences designed for editing the human IL-1β gene.

[0076] Fig.41A and 41B Shown together are exemplary CRISPR / Cas9 crRNA sequences designed to edit the canine IL-1α gene.

[0077] Fig.42A and 42B Shown together are exemplary CRISPR / Cas9 crRNA sequences designed to edit the canine IL-1β gene.

[0078] Fig.43A and 43B Shown together are exemplary CRISPR / Cas9 crRNA sequences designed for editing the equine IL-1α gene.

[0079] Fig.44A and 44B Shown together are exemplary CRISPR / Cas9 crRNA sequences designed for editing the equine IL-1β gene.

[0080] Fig.45A and 45B Shown together are exemplary CRISPR / Cas9 crRNA sequences designed to edit the canine IL-1α gene.

[0081] Fig.46A and 46B Shown together are exemplary CRISPR / Cas9 crRNA sequences designed to edit the canine IL-1β gene.

[0082] Although the above-mentioned drawings illustrate the embodiments disclosed by the present invention, other embodiments may be envisioned as mentioned in the discussion. The present disclosure presents illustrative embodiments by way of representation and not limitation. Those skilled in the art may devise numerous other modifications and embodiments that fall within the scope and spirit of the principles of the embodiments disclosed by the present invention. DETAILED DESCRIPTION

[0083] As described herein, embodiments of the present disclosure provide compositions and methods for improving joint function and treating joint diseases. In a specific embodiment, there is provided a composition and method for gene editing synovial fibroblasts, synovial cells, chondrocytes or synovial macrophages to reduce the expression of inflammatory cytokines, such as IL-1α, IL-1β, TNF-α, IL-6, IL-8, IL-18, one or more matrix metalloproteinases (MMP), or one or more components of NLRP3 inflammasomes. The embodiment is used to treat osteoarthritis and other inflammatory joint diseases. The embodiment can further be used to treat canine lameness caused by osteoarthritis. The embodiment can further be used to treat equine lameness caused by joint diseases. The embodiment can further be used to treat post-traumatic arthritis, gout, pseudogout and other inflammatory-mediated or immune-mediated joint diseases.

[0084] definition

[0085] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs.All patents and publications mentioned herein are incorporated by reference in their entirety.

[0086] The term "in vivo" refers to events occurring within the body of a subject.

[0087] The term "in vitro" refers to events that occur outside the body of a subject. In vitro assays encompass cell-based assays in which live or dead cells are used, and may also encompass cell-free assays in which intact cells are not used.

[0088] The term "ex vivo" refers to events involving treatment of cells, tissues and / or organs that have been removed from a subject's body or procedures performed on such cells, tissues and / or organs. Suitably, the cells, tissues and / or organs may be returned to the subject's body by surgical or therapeutic methods.

[0089] The term "IL-1" (also referred to herein as "IL1") refers to the proinflammatory cytokine known as interleukin-1, and includes all forms of IL-1, including IL1-α and IL-1β, human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-1α and IL-1β bind to the same receptor molecule, which is called the type I IL-1 receptor (IL-1R1). There is a third ligand for this receptor: interleukin 1 receptor antagonist (IL-1Ra), which does not activate downstream signaling; therefore, it acts as an inhibitor of IL-1α and IL-1β signaling by competing with IL-1α and IL-1β for the binding site of the receptor. See, e.g., Dinarello, Blood 117:3720–32 (2011) and Weber et al., Science Signaling, 3(105):cm1, doi:10.1126 / scisignal.3105cm1. IL-1 is described in, for example, Dinarello, Cytokine Growth Factor Rev., 8:253-65 (1997), the disclosure of which is incorporated herein by reference. For example, the term IL-1 encompasses a human recombinant form of IL-1.

[0090] Table 1. Exemplary amino acid sequences.

[0091]

[0092]

[0093] The term "NLRP3 inflammasome" refers to a multiprotein complex responsible for the activation of certain inflammatory responses. The NLRP3 inflammasome promotes the production of functional proinflammatory cytokines such as IL-1β and IL-18. The core components of the NLRP3 inflammasome are NLRP3, apoptosis-associated speck-like protein containing CARD (ASC), and caspase-1, as described by Lee et al., Lipids Health Dis. 16:271 (2017) and Groslambet and Py, J. Inflamm. Res., 11:359-374 (2018).

[0094] The terms "matrix metalloproteinase" and "MMP" are defined as any of the members of the matrix metalloproteinase family, such as zinc endopeptidases, as characterized by Fanjul-Fernandez et al., Biochem. Biophys. Acta 1803: 3-19 (2010). In the art, family members are often referred to as prototypical MMPs, gelatinases, stromelysins, and / or furin-activated MMPs. As used herein, "matrix metalloproteinase" and "MMP" encompass the entire MMP family, including but not limited to MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-18, MMP-19, MMP-20, MMP-21, MMP-23, MMP-25, MMP-26, MMP-27 and MMP-28.

[0095] As used herein, the terms "co-administration," "co-administering," "administered in combination with," "administering in combination with," "simultaneous," and "concurrent" encompass the administration of two or more active pharmaceutical ingredients to a subject (in a preferred embodiment of the present disclosure, for example, at least one anti-inflammatory compound in combination with a viral vector functionally engineered to deliver a gene-edited nucleic acid as described herein) such that the active pharmaceutical ingredients and / or their metabolites are present in the subject at the same time. Co-administration includes simultaneous administration in separate compositions, administration in separate compositions at different times, or administration in a composition in which two or more active pharmaceutical ingredients are present. Simultaneous administration in separate compositions and administration in a composition in which two agents are present simultaneously are preferred.

[0096] The term "effective amount" or "therapeutically effective amount" refers to an amount of a composition or combination of compositions as described herein that is sufficient to achieve the intended application, including but not limited to disease treatment. The therapeutically effective amount can vary depending on the intended application (in vitro or in vivo), or the subject and disease condition being treated (e.g., the subject's weight, age, and sex), the severity of the disease condition, or the mode of administration. The term also applies to a dose that will induce a specific response in the target cell (e.g., reduction in platelet adhesion and / or cell migration). The specific dose will vary depending on the specific composition selected, the dosing regimen followed, whether the composition is administered in combination with other compositions or compounds, the timing of administration, the tissue to be administered, and the physical delivery system in which the composition is carried.

[0097] The terms "treatment", "treating", "treat", and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or adverse effects attributable to the disease. For example, the compositions, methods, or systems of the present disclosure may be administered as a preventive treatment to a subject who is predisposed to a given condition (e.g., arthritis). As used herein, "treatment" encompasses any treatment of a disease in a mammal (particularly in humans, canines, felines, or equines), and includes: (a) preventing the occurrence of the disease in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., preventing the development or progression of the disease; and (c) alleviating the disease, i.e., causing regression of the disease and / or alleviating one or more symptoms of the disease. "Treatment" is also meant to encompass the delivery of an agent so as to provide a pharmacological effect even in the absence of a disease or condition. For example, "treatment" encompasses the delivery of a composition that can elicit an immune response or confer immunity in the absence of a disease condition, such as in the case of a vaccine. It should be understood that the compositions and methods of the present disclosure are suitable for treating all mammals, including but not limited to human, canine, feline, equine, and bovine subjects.

[0098] The term "heterologous" when used with reference to portions of a nucleic acid or protein indicates that the nucleic acid or protein includes two or more subsequences that are not found in the same relationship to each other in nature. For example, nucleic acids are often recombinantly produced, with two or more sequences from unrelated genes arranged to make a new functional nucleic acid, such as a promoter from one source and a coding region from another source, or coding regions from different sources. Similarly, a heterologous protein indicates that the protein includes two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0099] The terms "polynucleotide", "nucleotide" and "nucleic acid" are used interchangeably herein and refer to all forms of nucleic acids, oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Polynucleotides include genomic DNA, cDNA and antisense DNA, as well as spliced ​​or unspliced ​​mRNA, rRNA, tRNA, lncRNA, RNA antagomirs and inhibitory DNA or RNA (RNAi, for example, small or short hairpin (sh) RNA, microRNA (miRNA), aptamer, small or short interfering (si) RNA, trans-splicing RNA or antisense RNA). Polynucleotides also include non-coding RNA, which includes but is not limited to RNAi, miRNA, lncRNA, RNA antagomirs, aptamers and any other non-coding RNA known to those skilled in the art. Polynucleotides include naturally occurring, synthetic and intentionally changed or modified polynucleotides and analogs and derivatives. The term "polynucleotide" also refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides or their analogs, and is synonymous with nucleic acid sequences. Polynucleotides can include modified nucleotides, such as methylated nucleotides and nucleotide analogs, and can be interrupted by non-nucleotide components. If present, the modification of the nucleotide structure can be given before or after the assembly of the polymer. As used herein, the term polynucleotide refers to double-stranded molecules and single-stranded molecules interchangeably. Unless otherwise specified or required, any embodiment of polynucleotides as described herein covers double-stranded forms and each of the two complementary single-stranded forms known or predicted to constitute double-stranded forms. Polynucleotides can be single-stranded, double-stranded or triple-stranded, linear or circular, and can be any length. When discussing polynucleotides, this article can describe the sequence or structure of specific polynucleotides according to the convention of providing sequences in the 5' to 3' direction.

[0100] The term "gene" or "nucleotide sequence encoding a polypeptide" refers to a segment of DNA involved in producing a polypeptide chain. The DNA segment may include regions before and after the coding region (leader and trailer) involved in the transcription / translation and regulation of transcription / translation of the gene product, as well as intervening sequences (introns) between the various coding segments (exons). For example, a gene comprises a polynucleotide containing at least one open reading frame that is capable of encoding a specific protein or polypeptide after being transcribed and translated.

[0101] In terms of nucleotide sequences, the term "homologous" comprises nucleotide (nucleic acid) sequences that are identical or substantially similar to a known reference sequence. In one embodiment, the term "homologous nucleotide sequence" is used to characterize a sequence having a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a known reference sequence.

[0102] "Heterologous" means an entity that is derived from a different genotype than the rest of the entity being compared. For example, a polynucleotide introduced into a plasmid or vector derived from a different species by genetic engineering techniques is a heterologous polynucleotide. A promoter removed from its native coding sequence and operably linked to the coding sequence is not naturally found linked to a heterologous promoter. Although the term "heterologous" is not always used herein to refer to a polynucleotide, even in the absence of the modifier "heterologous", reference to a polynucleotide is intended to include a heterologous polynucleotide, albeit omitted.

[0103] In the context of two or more nucleic acids or polypeptides, the terms "sequence identity", "percentage of identity" and "percentage of sequence identity" (or their synonyms, e.g., "99% identity") refer to two or more identical sequences or subsequences, or to nucleotides or amino acid residues having the same specified percentage, without considering any conservative amino acid substitutions as part of the sequence identity when compared and aligned (if necessary, introducing gaps) for maximum correspondence. Percentage of identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences. Suitable programs for determining percent sequence identity include, for example, the BLAST suite of programs available from the BLAST website of the National Center for Biotechnology Information of the U.S. Government. Comparisons between two sequences can be obtained using the BLASTN or BLASTP algorithms. BLASTN is used for comparing nucleic acid sequences, while BLASTP is used for comparing amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, California), or MegAlign, available from DNASTAR, are additional publicly available software programs that can be used to align sequences. ClustalW and ClustalX can be used to generate alignments, Larkin et al., Bioinformatics, 23:2947-2948 (2007); Goujon et al., Nucleic Acids Research, 38 Suppl: W 695-9 (2010); and McWilliam et al., Nucleic Acids Research, 41 (WebServer issue): W 597-600 (2013). One skilled in the art can determine appropriate parameters for maximum alignment by a particular alignment software. In certain embodiments, the default parameters of the alignment software are used.

[0104] As used herein, the term "variant" encompasses, but is not limited to, antibodies or fusion proteins comprising an amino acid sequence that differs from the amino acid sequence of a reference antibody by one or more substitutions, deletions and / or additions at certain positions within or near the amino acid sequence of the reference antibody. Variants may include one or more conservative substitutions in the amino acid sequence compared to the amino acid sequence of the reference antibody. Conservative substitutions may involve, for example, substitutions of similarly charged or uncharged amino acids. Variants retain the ability to specifically bind to the antigen of the reference antibody. The term variant also encompasses pegylated antibodies or proteins.

[0105] "Joint disease" is defined as measurable abnormalities in the cells or tissues of the joint that may lead to disease, e.g., metabolic and molecular disturbances that trigger anatomical and / or physiological changes in the joint. Radiographic findings including, but not limited to, joint space narrowing, subchondral sclerosis, subchondral cysts, and osteophyte formation.

[0106] "Joint disorder" in human subjects is defined as symptoms that drive the subject to seek medical intervention, e.g., subject-reported pain, stiffness, swelling, or immobility. For non-human mammals, "joint disorder" is defined as, e.g., lameness, observable changes in gait, weight bearing, allodynia, or exploratory behavior.

[0107] As used herein, sgRNA (single-stranded guide RNA) is an RNA composed of a targeting sequence and a scaffold, preferably a synthetic RNA. It is used to guide Cas9 to a specific genomic locus in a genome engineering experiment. sgRNA can be applied or formulated, for example as a synthetic RNA, or as a nucleic acid comprising a sequence encoding a gRNA, which is then expressed in a target cell. As will be apparent to those of ordinary skill in the art, various tools can be used to design and / or optimize the sequence of sgRNA, for example to increase the specificity and / or accuracy of genome editing. In general, candidate sgRNAs can be designed based on any available web-based tools by identifying sequences with high predicted on-target efficiency and low off-target efficiency in the target region. Candidate sgRNAs can be further evaluated by manual inspection and / or experimental screening. Examples of web-based tools include, but are not limited to, CRISPR seek, CRISPR design tools, Cas-OFFinder, E-CRISP, ChopChop, CasOT, CRISPR direct, CRISPOR, BREAKING-CAS, CrispRGold, and CCTop. See, for example, Safari et al., Current Pharma. Biotechol. (2017) 18 (13), which is incorporated herein by reference in its entirety for all purposes. Such tools are also described in, for example, PCT Publication No. WO2014093701A1 and Liu et al., "Computational approached for effective CRISPR guide RNA design and evaluation", Comput Struct Biotechnol J., 2020; 18: 35-44, each of which is incorporated herein by reference in its entirety for all purposes.

[0108] As used herein, "Cas9" refers to CRISPR-associated proteins; Cas9 nucleases are active enzymes of type II CRISPR systems. "nCas9" refers to Cas9 with one of the two inactivated nuclease domains (i.e., RuvC or HNH domains). nCas9 can only cut one strand of the target DNA (a "nickase"). The term "Cas9" refers to an RNA-guided double-stranded DNA binding nuclease protein or a nickase protein or a variant thereof. In this article, "Cas9" refers to both naturally occurring and recombinant Cas9. Wild-type Cas9 nucleases have two functional domains, such as RuvC and HNH, that cut different DNA chains. The Cas9 enzyme described herein may include an HNH or HNH-like nuclease domain and / or a RuvC or RuvC-like nuclease domain. When both functional domains are active, Cas9 can induce double-strand breaks in genomic DNA (target locus). The Cas9 enzyme may include one or more catalytic domains of a Cas9 protein derived from a bacterium belonging to the group consisting of Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, In some embodiments, the two catalytic domains are derived from different bacterial species.

[0109] As used herein, "PAM" refers to the protospacer adjacent motif, and is necessary for Cas9 to bind to the target DNA, and is immediately after the target sequence. Cas9 can be applied or formulated, for example, as a protein (e.g., a recombinant protein), or as a nucleic acid comprising a sequence encoding the Cas9 protein, which is then expressed in the target cell. Naturally occurring Cas9 molecules recognize specific PAM sequences (e.g., PAM recognition sequences of Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus mutans, Staphylococcus aureus, and Neisseria meningitidis). In one embodiment, the Cas9 molecule has the same PAM specificity as the naturally occurring Cas9 molecule. In other embodiments, the Cas9 molecule has a PAM specificity that is unrelated to the naturally occurring Cas9 molecule. In other embodiments, the PAM specificity of the Cas9 molecule is independent of the naturally occurring Cas9 molecule to which it has the closest sequence homology. For example, the naturally occurring Cas9 molecule can be changed so that the PAM sequence recognition is changed to reduce off-target sites, improve specificity, or eliminate PAM recognition requirements. In one embodiment, the Cas9 molecule can be altered (e.g., to extend the PAM recognition sequence, improve Cas9 specificity to a high level of identity, reduce off-target sites, and / or increase specificity). In one embodiment, the PAM recognition sequence is at least 4, 5, 6, 7, 8, 9, 10, or 15 amino acids in length. In some embodiments, the Cas9 molecule can be altered to ablate PAM recognition.

[0110] An "expression cassette" is a recombinant or synthetically produced nucleic acid construct having a series of specified nucleic acid elements that allow a specific polynucleotide sequence to be transcribed in a host cell. An expression cassette or vector can be part of a plasmid, a viral genome, or a nucleic acid fragment. Typically, an expression cassette or vector comprises a polynucleotide to be transcribed, which is operably linked to a promoter.

[0111] The term "promoter" is used herein to refer to an array of nucleic acid control sequences that direct the transcription of nucleic acids. As used herein, a promoter comprises a necessary nucleic acid sequence near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. The promoter also optionally comprises a distal enhancer or repressor element, which may be located at a position up to several thousand base pairs from the start site of transcription. Other elements that may be present in the expression vector include elements that enhance transcription (e.g., enhancers) and terminate transcription (e.g., terminators), as well as elements that confer certain binding affinity or antigenicity to the recombinant protein produced by the expression vector.

[0112] The term "operably linked" refers to the juxtaposition of genetic elements in which the elements are in a relationship that allows them to operate in the intended manner. For example, a promoter is operably linked to a coding region if the promoter helps to initiate transcription of a coding sequence. Intervening residues may exist between the promoter and the coding region as long as this functional relationship is maintained.

[0113] An "isolated" plasmid, nucleic acid, vector, virus, virion, host cell or other substance refers to a preparation of a substance that is free of at least some other components present when the substance or a similar substance is naturally present or originally prepared therefrom. Thus, for example, an isolated substance can be prepared by using a purification technique to enrich the isolated substance from a source mixture. Enrichment can be measured on an absolute basis, such as weight per volume of solution, or can be measured relative to a second potentially interfering substance present in the source mixture. The increased enrichment of the embodiments of the present disclosure is increasingly isolated. In some embodiments, the isolated plasmid, nucleic acid, vector, virus, host cell or other substance is purified, for example, about 80% to about 90% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, or at least about 99% or more pure.

[0114] As used herein, "AAV vector" refers to an AAV vector nucleic acid sequence encoding various nucleic acid sequences, including variants or chimeric capsid polypeptides in some embodiments (i.e., the AAV vector includes a nucleic acid sequence encoding a variant or chimeric capsid polypeptide). The AAV vector may also include a heterologous nucleic acid sequence from a non-AAV source as a part of a nucleic acid insert. This heterologous nucleic acid sequence generally includes a sequence of interest for genetic transformation of cells. Typically, the flank of the heterologous nucleic acid sequence is flanked by at least one AAV inverted terminal repeat (ITR), and there are generally two AAV inverted terminal repeats (ITR). In certain embodiments, when applied to a subject, the Cas sequence, guide RNA sequence, and any other genetic element (e.g., promoter sequence, PAM sequence, etc.) may be on the same AAV vector, or on two or more different AAV vectors. In certain embodiments, when applied to a subject, the Cas sequence, guide RNA sequence, and any other genetic element (e.g., promoter sequence, PAM sequence, etc.) may be on two or more different AAV vectors, and the AAV may be the same serotype, or the AAV may be two or more different serotypes (e.g., AAV5 and AAV6).

[0115] "AAV virion" or "AAV virus" or "AAV viral particle" or "AAV vector particle" refers to a viral particle composed of at least one AAV capsid polypeptide and an encapsulated polynucleotide AAV transfer vector. If the particle includes a heterologous nucleic acid (i.e., a polynucleotide different from the wild-type AAV genome, such as a transgene to be delivered to a cell), the particle may be referred to as an "AAV vector particle" or simply "AAV vector". Therefore, the production of AAV virions or AAV particles necessarily includes the production of AAV vectors, because such vectors are contained within AAV virions or AAV particles.

[0116] As used herein, "carrier" or "vehicle" refers to a carrier material suitable for drug administration. Carriers and vehicles useful herein include any such materials known in the art, such as any liquid, gel, solvent, liquid diluent, solubilizer, surfactant, etc., which are non-toxic and do not interact with other components of the composition in a deleterious manner.

[0117] The phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0118] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inert ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Except where any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the present disclosure is contemplated. Additional active pharmaceutical ingredients, such as other medicaments, may also be incorporated into the described compositions and methods.

[0119] The term "pharmaceutically acceptable excipient" is intended to include media and carriers that can be co-administered with a compound to promote its intended function. The use of such media for pharmaceutically active substances is well known in the art. Examples of such media and carriers include solutions, solvents, dispersion media, delay agents, emulsions, etc. Any other conventional carriers suitable for use with multi-binding compounds also fall within the scope of the present disclosure.

[0120] As used herein, the terms "a," "an," or "the" are generally construed to cover both the singular and the plural.

[0121] The terms "about" and "approximately" mean within a statistically significant range of values. This range can be within the magnitude of a given value or range, preferably within 50%, more preferably within 20%, more preferably still within 10%, and even more preferably within 5%. The allowable variation of the terms "about" or "approximately" encompassed by the term depends on the specific system under study, and can be easily understood by those of ordinary skill in the art. In addition, as used herein, the terms "about" and "approximately" mean that the composition, amount, formula, parameter, shape and other quantities and features are not precise and do not need to be precise, but can be approximate and / or larger or smaller as needed, reflecting tolerances, conversion factors, rounding, measurement errors, etc. and other factors known to those skilled in the art. In general, size, size, formula, parameter, shape or other quantity or feature is "about" or "approximate", whether or not it is explicitly stated as such. It should be noted that embodiments of very different sizes, shapes and sizes can adopt the described arrangements.

[0122] As used herein, the term "substantially" may refer to a majority or majority, such as at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more.

[0123] When used in the appended claims, in both original and amended forms, the transitional terms "comprising," "consisting essentially of," and "consisting of" define the scope of the claims with respect to which unrecited appended claim elements or steps, if any, are excluded from the scope of the claims. The term "comprising" is intended to be inclusive or open-ended, and does not exclude any additional, unrecited elements, methods, steps, or materials. The term "consisting of" excludes any elements, steps, or materials other than those specified in the claim, and in the latter case, excludes impurities normally associated with the specified materials. The term "consisting essentially of" limits the scope of the claim to the specified elements, steps, or materials, as well as those elements, steps, or materials that do not materially affect the basic and novel features of the claimed methods and compositions. In alternative embodiments, all compositions, methods, and kits described herein that embody the present disclosure may be more specifically defined by any of the transitional terms "comprising," "consisting essentially of," and "consisting of."

[0124] The subject treated by any of the methods or compositions described herein can be of any age, and can be an adult, infant, or child. In some cases, the subject is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 , 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 years old or within a range therein (e.g., but not limited to, between 2 and 20 years old, between 20 and 40 years old, or between 40 and 90 years old). The subject can be a human or non-human subject. A particular class of subjects that may benefit from the compositions and methods of the present disclosure includes subjects that are over 40, 50 or 60 years old. Another class of subjects that may benefit from the compositions and methods of the present disclosure are subjects with arthritis (e.g., osteoarthritis).

[0125] Any of the compositions disclosed herein can be administered to non-human subjects, such as laboratory or farm animals. Non-limiting examples of non-human subjects include laboratory or research animals, pets, wild or domestic animals, farm animals, etc., for example, dogs, goats, guinea pigs, hamsters, mice, pigs, non-human primates (e.g., gorillas, apes, orangutans, lemurs, baboons, etc.), rats, sheep, horses, cattle, etc.

[0126] CRISPR / Cas systems – minimum requirements

[0127] In one aspect, clustered regularly interspaced short palindromic repeats and CRISPR-associated RNA-guided nuclease-related methods, components, and compositions of the present disclosure (hereinafter referred to as CRISPR / Cas systems) maximally require at least one isolated or non-naturally occurring protein component (e.g., a Cas protein) and at least one isolated or non-naturally occurring nucleic acid component (e.g., a guide RNA (gRNA)) to achieve amplification of a nucleic acid sequence (e.g., genomic DNA).

[0128] In some embodiments, the CRISPR / Cas system effects alteration of a targeted gene or locus in a eukaryotic cell by effecting an alteration of the sequence at the target location (e.g., by creating insertions or deletions (collectively referred to as indels) that result in loss of function (i.e., knockout) of the affected gene or allele; for example, nucleotide substitutions that result in truncation, nonsense mutations, or other types of loss of function of the encoded gene product (e.g., the encoded IL1A or IL1B mRNA or protein); deletions of one or more nucleotides that result in truncation, nonsense mutations, or other types of loss of function of, for example, the encoded IL1A or IL1B gene product; loss of function of the encoded mRNA or protein, such as caused by single-nucleotide, dinucleotide, or other frameshift deletions or deletions that result in premature stop codons; or deletions that result in a gene product (e.g., such as the encoded IL1A or IL1B mRNA or protein) truncation, nonsense mutation or other types of functional loss insertion; such as single nucleotide, dinucleotide or other frameshift insertion, or insertion leading to premature stop codon. In some embodiments, the CRISPR / Cas system of the present disclosure provides for the alteration (e.g., knockout) of genes associated with inflammatory joint diseases (e.g., rheumatoid arthritis or osteoarthritis) by changing the sequence at the target position, such as creating an insertion deletion that leads to nonsense-mediated decay of the encoded gene product (e.g., encoded transcript).

[0129] In one aspect, the CRISPR / Cas system achieves changes in nucleic acid sequences through nuclease activity. For example, in the case of genomic DNA, the nuclease - guided by a protein-related exogenous nucleic acid that localizes the target position within a targeted gene or locus by sequence complementarity with a target genomic sequence (e.g., a complementary component of a CRISPR RNA (crRNA) or a synthetic single guide RNA (sgRNA)) - cuts the genomic DNA upon recognition of a specific nuclease-specific motif (called a protospacer adjacent motif (PAM). See generally Collias, D., & Beisel, CL (2021), Nature Communications, 12(1), 1-12.

[0130] In the case of genomic DNA, nuclease activity (i.e., cutting) induces double-strand breaks (DSBs). Due to the interference of the components from the CRISPR / Cas system, the endogenous cell mechanism of DSB repair, i.e., non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ) and homologous recombination, results in error repair at a given target position with a certain calculable frequency, thereby introducing substitution or insertion and deletion into genomic DNA. Generally referring to Scully, R. et al., (2019), "Nature Reviews Molecular Cell Biology (Nature Reviews Molecular Cell Biology)", 20 (11), 698-714. At a certain frequency, these insertion and deletion and / or substitution may cause frameshifts, nonsense mutations (i.e., early stop codons) or truncations that affect the availability of gene products such as mRNA and / or proteins. In certain embodiments, the CRISPR / Cas system can be provided to cells as part of the system together with nucleases and gRNA by using a template (e.g., HDR template) to induce homology-directed repair (HDR) mechanism, resulting in the insertion of non-random sequences at the target position. See Bloh, K., & Rivera-Torres, N. (2021), International Journal of Molecular Sciences, 22(8), 3834.

[0131] In general, the minimum requirements of a CRISPR / Cas system will depend on the nuclease (i.e., Cas protein) provided with it. In this regard, these bacterially derived nucleases have been functionally classified into types I, III, and V (all of which belong to class 1) and II, IV, and VI (which are grouped into class 2).

[0132] Type 1 CRISPR / Cas system:

[0133] The exact components, compositions and methods for using Class 1 CRISPR / Cas systems to achieve changes in the targeted nucleic acid sequence will vary, but should minimally include: a nuclease (selected from at least Type I and Type III), at least one guide RNA selected from a combination of 1) sgRNA or 2) crRNA and tracrRNA. These CRISPR / Cas systems are classified together as Class 1 CRISPR / Cas systems due to their similarities in requirements and modes of action within eukaryotic cells. For this reason, the compositions, components and methods in Class 1 components can be considered functionally interchangeable, and the following details provided for exemplary purposes only do not represent an exhaustive list of class members:

[0134] Cas3 (see Table 1) is a prototype type I DNA nuclease that is used as an effector protein as part of a larger complex (a cascade complex containing Cse1 and Cse2) capable of genome editing. See generally He, L. et al. (2020), Genes, 11 (2), 208. Unlike other CRISPR / Cas systems, the type I system is located at a DNA target without a Cas3 nuclease via a cascade complex, and then the cascade complex recruits Cas3 to cut DNA when it binds and locates 3'PAM. The cascade complex is also responsible for processing crRNA so that they can be used to guide it to the target position. Due to this function, the cascade has the ability to process multiple array crRNAs from a single molecule. See Luo, M. (2015), Nucleic Acids Research, 43 (1), 674-681. Therefore, the type I system can be used to edit multiple targeted genes or loci from a single molecule.

[0135] Because the natural Cas3 substrate is ssDNA, its function in genome editing is thought to be a nickase; however, when targeted in tandem, the resulting edit is a blunt-end cleavage of the opposite strand to approximate the result of a blunt-cutting nuclease such as Cas9. See Pickar-Oliver, A., & Gersbach, CA (2019), Nature Reviews Molecular Cell Biology, 20(8), 490-507.

[0136] Like type I nucleases, type III systems rely on protein complexes to achieve nucleic acid cleavage. In particular, Cas10 has the nuclease activity of cutting ssDNA in prokaryotes. See Tamulaitis, G. Trends in Microbiology, 25 (1), 49-61. Interestingly, the CRISPR / Cas system (natural to archaea) exhibits dual specificity and targets both ssDNA and ssRNA. In addition to this change, the function of the system is very similar to type I, because crRNA targets the effector complex in a sequence-dependent manner (similar to cascade). Similarly, the effector complex processes crRNA before association. The dual nature of this nuclease makes its application in genome editing potentially more powerful, because genomic DNA and mRNA with the same sequence in some cases can be targeted to silence specific targeted genes.

[0137] Type 2 CRISPR / Cas systems:

[0138] The exact components, compositions and methods for using Class 2 CRISPR / Cas systems to achieve changes in the targeted nucleic acid sequence will vary, but should minimally include: a nuclease (selected from at least Type II and Type V), at least one guide RNA selected from a combination of 1) sgRNA or 2) crRNA and tracrRNA. These CRISPR / Cas systems are classified together as Class 2 CRISPR / Cas systems due to their similarities in requirements and modes of action within eukaryotic cells. For this reason, the compositions, components and methods in Class 2 components can be considered functionally interchangeable, and the following details provided for exemplary purposes only do not represent an exhaustive list of class members:

[0139] Type II nucleases are the best characterized CRISPR / Cas systems, specifically the prototypical genome editing nuclease Cas9 (see Table 1). A variety of Cas9 proteins have been isolated from various bacterial species. The main difference between these nucleases is the PAM, a recognition site within the desired dsDNA for targeting. Upon association with the gRNA molecule, the crRNA (or the targeting domain of the sgRNA) directs the nuclease to the appropriate location, but recognition of the PAM by the protein is what induces the cleavage event near this site, resulting in a blunt DSB.

[0140] In addition to the Cas9 protein of natural origin, several engineered variants are also reported similarly. These range from Cas9 with enhanced specificity (i.e., lower off-target activity), such as espCas9. Others have been catalytically modified via point mutations in RuvC (e.g., D10A) and HNH (e.g., H840A) domains, so that they only induce single-strand breaks (i.e., Cas9 nickase). See Frock, R. et al., (2015), "Nature Biotechnology (Nature Biotechnology)", 33 (2), 179-186. These variants, collectively referred to herein as enhanced specific Cas9 variants (spCas9), have also been shown to be less prone to error in editing. When selecting the desired insertion (i.e., knock-in mutation, wherein the desired nucleotide sequence is introduced into the target nucleic acid molecule) rather than missing, such relief of off-target effects becomes crucial. In fact, less off-target effects can contribute to preferred DNA repair mechanisms (HDR, in most cases for knock-in mutations). See generally Naeem, M. et al. (2020), Cells, 9(7), 1608.

[0141] Typical Cas proteins (e.g., mutants, chimeras, and additional exemplary additional engineered variants including the following (which are hereby incorporated by reference): WO2015035162A2, WO2019126716A1, WO2019126774A1, WO2014093694A1, and WO2014150624A1.

[0142] For the avoidance of doubt, spCas9 is collectively referred to as any one of the group consisting of: espCas9 (also referred to herein as ESCas9 or esCas9), HFCas9, PECas9, arCas9.

[0143] Like the typical Cas9 system, the V-type nuclease only requires a synthetic sgRNA with a targeting domain complementary to the genomic sequence for genome editing. These nucleases contain the RuvC domain, but lack the HNH domain of the II-type nuclease. In addition, for example, compared with the blunt incision of Cas9 next to the PAM, Cas12 leaves a staggered incision in the dsDNA substrate far from the PAM. Both Cas12a (also known as Cpf1) and Cas12b (also known as C2c1) (see Table 1) act as part of a larger complex of two gRNA-related nucleases, which act on dsDNA with a quaternary structure and cut each chain at the same time. See Zetsche B et al., Cell, 2015; 163 (3): 759-771.; See also Liu L, Chen P, Wang M et al., Molecular Cell, 2017; 65 (2): 310-322. In addition, Cas12b (C2c1) is a highly accurate nuclease with little tolerance to mismatches. See Yang H et al., Cell, 2016; 167(7): 1814-1828.e12.

[0144] Table 1. Exemplary list of Cas nucleases and their requirements

[0145]

[0146] See generally Wang, J., Zhang, C., & Feng, B. (2020), Journal of Cellular and Molecular Medicine, 24(6), 3256-3270, wherein N = any nucleotide; R = any purine (A or G); Y = any pyrimidine (C or T); W = A or T; V = A, C or G.

[0147] In one aspect, the CRISPR / Cas system of the present disclosure comprises at least one Cas protein derived from one or more of the following selected bacterial genera: Corynebacterium, Sutterella, Legionella, Treponema, Filamentosa, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flavobacterium, Spirochete, Azospirillum, Gluconacetobacter, Neisseria, Rose, Parvibacillum, Nitrobacter, Campylobacter, Pseudomonas, Streptomyces, Staphylococcus, Francisella, Acidobacterium, Lachnospira, Leptotrichia, and Prevotella. In some embodiments, the Cas protein is derived from a δ-Proteobacteria or Planctomycete bacterial species.

[0148] Some aspects of the present disclosure provide strategies, methods, compositions and treatment modes for changing the targeting sequence within the locus (e.g., changing the sequence of wild-type and / or mutants in cells or patients with or experiencing rheumatoid arthritis, osteoarthritis, or other inflammatory diseases of the joints or cells therein) by inserting or deleting one or more nucleotides mediated by RNA-guided nucleases and one or more guide RNAs (gRNAs), thereby resulting in loss of function of the targeted gene product. Such changes may alternatively be referred to as "knockouts" of the gene of interest (i.e., "knockouts" generation).

[0149] In certain embodiments, any region of the IL1A gene (e.g., 5' untranslated region [UTR], exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, intronic region, intron / exon junction, 3'UTR, or polyadenylation signal) is targeted by an RNA-guided nuclease to alter the gene. In certain embodiments, any region of the IL1B gene (e.g., 5' untranslated region [UTR], exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, intronic region, intron / exon junction, 3'UTR, or polyadenylation signal) is targeted by an RNA-guided nuclease to alter the gene. In certain embodiments, the targeted gene is selected from inflammatory effectors (e.g., IL1A, IL1B, IL1R1, IL1R2, IL6, IL18, TNF, TGFB1). In some embodiments, any region of the targeted gene (e.g., promoter region, 5' untranslated region, 3' untranslated region, exon, intron, or exon / intron boundary) is targeted by an RNA-guided nuclease to alter the gene. In some embodiments, a non-coding region of the targeted gene (e.g., enhancer region, promoter region, intron, 5'UTR, 3'UTR, polyadenylation signal) is targeted to alter the gene.

[0150] CRISPR Guide RNA:

[0151] In one aspect, the CRISPR / Cas system of the present disclosure further provides a gRNA molecule (e.g., an isolated or non-naturally occurring RNA molecule) interacting with a Cas protein. In certain embodiments, gRNA is sgRNA, wherein the targeting (i.e., complementary) domain comprising a nucleotide sequence complementary to the target domain from a targeted gene is incorporated into a single RNA molecule with a protein interaction domain. In certain embodiments, the targeting domain is crRNA, which is provided to a eukaryotic cell together with tracrRNA, which acts as a scaffold by interacting with crRNA and nuclease. In some embodiments, the system is further optionally composed of oligonucleotides, which are HDR templates with homology to either side of the target position. See Bloh, K., & Rivera-Torres, N, at 3836.

[0152] In one embodiment, the targeting domain of the gRNA molecule is configured to direct the associated nuclease so that a cutting event (e.g., a double-strand break or a single-strand break) occurs in a targeted gene or locus close enough to the target position to promote changes in the nucleic acid sequence. In some embodiments, the length of the targeting domain is 20 nucleotides. In some embodiments, the length of the targeting domain is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides.

[0153] In some embodiments, the targeting domain directs the nuclease so that the cleavage event occurs within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, or 200 nucleotides of the target position. The double-stranded or single-stranded break can be located upstream or downstream of the target position and within or upstream of the functional domain cluster within the targeted gene.

[0154] In certain embodiments, the second gRNA molecule comprising the second targeting domain directs the second related nuclease so that the cleavage event occurs in the targeted gene or locus close enough to the target position, thereby promoting the change of the nucleic acid sequence. In one embodiment, the second gRNA molecule targets the same targeted gene or locus as the first gRNA molecule. In other embodiments, the second gRNA molecule targets a targeted gene or locus different from the first gRNA molecule. In some embodiments, the length of the second targeting domain is 20 nucleotides. In some embodiments, the length of the second targeting domain is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides.

[0155] In some embodiments, the second targeting domain directs the nuclease so that the cleavage event occurs within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, or 200 nucleotides of the target position. The double-stranded or single-stranded break can be located upstream or downstream of the target position and within or upstream of the functional domain cluster within the targeted gene.

[0156] In one embodiment, the targeting domains of the first gRNA molecule and the second gRNA molecule are configured so that the cleavage event is independently positioned within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175 or 200 nucleotides of the corresponding target position for each gRNA molecule. In one embodiment, the first gRNA molecule and the second gRNA molecule change the targeting nucleic acid sequence simultaneously. In one embodiment, the first gRNA molecule and the second gRNA molecule change the targeting nucleic acid sequence in sequence.

[0157] In one embodiment, the single-strand break is accompanied by the second single-strand break, which is located by the targeting domain of the first gRNA molecule and the second gRNA molecule, respectively. For example, the targeting domain can direct the relevant nuclease so that the cutting event (e.g., two single-strand breaks) is located within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175 or 200 nucleotides of the target position. In one embodiment, the targeting domain of the first gRNA molecule and the second gRNA molecule is configured to direct the relevant nuclease so that, for example, two single-strand breaks occur in the same target position on the relative chain of the genomic DNA, or within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides from each other, thereby substantially approximating a double-strand break.

[0158] In one embodiment, the nucleic acid encoding the Figure 5In one embodiment, the nucleic acid encodes the targeting domain of the first gRNA molecule and the targeting domain of the second gRNA molecule selected from the sequence in Figure 7. In one embodiment, the nucleic acid encodes the first sgRNA molecule. In one embodiment, the nucleic acid encodes the second sgRNA molecule. In one embodiment, the nucleic acid encodes the third sgRNA molecule. In one embodiment, the nucleic acid encodes the fourth sgRNA molecule.

[0159] In one embodiment, the nucleic acid encoding the Figure 5 In one embodiment, the nucleic acid encodes a crRNA sequence of the first gRNA molecule of the sequence in Figure 7 and a crRNA sequence of the second gRNA molecule. In one embodiment, the nucleic acid encodes a crRNA sequence of the first gRNA molecule of the sequence in Figure 7 and a crRNA sequence of the second gRNA molecule. In one embodiment, the nucleic acid encodes a first gRNA molecule consisting of a crRNA sequence and tracrRNA. In one embodiment, the nucleic acid encodes a second gRNA molecule consisting of a crRNA sequence and tracrRNA. In one embodiment, the nucleic acid encodes a third gRNA molecule consisting of a crRNA sequence and tracrRNA. In one embodiment, the nucleic acid encodes a fourth gRNA molecule consisting of a crRNA sequence and tracrRNA.

[0160] In certain embodiments, the nucleic acid encodes a first gRNA molecule and a second gRNA molecule comprising a chimeric gRNA molecule. In other embodiments, the nucleic acid encodes a first gRNA molecule, a second gRNA molecule, and a third gRNA molecule comprising a chimeric gRNA molecule. In some embodiments, the nucleic acid encodes a first gRNA molecule, a second gRNA molecule, a third gRNA molecule, and a fourth gRNA molecule comprising a chimeric gRNA molecule.

[0161] In certain embodiments, the nucleic acid may include (a) a sequence encoding a first gRNA molecule comprising a targeting domain complementary to a target position in a targeted gene or locus; (b) a sequence encoding a second gRNA molecule comprising a targeting domain complementary to a target position in a second targeted gene or locus; and (c) a sequence encoding an RNA-guided nuclease (e.g., Cas9 or other Cas proteins). Optionally, (d) and (e) are sequences encoding a third gRNA molecule and a fourth gRNA molecule, respectively. In some embodiments, the second targeted gene or locus is the same as the first targeted gene or locus. In other embodiments, the second targeted gene or locus is different from the first targeted gene or locus. In some embodiments, (a), (b) and (c) are encoded in the same nucleic acid molecule (i.e., the same vector, the same viral vector, the same adeno-associated virus (AAV) vector). In some embodiments, (a) and (b) are encoded in the same nucleic acid molecule. In some embodiments, (a), (b) and (d) are encoded in the same nucleic acid molecule. In some embodiments, (a), (b), and (e) are encoded in the same nucleic acid molecule. In some embodiments, (a), (b), (d), and (e) are encoded in the same nucleic acid molecule. In some embodiments, (a), (b), and (c) are encoded in separate nucleic acid molecules. When more than two gRNAs are used, any combination of (a), (b), (c), (d), and (e) can be encoded in a single or separate nucleic acid molecule.

[0162] In one embodiment, the nucleic acid molecule is an AAV vector. Exemplary AAV vectors that can be used with any CRISPR / Cas system of the present disclosure include AAV1 vectors, AAV2 vectors, AAV3 vectors, AAV4 vectors, AAV5 vectors, AAV6 vectors, AAV7 vectors, AAV8 vectors, AAV9 vectors, AAV10 vectors.

[0163] In some embodiments, (a), (b), and (c) are encoded in the same vector. In some embodiments, (a) and (b) are encoded in the same vector. In some embodiments, (a), (b), and (d) are encoded in the same vector. In some embodiments, (a), (b), and (e) are encoded in the same vector. In some embodiments, (a), (b), (d), and (e) are encoded in the same vector. In some embodiments, (a), (b), and (c) are encoded in separate vectors. When more than two gRNAs are used, any combination of (a), (b), (c), (d), and (e) can be encoded in a single or separate vector.

[0164] In one aspect, nucleic acid molecules (i.e., those encoding (a), (b), (c), (d) or (e)) are delivered to a target cell (i.e., any combination of an RNA-guided nuclease encoded by (c) and at least one gRNA molecule encoded by (a), (b), (d) or (e) contacts the target cell). In some embodiments, the nucleic acid molecules are delivered to the target cell in vivo. In other embodiments, the nucleic acid molecules are delivered to the target cell ex vivo. In some embodiments, the nucleic acid molecules are delivered to the target cell in vitro. In certain embodiments, the nucleic acid molecules are delivered to the target cell as DNA. In other embodiments, the nucleic acid molecules are delivered to the target cell as RNA (e.g., mRNA). In some embodiments, the products of the nucleic acid molecules are delivered as assembled ribonucleoproteins (RNPs).

[0165] In one embodiment, contacting the target cell comprises delivering the encoded RNA-guided nuclease of (c) as a protein or mRNA together with at least one of the nucleic acid molecules selected from (a), (b), (d) and (e).

[0166] In one embodiment, contacting the target cell comprises delivering the encoded RNA-guided nuclease of (c) as DNA together with at least one of the nucleic acid molecules selected from (a), (b), (d) and (e).

[0167] In certain embodiments, CRISPR components are delivered to target cells via nanoparticles. Exemplary nanoparticles that can be used with all CRISPR / Cas systems disclosed herein include at least lipid nanoparticles or liposomes, hydrogel nanoparticles, metal organic nanoparticles, gold nanoparticles, and magnetic nanoparticles. See generally Xu, CF et al., (2021), Advanced Drug Delivery Reviews, 168, 3-29.

[0168] Unless otherwise defined, all technical terms and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present disclosure belongs. Although methods and materials similar or equivalent to the methods and materials described herein can be used to practice or test the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In addition, materials, methods and examples are illustrative only and are not intended to be limiting.

[0169] The present disclosure provides compositions useful for treating joint disorders with an inflammatory component. In some aspects, the compositions are useful for preventing the progression of osteoarthritis in a mammal's joint and for treating osteoarthritis.

[0170] In some aspects, the pharmaceutical composition comprises a gene editing system, wherein the gene editing system silences or reduces the expression of at least one gene locus associated with joint function in at least a portion of cells comprising the joint.

[0171] In one aspect, the pharmaceutical composition includes a gene editing system, wherein the gene editing system targets one or more of IL-1α and IL-1β. In some aspects, the pharmaceutical composition includes a gene editing system, wherein the gene editing system targets one or more of components of TNF-α, IL-6, IL-8, IL-18, matrix metalloproteinases (MMPs) or NLRP3 inflammasomes.

[0172] In some aspects, the pharmaceutical composition includes a gene editing system, wherein the gene editing includes the use of a programmable nuclease that mediates the generation of double-stranded or single-stranded breaks at at least one locus associated with joint function. In some embodiments, the gene editing system reduces gene expression of one or more targeted loci. In some embodiments, at least one locus associated with joint tissue is silenced or reduced in at least a portion of cells comprising a joint.

[0173] In some aspects, the cells comprising the joint are synovial cells. In some aspects, the cells are synovial macrophages. In some aspects, the cells are synovial fibroblasts. In some aspects, at least a portion of the synovial cells are edited. In some aspects, the cells comprising the joint are chondrocytes.

[0174] In one aspect, the pharmaceutical composition targets one or more cytokines and / or growth factor genes selected from the group consisting of IL-1α, IL-1β, TNF-α, IL-6, IL-8, IL-18, matrix metalloproteinases (MMPs) or components of the NLRP3 inflammasome. In some embodiments, the components of the NLRP3 inflammasome include NLRP3, apoptosis-associated speck-like protein (ASC) containing CARD, caspase-1, and combinations thereof.

[0175] Also provided is a pharmaceutical composition, wherein the gene editing enhances the expression of one or more cytokine and / or growth factor genes in at least a portion of the cells comprising the joint, the cytokine and / or growth factor genes being selected from the group consisting of IL-1Ra, TIMP-1, TIMP-2, TIMP-3, TIMP-4, and combinations thereof.

[0176] In some embodiments, the pharmaceutical composition provides gene editing, wherein the gene editing comprises the use of a programmable nuclease that mediates the generation of double-stranded or single-stranded breaks at the one or more cytokine and / or growth factor genes. In some embodiments, gene editing comprises one or more methods selected from CRISPR methods, TALE methods, zinc finger methods, and combinations thereof.

[0177] In one aspect, gene editing includes CRISPR methods. In yet other aspects, the CRISPR method is a CRISPR-Cas9 method. In some aspects, Cas9 is mutated to enhance function.

[0178] Animal Models of Osteoarthritis

[0179] Several animal models of osteoarthritis are known in the art. Exemplary non-limiting animal models are summarized; however, it should be understood that various models can be used. Animals of many different species are used to simulate OA, for example, mice, rats, rabbits, guinea pigs, dogs, pigs, horses, and even other animals have been studied. See, for example, Kuyinu et al., Journal of Orthopedics and Research (J Orthop Surg Res.) 11:19 (2016) (hereinafter referred to as "Kuyinu, 2016").

[0180] It should be understood that various methods for inducing OA can be used in any mammal. In mice, spontaneous, chemically induced, surgically induced, and non-invasive inductions are commonly used. For example, Kuyinu, 2016; Bapat et al., Clin Transl Med, 7:36 (2018) (hereinafter referred to as "Bapat, 2018"); and Poulet, Curr Rheumatol Rep, 18:40 (2016). In horses, osteochondral fragment-exercise model, chemical induction, trauma induction, and induction by overuse are commonly used. In sheep, surgical induction is the most common; in guinea pigs, surgical induction, chemical induction, and spontaneous (Durkin Hartley) methods are often used. For example, Bapat, 2018.

[0181] The unstable medial meniscus (DMM) is often used in mice to model post-traumatic osteoarthritis, for example, Culley et al., Methods Mol Biol., 1226:143-73 (2015). The DMM model mimics clinical meniscal injury, a known susceptibility factor for the development of human OA, and allows the study of structural and biological changes during the disease process. Mice are attractive model organisms because mouse strains with defined genetic backgrounds can be used. In addition, knockout or other genetically manipulated mouse strains can be used to evaluate the importance of various molecular pathways in responding to various OA treatment modes and regimens. For example, STR / ort mice have characteristics that make the strain particularly susceptible to developing OA, including increased levels of the inflammatory cytokine IL1β, Bat et al., Clinical Metastasis Methods, 7:36 (2018). These mice typically develop OA in the knee, ankle, elbow, and temporomandibular joints, Jaeger et al., Osteoarthritis Cartilage 16:607–614 (2008). Other useful mutant strains of mice are known to the skilled artisan, such as the Col9a1 (- / -) mouse, Allen et al., Arthritis Rheum, 60:2684-2693 (2009).

[0182] Another commonly used surgical model for OA is the anterior cruciate ligament transection (ACLT) model Little and Hunter, Nat Rev Rheumatol., 9(8):485–497 (2013). The subject's ACL is surgically transected, resulting in joint instability. The anterior drawer test with the joint flexed is used to confirm that transection of the ligament has occurred. In some cases, other ligaments such as the posterior cruciate ligament, medial collateral ligament, lateral collateral ligament, and / or either meniscus may be transected. As with the DMM model, a variety of mouse strains can be used to study a variety of molecular pathways.

[0183] Animals of different sizes can be selected for use, depending on the technical goals. Rodents are useful because the time required for skeletal maturity is shorter, and therefore the time to develop OA after surgery or other techniques that induce OA is shorter. Larger animals are particularly useful for evaluating therapeutic interventions. The anatomical structures in larger animals are very similar to those in humans; for example, in dogs, the cartilage thickness is less than about half of the thickness in humans; this striking similarity is an example of why such cartilage degradation and osteochondral defect studies are more useful in large animal models. For example, McCoy, Vet. Pathol., 52: 803-18 (2015); and Pelletier et al., Therapy, 7: 621–34 (2010).

[0184] Gene editing process

[0185] Overview: Composition of gene-edited synoviocytes

[0186] Embodiments of the present disclosure relate to methods for gene editing synovial cells (synoviocytes), the methods comprising one or more steps of gene editing at least a portion of synovial cells in a joint to treat osteoarthritis or other joint conditions. As used herein, "gene editing", "gene editing" and "genome editing" refer to the type of genetic modification in which DNA is permanently modified in the genome of a cell, such as DNA inserted, deleted, modified or replaced in the genome of a cell. In some embodiments, gene editing silences the expression of a DNA sequence (sometimes referred to as gene knockout) or inhibits / reduces (sometimes referred to as gene knockout). In other embodiments, gene editing results in enhanced expression of a DNA sequence (e.g., by causing overexpression). According to embodiments of the present disclosure, gene editing techniques are used to reduce the expression of proinflammatory genes or silence proinflammatory genes and / or enhance the expression of regenerative genes.

[0187] Interleukin

[0188] According to another embodiment, the gene editing method of the present disclosure can be used to increase the expression of certain interleukins, such as one or more of IL-1α, IL-1β, IL-4, IL-6, IL-8, IL-9, IL-10, IL-13, IL-18 and TNF-α. Certain interleukins have been shown to enhance inflammatory responses in joint tissues and are associated with disease progression.

[0189] Expression constructs

[0190] The expression construct encoding one or both of the guide RNA and / or Cas9 editing enzyme can be administered in any effective carrier, such as any formulation or composition capable of effectively delivering the component genes to cells in vivo. The method includes, for example, electroporation and / or insertion of genes in viral vectors, which include recombinant retroviruses, adenoviruses, adeno-associated viruses, lentiviruses, and herpes simplex virus-1 or recombinant bacteria or eukaryotic plasmids. Viral vectors directly transfect cells; plasmid DNA can be delivered naked or with the aid of, for example, cationic liposomes (lipofectamine) or derivatives (e.g., antibody conjugated), polylysine conjugates, gram-positive bacteria S (gramacidin S), artificial viral envelopes or other such intracellular carriers, as well as direct injection of gene constructs or CaPO in vivo. 4 Delivered by precipitation.

[0191] A preferred method for introducing nucleic acid into cells in vivo is to use a viral vector containing nucleic acid, such as cDNA. Infecting cells with a viral vector has the advantage that most targeted cells can accept nucleic acid. In addition, molecules encoded in a viral vector, such as molecules encoded by a cDNA contained in a viral vector, are effectively expressed in cells that have taken up the viral vector nucleic acid.

[0192] Retroviral vectors and adeno-associated virus vectors can be used as recombinant gene delivery systems for transferring foreign genes in vivo, particularly in humans. These vectors provide effective delivery of genes to cells. In some cases, the nucleic acid transferred is stably integrated into the host's chromosomal DNA. In other cases, particularly for adeno-associated virus vectors, stable integration into host DNA can be a rare event, resulting in episomal expression of transgenics and transient expression of transgenics.

[0193] The development of specialized cell lines (referred to as "packaging cells") that produce only replication-defective retroviruses has increased the utility of retroviruses for gene therapy, and defective retroviruses are characterized by gene transfer for gene therapy purposes (for review, see Miller, Blood, 76:271 (1990)). Replication-defective retroviruses can be packaged into virions that can be used to infect target cells using helper viruses by standard techniques. Protocols for producing recombinant retroviruses and infecting cells with such viruses in vitro or in vivo can be found in Ausubel et al., eds., Current Protocols in Molecular Biology, Greene Publishing Associates, (1989), Sections 9.10-9.14, and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE, and pEM known to those skilled in the art. Examples of suitable packaging virus lines for preparing ecotropic and amphoteric retroviral systems include ΨCrip, ΨCre, Ψ2, and ΨAm.Retroviruses have been used to introduce a variety of genes into many different cell types in vitro and / or in vivo, including epithelial cells (see, e.g., Eglitis et al. (1985) Science, 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA, 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA, 85:6460-6464). Proc Natl Acad Sci U S A, 85:3014-3018; Armentano et al. (1990), Proc Natl Acad Sci U S A, 87:6141-6145; Huber et al. (1991), Proc Natl Acad Sci U S A, 88:8039-8043; Ferry et al. (1991), Proc Natl Acad Sci U S A, 88:8377-8381; Chowdhury et al. (1991), Science, 254:1802-1805; van Beusechem et al. (1992), Proc. Natl. Acad. Sci. USA, 89:7640-7644; Kay et al. (1992), Human Gene Therapy, 3:641-647; Dai et al. (1992), Proc. Natl. Acad. Sci. USA, 89:10892-10895; Hwu et al. (1993), J. Immunol., 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCT Application WO 89 / 07136; PCT Application WO 89 / 02468; PCT Application WO 89 / 05345; and PCT Application WO 92 / 07573, each of which is incorporated herein by reference in its entirety for all purposes).

[0194] Another viral gene delivery system that can be used for the method of the present invention utilizes adenovirus-derived vectors. The genome of the adenovirus can be manipulated so that it encodes and expresses a gene product of interest, but is inactivated in terms of its ability to replicate in the normal lytic virus life cycle. See, for example, Berkner et al., BioTechniques, 6:616 (1988); Rosenfeld et al., Science, 252:431-434 (1991); and Rosenfeld et al., Cell, 68:143-155 (1992). Suitable adenoviral vectors can be derived from any strain of adenovirus (e.g., Ad2, Ad3, Ad5 or Ad7, etc.), including adenoviral serotypes from other species (e.g., mice, dogs, humans, etc.) known to those skilled in the art. Viral particles are relatively stable and suitable for purification and concentration, and as described above, can be modified to affect the spectrum of infectivity. In addition, the introduced adenoviral DNA (and the foreign DNA contained therein) is not integrated into the genome of the host cell, but remains free, thereby avoiding potential problems that may occur due to in situ insertional mutagenesis, in which the introduced DNA becomes integrated into the host genome (e.g., retroviral DNA). In addition, relative to other gene delivery vectors, the adenoviral genome has a large carrying capacity for foreign DNA (up to 8 kilobases) (Berkner et al., supra; Haj-Ahmand and Graham, J. Virol., 57:267 (1986).

[0195] Helper-dependent (HDAd) vectors can also be generated in which all adenoviral sequences are deleted except for the DNA replication origin at each end of the viral DNA and the packaging signal at the 5-priming end of the genome downstream of the left packaging signal. HDAd vectors are constructed and propagated in the presence of a replication-competent helper adenovirus that provides the required early and late proteins required for replication.

[0196] Another viral vector system that can be used for nucleic acid delivery is adeno-associated virus (AAV). Adeno-associated virus is a naturally occurring defective virus that requires another virus (such as adenovirus or herpes virus) as a helper virus to achieve effective replication and productive life cycle. (For review, see Muzyczka et al., Current Topics in Micro and Immunol., 158: 97-129 (1992). It is also one of the few viruses that can integrate its DNA into non-dividing cells, and shows a high frequency of stable integration (see, for example, Flotte et al., Am. J. Respir. Cell. Mol. Biol. 7: 349-356 (1992); Samulski et al., Journal of Virology, 63: 3822-38 28 (1989); and McLaughlin et al., Journal of Virology, 62:1963-1973 (1989). Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate. The space for foreign DNA is limited to about 4.5 kb. AAV vectors, such as those described in Tratschin et al., Mol. Cell. Biol., 5:3251-3260 (1985), can be used to introduce DNA into AAV vectors have been used to introduce a variety of nucleic acids into different cell types (see, for example, Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466-6470 (1984); Tratschin et al., Mol. Cell. Biol., 4:2072-2081 (1985); Wondisford et al., Mol. Endocrinol., 2:32-39 (1988); Tratschin et al., J. Virol., 51:611-619 (1984); and Flotte et al., J. Biol. Chem., 268:3781-3790 (1993). The identification of Staphylococcus aureus (SaCas9) and other smaller Cas9 enzymes has paved a new way for therapeutic gene editing, as these enzymes can be packaged into adeno-associated virus (AAV) vectors that are highly stable and effective in vivo, easy to produce, approved by the FDA, and tested in multiple clinical trials.

[0197] In some embodiments, nucleic acids encoding CRISPR IL-1α or IL-1β gene editing complexes (e.g., Cas9 or gRNA) are encapsulated in liposomes (e.g., lipofectins) with positive charges on their surfaces, which can be labeled with antibodies against cell surface antigens of target cells. These delivery vehicles can also be used to deliver Cas9 protein / gRNA complexes.

[0198] In a clinical setting, a gene delivery system encoding a nucleic acid of a CRISPR IL-1α or IL-1β gene editing complex can be introduced into a subject by any of a variety of methods, each of which is well known in the art. For example, a pharmaceutical preparation of a gene delivery system can be systemically introduced, for example, by intravenous injection, and the specific transduction of proteins in target cells will occur mainly from the specificity of the transfection provided by the gene delivery vehicle, the cell type or tissue type expression or a combination thereof caused by the transcriptional regulatory sequence controlling the expression of the receptor gene. In other embodiments, the initial delivery of nucleic acids encoding CRISPR L-1α or IL-1β gene editing complexes is more limited, wherein the introduction into the subject is quite limited. For example, nucleic acids encoding CRISPR IL-1α or IL-1β gene editing complexes can be introduced into joints exhibiting joint diseases (e.g., osteoarthritis) by intra-articular injection. In some embodiments, nucleic acid encoding CRISPR IL-1α or IL-1β gene editing complex is administered during or after surgery; in some embodiments, a hydrogel comprising a controlled release of nucleic acid encoding CRISPR IL-1α or IL-1β gene editing complex is administered at the end of surgery prior to closure to prevent, reduce, or eliminate osteoarthritis by providing a steady dose of nucleic acid encoding CRISPR IL-1α or IL-1β gene editing complex over time.

[0199] The pharmaceutical preparation of the nucleic acid encoding the CRISPR IL-1α or IL-1β gene editing complex can be essentially composed of a gene delivery system (e.g., a viral vector) in an acceptable diluent, or can include a slow-release matrix in which a gene delivery vehicle is embedded. Alternatively, when the complete gene delivery system can be completely produced from recombinant cells (e.g., an adeno-associated viral vector), the pharmaceutical preparation can include one or more cells that produce the gene delivery system.

[0200] Preferably, the CRISPR IL-1α or IL-1β editing complex is specific, that is, preferentially induces genomic changes at the target site (IL-1α or IL-1β), and does not induce changes at other sites, or only rarely induces changes at other sites. In certain embodiments, the CRISPR IL-1α or IL-1β editing complex has an editing efficiency of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99%.

[0201] The sgRNA in the CRISPR / Cas system for HR generally comprises a guide sequence (e.g., crRNA) complementary to the target nucleic acid sequence (target locus) and a scaffold sequence (e.g., tracrRNA) that interacts with the Cas nuclease (e.g., Cas9 polypeptide) or a variant or fragment thereof. The single-stranded guide RNA (sgRNA) may comprise crRNA and tracrRNA.

[0202] Exemplary target sequences for inducing genomic alterations in IL-1α or IL-1β genes by CRISPR-Cas editing complexes are provided in Tables 2 and 12. Exemplary guide RNAs for use in the compositions, methods, and systems of the present disclosure are provided in Tables 3 and 13.

[0203] Table 12. Exemplary target IL-1α and IL-1β gene sequences

[0204]

[0205]

[0206]

[0207]

[0208] Table 13. Exemplary crRNA sequences of CRISPR sgRNAs

[0209]

[0210]

[0211]

[0212]

[0213]

[0214] In certain embodiments, the sequence of guide RNA (e.g., single-stranded guide RNA or sgRNA) can be modified to increase editing efficiency and / or reduce off-target effects. In certain embodiments, the sequence of guide RNA can differ from the target sequence by about 1 base, about 2 bases, about 3 bases, about 4 bases, about 5 bases, about 5 bases, about 6 bases, about 7 bases, about 8 bases, about 9 bases, about 10 bases, about 15 bases or more than about 15 bases. In certain embodiments, the sequence of guide RNA can differ from the target sequence by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20% or more than about 20%. As used herein, differences from a target sequence can refer to the degree of complementarity.

[0215] In certain embodiments, the guide RNA used with the compositions, methods, or systems of the present disclosure is identical to the sequence shown in any one of SEQ ID NO.: 21-34 and SEQ ID NO.: 168-297. In certain embodiments, the guide RNA used with the compositions, methods, or systems of the present disclosure is at least about 95% identical to the sequence shown in any one of SEQ ID NO.: 21-34 and SEQ ID NO.: 168-297. In certain embodiments, the guide RNA used with the compositions, methods, or systems of the present disclosure is at least about 90% identical to the sequence shown in any one of SEQ ID NO.: 21-34 and SEQ ID NO.: 168-297. In certain embodiments, the guide RNA used with the compositions, methods, or systems of the present disclosure is at least about 85% identical to the sequence shown in any one of SEQ ID NO.: 21-34 and SEQ ID NO.: 168-297. In certain embodiments, the guide RNA used with the compositions, methods, or systems of the present disclosure is at least about 80% identical to the sequence shown in any one of SEQ ID NOs.: 21-34 and SEQ ID NOs.: 168-297. In certain embodiments, the guide RNA used with the compositions, methods, or systems of the present disclosure is at least about 75% identical to the sequence shown in any one of SEQ ID NOs.: 21-34 and SEQ ID NOs.: 168-297. In certain embodiments, the guide RNA used with the compositions, methods, or systems of the present disclosure is at least about 70% identical to the sequence shown in any one of SEQ ID NOs.: 21-34 and SEQ ID NOs.: 168-297. In certain embodiments, the guide RNA used with the compositions, methods, or systems of the present disclosure is at least about 65% identical to the sequence shown in any one of SEQ ID NOs.: 21-34 and SEQ ID NOs.: 168-297. In certain embodiments, the guide RNA used with the compositions, methods, or systems of the present disclosure is at least about 60% identical to the sequence shown in any one of SEQ ID NOs.: 21-34 and SEQ ID NOs.: 168-297. In certain embodiments, the guide RNA used with the compositions, methods, or systems of the present disclosure is at least about 55% identical to the sequence shown in any one of SEQ ID NOs.: 21-34 and SEQ ID NOs.: 168-297. In certain embodiments, the guide RNA used with the compositions, methods, or systems of the present disclosure is at least about 50% identical to the sequence shown in any one of SEQ ID NOs.: 21-34 and SEQ ID NOs.: 168-297.In certain embodiments, the guide RNA used with the compositions, methods, or systems of the present disclosure is at least about 45% identical to the sequence shown in any one of SEQ ID NOs.: 21-34 and SEQ ID NOs.: 168-297. In certain embodiments, the guide RNA used with the compositions, methods, or systems of the present disclosure is at least about 40% identical to the sequence shown in any one of SEQ ID NOs.: 21-34 and SEQ ID NOs.: 168-297. In certain embodiments, the guide RNA used with the compositions, methods, or systems of the present disclosure is at least about 35% identical to the sequence shown in any one of SEQ ID NOs.: 21-34 and SEQ ID NOs.: 168-297. In certain embodiments, the guide RNA used with the compositions, methods, or systems of the present disclosure is at least about 35% identical to the sequence shown in any one of SEQ ID NOs.: 21-34 and SEQ ID NOs.: 168-297.

[0216] In certain embodiments, the guide RNA used with the compositions, methods, or systems of the present disclosure has 1 base substitution in the sequence shown in any one of SEQ ID NO.: 21-34 and SEQ ID NO.: 168-297. In certain embodiments, the guide RNA used with the compositions, methods, or systems of the present disclosure has 2 base substitutions in the sequence shown in any one of SEQ ID NO.: 21-34 and SEQ ID NO.: 168-297. In certain embodiments, the guide RNA used with the compositions, methods, or systems of the present disclosure has 3 base substitutions in the sequence shown in any one of SEQ ID NO.: 21-34 and SEQ ID NO.: 168-297. In certain embodiments, the guide RNA used with the compositions, methods, or systems of the present disclosure has 4 base substitutions in the sequence shown in any one of SEQ ID NO.: 21-34 and SEQ ID NO.: 168-297. In certain embodiments, the guide RNA used with the compositions, methods, or systems of the present disclosure has 4 base substitutions in the sequence shown in any one of SEQ ID NO.: 21-34 and SEQ ID NO.: 168-297. In certain embodiments, the guide RNA used with the compositions, methods, or systems of the present disclosure has 6 base substitutions in the sequence shown in any one of SEQ ID NO.: 21-34 and SEQ ID NO.: 168-297. In certain embodiments, the guide RNA used with the compositions, methods, or systems of the present disclosure has 7 base substitutions in the sequence shown in any one of SEQ ID NO.: 21-34 and SEQ ID NO.: 168-297. In certain embodiments, the guide RNA used with the compositions, methods, or systems of the present disclosure has 8 base substitutions in the sequence shown in any one of SEQ ID NO.: 21-34 and SEQ ID NO.: 168-297. In certain embodiments, the guide RNA used with the compositions, methods, or systems of the present disclosure has 9 base substitutions in the sequence shown in any one of SEQ ID NOs.: 21-34 and SEQ ID NOs.: 168-297. In certain embodiments, the guide RNA used with the compositions, methods, or systems of the present disclosure has 10 base substitutions in the sequence shown in any one of SEQ ID NOs.: 21-34 and SEQ ID NOs.: 168-297. In certain embodiments, the guide RNA used with the compositions, methods, or systems of the present disclosure has 11 base substitutions in the sequence shown in any one of SEQ ID NOs.: 21-34 and SEQ ID NOs.: 168-297.In certain embodiments, the guide RNA used with the compositions, methods, or systems of the present disclosure has 12 base substitutions in the sequence shown in any one of SEQ ID NO.: 21-34 and SEQ ID NO.: 168-297. In certain embodiments, the guide RNA used with the compositions, methods, or systems of the present disclosure has 13 base substitutions in the sequence shown in any one of SEQ ID NO.: 21-34 and SEQ ID NO.: 168-297. In certain embodiments, the guide RNA used with the compositions, methods, or systems of the present disclosure has 14 base substitutions in the sequence shown in any one of SEQ ID NO.: 21-34 and SEQ ID NO.: 168-297. In certain embodiments, the guide RNA used with the compositions, methods, or systems of the present disclosure has 15 base substitutions in the sequence shown in any one of SEQ ID NO.: 21-34 and SEQ ID NO.: 168-297.

[0217] In certain embodiments, the guide RNA of the present disclosure is designed to and / or is capable of knocking down the expression of the target gene shown in any one of SEQ ID NO.: 7-20 and SEQ ID NO.: 37-167. In certain embodiments, the guide RNA of the present disclosure is designed to or is capable of knocking down the human IL-1α gene by binding to at least a portion of exon 1 of the human IL-1α gene. In certain embodiments, the guide RNA of the present disclosure is designed to or is capable of knocking down the human IL-1α gene by binding to at least a portion of exon 2 of the human IL-1α gene. In certain embodiments, the guide RNA of the present disclosure is designed to or is capable of knocking down the human IL-1α gene by binding to at least a portion of exon 3 of the human IL-1α gene. In certain embodiments, the guide RNA of the present disclosure is designed to or is capable of knocking down the human IL-1α gene by binding to at least a portion of exon 4 of the human IL-1α gene. In certain embodiments, the guide RNA of the present disclosure is designed to or is capable of knocking down the human IL-1α gene by binding to at least a portion of exon 5 of the human IL-1α gene. In certain embodiments, the guide RNA of the present disclosure is designed to or is capable of knocking down the human IL-1α gene by binding to at least a portion of exon 6 of the human IL-1α gene. In certain embodiments, the guide RNA of the present disclosure is designed to or is capable of knocking down the human IL-1α gene by binding to at least a portion of exon 7 of the human IL-1α gene. In certain embodiments, the guide RNA of the present disclosure is designed to or is capable of knocking down the human IL-1α gene by binding to at least a portion of exon 8 of the human IL-1α gene.

[0218] In certain embodiments, the guide RNA of the present invention is designed to or can be combined with at least a portion of exon 1 of the human IL-1β gene to knock down the human IL-1β gene. In certain embodiments, the guide RNA of the present invention is designed to or can be combined with at least a portion of exon 2 of the human IL-1β gene to knock down the human IL-1β gene. In certain embodiments, the guide RNA of the present invention is designed to or can be combined with at least a portion of exon 3 of the human IL-1β gene to knock down the human IL-1β gene. In certain embodiments, the guide RNA of the present invention is designed to or can be combined with at least a portion of exon 4 of the human IL-1β gene to knock down the human IL-1β gene. In certain embodiments, the guide RNA of the present invention is designed to or can be combined with at least a portion of exon 5 of the human IL-1β gene to knock down the human IL-1β gene. In certain embodiments, the guide RNA of the present invention is designed to or can be combined with at least a portion of exon 6 of the human IL-1β gene to knock down the human IL-1β gene. In certain embodiments, the guide RNA of the present disclosure is designed to or is capable of knocking down the human IL-1 β gene by binding to at least a portion of exon 7 of the human IL-1 β gene.

[0219] In certain embodiments, the guide RNA of the present disclosure is designed to and / or is capable of knocking down the expression of the target gene shown in any one of SEQ ID NO.: 7-20 and SEQ ID NO.: 37-167. In certain embodiments, the guide RNA of the present disclosure is designed to or is capable of knocking down the canine IL-1α gene by binding to at least a portion of exon 1 of the canine IL-1α gene. In certain embodiments, the guide RNA of the present disclosure is designed to or is capable of knocking down the canine IL-1α gene by binding to at least a portion of exon 2 of the canine IL-1α gene. In certain embodiments, the guide RNA of the present disclosure is designed to or is capable of knocking down the canine IL-1α gene by binding to at least a portion of exon 3 of the canine IL-1α gene. In certain embodiments, the guide RNA of the present disclosure is designed to or is capable of knocking down the canine IL-1α gene by binding to at least a portion of exon 4 of the canine IL-1α gene. In certain embodiments, the guide RNA of the present disclosure is designed to or is capable of knocking down the canine IL-1α gene by binding to at least a portion of exon 5 of the canine IL-1α gene. In certain embodiments, the guide RNA of the present disclosure is designed to or is capable of knocking down the canine IL-1α gene by binding to at least a portion of exon 6 of the canine IL-1α gene. In certain embodiments, the guide RNA of the present disclosure is designed to or is capable of knocking down the canine IL-1α gene by binding to at least a portion of exon 7 of the canine IL-1α gene.

[0220] In certain embodiments, the guide RNA of the present invention is designed to or is capable of knocking down the canine IL-1β gene by binding to at least a portion of exon 1 of the canine IL-1β gene. In certain embodiments, the guide RNA of the present invention is designed to or is capable of knocking down the canine IL-1β gene by binding to at least a portion of exon 2 of the canine IL-1β gene. In certain embodiments, the guide RNA of the present invention is designed to or is capable of knocking down the canine IL-1β gene by binding to at least a portion of exon 3 of the canine IL-1β gene. In certain embodiments, the guide RNA of the present invention is designed to or is capable of knocking down the canine IL-1β gene by binding to at least a portion of exon 4 of the canine IL-1β gene. In certain embodiments, the guide RNA of the present invention is designed to or is capable of knocking down the canine IL-1β gene by binding to at least a portion of exon 5 of the canine IL-1β gene. In certain embodiments, the guide RNA of the present disclosure is designed or capable of knocking down the canine IL-1β gene by binding to at least a portion of exon 6 of the canine IL-1β gene. In certain embodiments, the guide RNA of the present disclosure is designed or capable of knocking down the canine IL-1β gene by binding to at least a portion of exon 7 of the canine IL-1β gene. In certain embodiments, the guide RNA of the present disclosure is designed or capable of knocking down the canine IL-1β gene by binding to at least a portion of exon 8 of the canine IL-1β gene.

[0221] In certain embodiments, the guide RNA of the present invention is designed to and / or is capable of knocking down the expression of the target gene shown in any one of SEQ ID NO.: 7-20 and SEQ ID NO.: 37-167. In certain embodiments, the guide RNA of the present invention is designed to or is capable of knocking down the domestic horse IL-1α gene by binding to at least a portion of exon 1 of the domestic horse IL-1α gene. In certain embodiments, the guide RNA of the present invention is designed to or is capable of knocking down the domestic horse IL-1α gene by binding to at least a portion of exon 2 of the domestic horse IL-1α gene. In certain embodiments, the guide RNA of the present invention is designed to or is capable of knocking down the domestic horse IL-1α gene by binding to at least a portion of exon 3 of the domestic horse IL-1α gene. In certain embodiments, the guide RNA of the present invention is designed to or is capable of knocking down the domestic horse IL-1α gene by binding to at least a portion of exon 4 of the domestic horse IL-1α gene. In certain embodiments, the guide RNA of the present disclosure is designed to or is capable of knocking down the domestic horse IL-1α gene by binding to at least a portion of exon 5 of the domestic horse IL-1α gene. In certain embodiments, the guide RNA of the present disclosure is designed to or is capable of knocking down the domestic horse IL-1α gene by binding to at least a portion of exon 6 of the domestic horse IL-1α gene. In certain embodiments, the guide RNA of the present disclosure is designed to or is capable of knocking down the domestic horse IL-1α gene by binding to at least a portion of exon 7 of the domestic horse IL-1α gene.

[0222] In certain embodiments, the guide RNA of the present invention is designed to or can be combined with at least a portion of exon 1 of the domestic horse IL-1β gene to knock down the domestic horse IL-1β gene. In certain embodiments, the guide RNA of the present invention is designed to or can be combined with at least a portion of exon 2 of the domestic horse IL-1β gene to knock down the domestic horse IL-1β gene. In certain embodiments, the guide RNA of the present invention is designed to or can be combined with at least a portion of exon 3 of the domestic horse IL-1β gene to knock down the domestic horse IL-1β gene. In certain embodiments, the guide RNA of the present invention is designed to or can be combined with at least a portion of exon 4 of the domestic horse IL-1β gene to knock down the domestic horse IL-1β gene. In certain embodiments, the guide RNA of the present invention is designed to or can be combined with at least a portion of exon 5 of the domestic horse IL-1β gene to knock down the domestic horse IL-1β gene. In certain embodiments, the guide RNA of the present disclosure is designed to or is capable of knocking down the domestic horse IL-1β gene by binding to at least a portion of exon 6 of the domestic horse IL-1β gene. In certain embodiments, the guide RNA of the present disclosure is designed to or is capable of knocking down the domestic horse IL-1β gene by binding to at least a portion of exon 7 of the domestic horse IL-1β gene.

[0223] In certain embodiments, the guide RNA of the present disclosure is designed to and / or is capable of knocking down the expression of the target gene shown in any one of SEQ ID NO.: 7-20 and SEQ ID NO.: 37-167. In certain embodiments, the guide RNA of the present disclosure is designed to or is capable of knocking down the IL-1α gene of the house mouse by binding to at least a portion of exon 1 of the IL-1α gene of the house mouse. In certain embodiments, the guide RNA of the present disclosure is designed to or is capable of knocking down the IL-1α gene of the house mouse by binding to at least a portion of exon 2 of the IL-1α gene of the house mouse. In certain embodiments, the guide RNA of the present disclosure is designed to or is capable of knocking down the IL-1α gene of the house mouse by binding to at least a portion of exon 3 of the IL-1α gene of the house mouse. In certain embodiments, the guide RNA of the present disclosure is designed to or is capable of knocking down the IL-1α gene of the house mouse by binding to at least a portion of exon 4 of the IL-1α gene of the house mouse. In certain embodiments, the guide RNA of the present disclosure is designed to or is capable of knocking down the Mus musculus IL-1α gene by binding to at least a portion of exon 5 of the Mus musculus IL-1α gene. In certain embodiments, the guide RNA of the present disclosure is designed to or is capable of knocking down the Mus musculus IL-1α gene by binding to at least a portion of exon 6 of the Mus musculus IL-1α gene. In certain embodiments, the guide RNA of the present disclosure is designed to or is capable of knocking down the Mus musculus IL-1α gene by binding to at least a portion of exon 7 of the Mus musculus IL-1α gene. In certain embodiments, the guide RNA of the present disclosure is designed to or is capable of knocking down the Mus musculus IL-1α gene by binding to at least a portion of exon 8 of the Mus musculus IL-1α gene.

[0224] In certain embodiments, the guide RNA of the present invention is designed to or can be combined with at least a portion of exon 1 of the IL-1β gene of the house mouse to knock down the IL-1β gene of the house mouse. In certain embodiments, the guide RNA of the present invention is designed to or can be combined with at least a portion of exon 2 of the IL-1β gene of the house mouse to knock down the IL-1β gene of the house mouse. In certain embodiments, the guide RNA of the present invention is designed to or can be combined with at least a portion of exon 3 of the IL-1β gene of the house mouse to knock down the IL-1β gene of the house mouse. In certain embodiments, the guide RNA of the present invention is designed to or can be combined with at least a portion of exon 4 of the IL-1β gene of the house mouse to knock down the IL-1β gene of the house mouse. In certain embodiments, the guide RNA of the present invention is designed to or can be combined with at least a portion of exon 5 of the IL-1β gene of the house mouse to knock down the IL-1β gene of the house mouse. In certain embodiments, the guide RNA of the present disclosure is designed to or is capable of knocking down the Mus musculus IL-1β gene by binding to at least a portion of exon 6 of the Mus musculus IL-1β gene. In certain embodiments, the guide RNA of the present disclosure is designed to or is capable of knocking down the Mus musculus IL-1β gene by binding to at least a portion of exon 7 of the Mus musculus IL-1β gene.

[0225] In some cases, sgRNA is introduced into cells (e.g., in vitro cells, such as primary cells for ex vivo therapy, or in vivo cells, such as cells in a patient) using a recombinant expression vector that includes a nucleotide sequence encoding a Cas nuclease (e.g., a Cas9 polypeptide) or a variant or fragment thereof. In some embodiments, sgRNA is complexed with a Cas nuclease (e.g., a Cas9 polypeptide) or a variant or fragment thereof to form a ribonucleoprotein (RNP)-based delivery system for introduction into cells (e.g., in vitro cells, such as primary cells for ex vivo therapy, or in vivo cells, such as cells in a patient). In other cases, sgRNA is introduced into cells (e.g., in vitro cells, such as primary cells for ex vivo therapy, or in vivo cells, such as cells in a patient) having mRNA encoding a Cas nuclease (e.g., a Cas9 polypeptide) or a variant or fragment thereof.

[0226] Any heterologous or exogenous nucleic acid (e.g., a target locus-specific sgRNA and / or a polynucleotide encoding a Cas9 polynucleotide) can be introduced into a cell using any method known to those skilled in the art. Such methods include, but are not limited to, electroporation, nuclear transfection, transfection, lipofection, transduction, microinjection, electroinjection, electrofusion, nanoparticle bombardment, transformation, conjugation, and the like.

[0227] The nucleic acid sequence of the sgRNA can be any polynucleotide sequence that has sufficient complementarity to a target polynucleotide sequence (e.g., a target DNA sequence) to hybridize with the target sequence and guide sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between the guide sequence of the sgRNA and its corresponding target sequence is about or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more when optimally aligned using a suitable alignment algorithm. The optimal alignment can be determined by using any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, an algorithm based on the Burrows-Wheeler transform (e.g., Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, California (San Diego) Diego, Calif)), SOAP (available from soap.genomics.org.cn), and Maq (available from maq.sourceforge.net). In some embodiments, the guide sequence is about 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 ​​nucleotides, 49 nucleotides, 50 In some cases, the length of the guide sequence is about 20 nucleotides. In other cases, the length of the guide sequence is about 15 nucleotides. In other cases, the length of the guide sequence is about 25 nucleotides. The ability of the guide sequence to guide the sequence-specific binding of the CRISPR complex to the target sequence can be assessed by any suitable assay. For example, components of a CRISPR system sufficient to form a CRISPR complex (including the guide sequence to be tested) can be provided to a host cell with a corresponding target sequence, such as by transfection with a vector encoding the components of the CRISPR sequence, and then evaluating the preferential cutting within the target sequence.Similarly, cleavage of a target polynucleotide sequence can be assessed in a test tube by providing a target sequence, components of a CRISPR complex, including a guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing the rate of binding or cleavage at the target sequence between reactions of the test guide sequence and the control guide sequence.

[0228] The nucleic acid sequence of sgRNA can be selected using any of the above-mentioned web-based software. Considerations for selecting DNA-targeted RNA include PAM sequences for Cas nucleases (e.g., Cas9 polypeptides) to be used, and strategies for minimizing off-target modifications. Tools such as CRISPR design tools can provide sequences for preparing sgRNA, for evaluating target modification efficiency, and / or evaluating the cutting at off-target sites. Another consideration for selecting the sequence of sgRNA includes reducing the degree of secondary structure within the guide sequence. The secondary structure can be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimum Gibbs free energy. Examples of suitable algorithms include mFold (Zuker and Stiegler, "Nucleic Acids Res", 9 (1981), 133-148), UNAFold packaging (Markham et al., "Molecular Biology Methods (Methods Mol Biol)", 2008, 453: 3-31) and RNAfold packaged from ViennaRNa.

[0229] The sgRNA can be about 10 to about 500 nucleotides, for example, about 10 nucleotides, 15 nucleotides, 20 nucleotides, 25 nucleotides, 30 nucleotides, 35 nucleotides, 40 nucleotides, 45 nucleotides, 50 nucleotides, 55 nucleotides, 60 nucleotides, 65 nucleotides, 70 nucleotides, 75 nucleotides, 80 nucleotides, 85 nucleotides, 90 nucleotides, 95 nucleotides, 100 nucleotides, 105 nucleotides, 110 nucleotides, 120 nucleotides, 130 nucleotides, 140 nucleotides, 150 nucleotides, 160 nucleotides, 170 nucleotides, 180 nucleotides, 190 nucleotides, 200 nucleotides, nucleotides, 210 nucleotides, 220 nucleotides, 230 nucleotides, 240 nucleotides, 250 nucleotides, 260 nucleotides, 270 nucleotides, 280 nucleotides, 290 nucleotides, 300 nucleotides, 310 nucleotides, 320 nucleotides, 330 nucleotides, 340 nucleotides, 350 nucleotides, 360 nucleotides, 370 nucleotides, 380 nucleotides, 390 nucleotides, 400 nucleotides, 410 nucleotides, 420 nucleotides, 430 nucleotides, 440 nucleotides, 450 nucleotides, 460 nucleotides, 470 nucleotides, 480 nucleotides, 490 nucleotides, or about 500 nucleotides.In some embodiments, the sgRNA is about 20 to about 500 nucleotides, e.g., 20 nucleotides, 25 nucleotides, 30 nucleotides, 35 nucleotides, 40 nucleotides, 45 nucleotides, 50 nucleotides, 55 nucleotides, 60 nucleotides, 65 nucleotides, 70 nucleotides, 75 nucleotides, 80 nucleotides, 85 nucleotides, 90 nucleotides, 95 nucleotides, 100 nucleotides, 105 nucleotides, 110 nucleotides, 115 nucleotides, 120 nucleotides, 125 nucleotides, 130 nucleotides, , 135 nucleotides, 140 nucleotides, 145 nucleotides, 150 nucleotides, 155 nucleotides, 160 nucleotides, 165 nucleotides, 170 nucleotides, 175 nucleotides, 180 nucleotides, 185 nucleotides, 190 nucleotides, 195 nucleotides, 200 nucleotides, 205 nucleotides, 210 nucleotides, 215 nucleotides, 220 nucleotides, 225 nucleotides, 230 nucleotides, 235 nucleotides, 240 nucleotides, 245 nucleotides, 250 nucleotides, 255 nucleotides nucleotides, 260 nucleotides, 265 nucleotides, 270 nucleotides, 275 nucleotides, 280 nucleotides, 285 nucleotides, 290 nucleotides, 295 nucleotides, 300 nucleotides, 305 nucleotides, 310 nucleotides, 315 nucleotides, 320 nucleotides, 325 nucleotides, 330 nucleotides, 335 nucleotides, 340 nucleotides, 345 nucleotides, 350 nucleotides, 355 nucleotides, 360 nucleotides, 365 nucleotides, 370 nucleotides, 375 nucleotides, 380 nucleotides, 385 nucleotides, 390 nucleotides, 395 nucleotides, 400 nucleotides, 405 nucleotides, 410 nucleotides, 415 nucleotides, 420 nucleotides, 425 nucleotides, 430 nucleotides, 435 nucleotides, 440 nucleotides, 445 nucleotides, 450 nucleotides, 455 nucleotides, 460 nucleotides, 465 nucleotides, 470 nucleotides, 475 nucleotides, 480 nucleotides, 485 nucleotides, 490 nucleotides, 495 nucleotides, or about 500 nucleotides.In some embodiments, the sgRNA is about 20 to about 100 nucleotides, e.g., about 20 nucleotides, e.g., 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 ​​nucleotides, 49 nucleotides, 50 nucleotides, 51 nucleotides, 52 nucleotides, 53 nucleotides, 54 nucleotides, 55 nucleotides, 56 nucleotides, 57 nucleotides, nucleotides, 58 nucleotides, 59 nucleotides, 60 nucleotides, 61 nucleotides, 62 nucleotides, 63 nucleotides, 64 nucleotides, 65 nucleotides, 66 nucleotides, 67 nucleotides, 68 nucleotides, 69 nucleotides, 70 nucleotides, 71 nucleotides, 72 nucleotides, 73 nucleotides, 74 nucleotides, 75 nucleotides, 76 nucleotides, 77 nucleotides, 78 nucleotides, 79 nucleotides, 80 nucleotides, 81 nucleotides, 82 nucleotides, 83 nucleotides, 84 nucleotides, 85 nucleotides, 86 nucleotides, 87 nucleotides, 88 nucleotides, 89 nucleotides, 90 nucleotides, 91 nucleotides, 92 nucleotides, 93 nucleotides, 94 nucleotides, 95 nucleotides, 96 nucleotides, 97 nucleotides, 98 nucleotides, 99 nucleotides, or about 100 nucleotides.

[0230] The scaffold sequence can be about 10 to about 500 nucleotides, for example, about 10 nucleotides, 15 nucleotides, 20 nucleotides, 25 nucleotides, 30 nucleotides, 35 nucleotides, 40 nucleotides, 45 nucleotides, 50 nucleotides, 55 nucleotides, 60 nucleotides, 65 nucleotides, 70 nucleotides, 75 nucleotides, 80 nucleotides, 85 nucleotides, 90 nucleotides, 95 nucleotides, 100 nucleotides, 105 nucleotides, 110 nucleotides, 120 nucleotides, 130 nucleotides, 140 nucleotides, 150 nucleotides, 160 nucleotides, 170 nucleotides, 180 nucleotides, 190 nucleotides, 200 nucleotides, nucleotides, 210 nucleotides, 220 nucleotides, 230 nucleotides, 240 nucleotides, 250 nucleotides, 260 nucleotides, 270 nucleotides, 280 nucleotides, 290 nucleotides, 300 nucleotides, 310 nucleotides, 320 nucleotides, 330 nucleotides, 340 nucleotides, 350 nucleotides, 360 nucleotides, 370 nucleotides, 380 nucleotides, 390 nucleotides, 400 nucleotides, 410 nucleotides, 420 nucleotides, 430 nucleotides, 440 nucleotides, 450 nucleotides, 460 nucleotides, 470 nucleotides, 480 nucleotides, 490 nucleotides, or about 500 nucleotides.In some embodiments, the scaffold sequence is about 20 to about 500 nucleotides, e.g., 20 nucleotides, 25 nucleotides, 30 nucleotides, 35 nucleotides, 40 nucleotides, 45 nucleotides, 50 nucleotides, 55 nucleotides, 60 nucleotides, 65 nucleotides, 70 nucleotides, 75 nucleotides, 80 nucleotides, 85 nucleotides, 90 nucleotides, 95 nucleotides, 100 nucleotides, 105 nucleotides, 110 nucleotides, 115 nucleotides, 120 nucleotides, 125 nucleotides, 130 nucleotides, , 135 nucleotides, 140 nucleotides, 145 nucleotides, 150 nucleotides, 155 nucleotides, 160 nucleotides, 165 nucleotides, 170 nucleotides, 175 nucleotides, 180 nucleotides, 185 nucleotides, 190 nucleotides, 195 nucleotides, 200 nucleotides, 205 nucleotides, 210 nucleotides, 215 nucleotides, 220 nucleotides, 225 nucleotides, 230 nucleotides, 235 nucleotides, 240 nucleotides, 245 nucleotides, 250 nucleotides, 255 nucleotides nucleotides, 260 nucleotides, 265 nucleotides, 270 nucleotides, 275 nucleotides, 280 nucleotides, 285 nucleotides, 290 nucleotides, 295 nucleotides, 300 nucleotides, 305 nucleotides, 310 nucleotides, 315 nucleotides, 320 nucleotides, 325 nucleotides, 330 nucleotides, 335 nucleotides, 340 nucleotides, 345 nucleotides, 350 nucleotides, 355 nucleotides, 360 nucleotides, 365 nucleotides, 370 nucleotides, 375 nucleotides, 38 0 nucleotides, 385 nucleotides, 390 nucleotides, 395 nucleotides, 400 nucleotides, 405 nucleotides, 410 nucleotides, 415 nucleotides, 420 nucleotides, 425 nucleotides, 430 nucleotides, 435 nucleotides, 440 nucleotides, 445 nucleotides, 450 nucleotides, 455 nucleotides, 460 nucleotides, 465 nucleotides, 470 nucleotides, 475 nucleotides, 480 nucleotides, 485 nucleotides, 490 nucleotides, 495 nucleotides, or 500 nucleotides.In some embodiments, the scaffold sequence is about 20 to about 100 nucleotides, e.g., about 20 nucleotides, e.g., 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 ​​nucleotides, 49 nucleotides, 50 nucleotides, 51 nucleotides, 52 nucleotides, 53 nucleotides, 54 nucleotides, 55 nucleotides, 56 nucleotides, 57 nucleotides, nucleotides, 58 nucleotides, 59 nucleotides, 60 nucleotides, 61 nucleotides, 62 nucleotides, 63 nucleotides, 64 nucleotides, 65 nucleotides, 66 nucleotides, 67 nucleotides, 68 nucleotides, 69 nucleotides, 70 nucleotides, 71 nucleotides, 72 nucleotides, 73 nucleotides, 74 nucleotides, 75 nucleotides, 76 nucleotides, 77 nucleotides, 78 nucleotides, 79 nucleotides, 80 nucleotides, 81 nucleotides, 82 nucleotides, 83 nucleotides, 84 nucleotides, 85 nucleotides, 86 nucleotides, 87 nucleotides, 88 nucleotides, 89 nucleotides, 90 nucleotides, 91 nucleotides, 92 nucleotides, 93 nucleotides, 94 nucleotides, 95 nucleotides, 96 nucleotides, 97 nucleotides, 98 nucleotides, 99 nucleotides, or about 100 nucleotides.

[0231] The nucleotides of the sgRNA can include modifications in a ribose (eg, sugar) group, a phosphate group, a nucleobase, or any combination thereof. In some embodiments, the modification in the ribose group includes a modification at the 2' position of the ribose.

[0232] In some embodiments, the nucleotide comprises 2' fluoro-arabinose nucleic acid, tricyclic-DNA (tc-DNA), peptide nucleic acid, cyclohexene nucleic acid (CeNA), locked nucleic acid (LNA), ethylene-bridged nucleic acid (eNA), phosphodiamidate morpholino, or a combination thereof.

[0233] The modified nucleotides or nucleotide analogs may comprise sugar- and / or backbone-ribonucleotides (i.e., comprise modifications to the phosphate-sugar backbone). For example, the phosphodiester bonds of natural or native RNA may comprise at least one of a nitrogen or sulfur heteroatom. In some backbone-ribonucleotides, the phosphate group attached to the adjacent ribonucleotide may be replaced by, for example, a thiophosphate group. In some sugar-ribonucleotides, the 2' portion is selected from H, OR, R, halo, SH, SR, H2, HR, R 2 or ON, where R is C 1 -C6 alkyl, alkenyl or alkynyl, and halo is F, Cl, Br or I.

[0234] In some embodiments, the nucleotide contains a sugar modification. Non-limiting examples of sugar modifications include 2'-deoxy-2'-fluoro-oligoribonucleotides (2'-fluoro-2'-deoxycytidine-5'-triphosphate, 2'-fluoro-2'-deoxyuridine-5'-triphosphate), 2'-deoxy-2'-deamine oligoribonucleotides (2'-amino-2'-deoxycytidine-5'-triphosphate, 2'-amino-2'-deoxyuridine-5'-triphosphate), 2'-O-alkyl oligoribonucleotides, 2'-deoxy-2'-deoxyuridine-5'-triphosphate ... '-C-alkyl oligoribonucleotides (2'-O-methylcytidine-5'-triphosphate, 2'-methyluridine-5'-triphosphate), 2'-C-alkyl oligoribonucleotides and isomers thereof (2'-arabinocytidine-5'-triphosphate, 2'-arabinuridine-5'-triphosphate), azidotriphosphates (2'-azido-2'-deoxycytidine-5'-triphosphate, 2'-azido-2'-deoxyuridine-5'-triphosphate) and combinations thereof.

[0235] In some embodiments, the sgRNA contains one or more 2'-fluoro modifications, 2'-amino modifications, and / or 2'-sulfenyl modifications. In some cases, the modification is 2'-fluorocytidine, 2'-fluorouridine, 2'-fluoroadenosine, 2'-fluoroguanosine, 2'-aminocytidine, 2'-aminouridine, 2'-aminoadenosine, 2'-aminoguanosine, 2,6-diaminopurine, 4-thiouridine, 5-amino-allyluridine, 5-bromouridine, 5-iodouridine, 5-methylcytidine, ribo-thymidine, 2-aminopurine, 2'-amino-butyryl-pyrenouridine, 5-fluorocytidine, and / or 5-fluorouridine.

[0236] More than 96 naturally occurring nucleoside modifications have been found on mammalian RNA. See, for example, Limbach et al., Nucleic Acids Research, 22 (12): 2183-2196 (1994). The preparation of nucleotides and nucleotides and nucleosides is well known in the art and is described in, for example, U.S. Patent Nos. 4,373,071, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530 and 5,700,642. Many nucleosides and nucleotides suitable for use as described herein are commercially available. Nucleosides can be analogs of naturally occurring nucleosides. In some cases, the analog is dihydrouridine, methyladenosine, methylcytidine, methyluridine, methylpseudouridine, thiouridine, deoxycytidine, and deoxyuridine.

[0237] In some cases, the sgRNA described herein comprises a nucleobase-ribonucleotide, i.e., a ribonucleotide containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase. Non-limiting examples of nucleobases that can be incorporated into nucleosides and nucleotides include m5C (5-methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), s2U (2-thiouridine), Um (2'-O-methyluridine), mlA (1-methyladenosine), m2A (2-methyladenosine), Am (2-1-O-methyladenosine), ms2m6A (2-methylthio-N6-methyladenosine), i6A (N6-isopentenyladenosine), ms2i6A (2-methylthio-N6-isopentenyladenosine), io 6A (N6-(cis-hydroxyisopentenyl)adenosine), ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine), g6A (N6-glycylaminoformyladenosine), t6A (N6-threonylaminoformyladenosine), ms2t6A (2-methylthio-N6-threonylaminoformyladenosine), m6t6A (N6-methyl-N6-threonylaminoformyladenosine), hn6A (N6.-hydroxynorvalylaminoformyladenosine), ms2hn6A (2-methylthio-N 6-Hydroxynorvalylaminoformyl adenosine), Ar(p)(2'-O-riboadenosine (phosphate)), I(inosine), mil(1-methylinosine), m'lm(l,2'-O-dimethylinosine), m3C(3-methylcytidine), Cm(2T-o-methylcytidine), s2C(2-thiocytidine), ac4C(N4-acetylcytidine), f5C(5-formylcytidine), m5Cm(5,2-O-dimethylcytidine), ac4Cm(N4 acetyl 2TO-methylcytidine), k2C (lysine), mlG (1-methylguanosine), m2G (N2-methylguanosine), m7G (7-methylguanosine), Gm (2'-O-methylguanosine), m22G (N2, N2-dimethylguanosine), m2Gm (N2, 2'-O-dimethylguanosine), m22Gm (N2, N2, 2'-O-trimethylguanosine), Gr(p) (2'-O-riboguanosine (phosphate)), yW (wybutosine), o2yW (peroxywybutosine), OHyW (hydroxywybutosine), guanosine), OHyW* (undermodified hydroxy wyosine), imG (wyosine), mimG (methyl wyosine), Q (queuosine), oQ (epoxyqueuosine), galQ (galactosyl-queuosine), manQ (mannosyl-queuosine), preQo (7-cyano-7-deazaguanosine), preQi (7-aminomethyl-7-deazaguanosine), G (archaeosine), D (dihydrouridine), m5Um (5,2'-O-dimethyluridine), s4U (4-thiouridine), m5s2U (5-methyl-2-thiouridine), s2Um (2-thio-2'-O-methyluridine), acp3U (3-(3-amino-3-carboxypropyl) uridine), ho5U (5-hydroxyuridine), mo5U (5-methoxyuridine), cmo5U (uridine 5-oxyacetic acid), mcmo5U (uridine 5-oxyacetic acid methyl ester), chm5U (5-(carboxyhydroxymethyl) uridine)), mchm5U (5-(carboxyhydroxymethyl) uridine methyl ester), mcm5U (5-methoxycarbonylmethyl uridine), mcm5Um (S-methoxycarbonylmethyl -2-O-methyluridine), mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine), nm5s2U (5-aminomethyl-2-thiouridine), mnm5U (5-methylaminomethyluridine), mnm5s2U (5-methylaminomethyl-2-thiouridine), mnm5se2U (5-methylaminomethyl-2-seleno-uridine), ncm5U (5-carbamoylmethyluridine), ncm5Um (5-carbamoylmethyl-2'-O-methyluridine), cmnm5U (5-carboxymethylaminomethyluridine), cmnm5Um (5-carboxymethylaminomethyl-2-LO-methyluridine), cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine), m62A (N6, N6-dimethyladenosine), Tm (2'-O-methylinosine), m4C (N4-methylcytidine), m4Cm (N4, 2-O-dimethylcytidine), hm5C (5-hydroxymethylcytidine), m3U (3-methyluridine), cm5U (5-carboxymethyluridine), m6Am (N6, TO-dimethyladenosine), rn62Am (N6, N6, 0-2-trimethyladenosine), m2'7G (N2, 7-dimethylguanosine), m2'2'7G (N2, N2, 7-trimethylguanosine), m3Um (3, 2T-O-dimethyluridine), m5 D (5-methyldihydrouridine), f5Cm (5-formyl-2'-O-methylcytidine), mlGm (l,2'-O-dimethylguanosine), m'Am (1,2-O-dimethyladenosine), irinomethyluridine), tm5s2U (S-taurine methyl-2-thiouridine), imG-14 (4-demethylguanosine), imG2 (isoguanosine), or ac6A (N6-acetyladenosine), hypoxanthine, inosine, 8-oxo-adenine, its 7-substituted derivatives, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5-(C, 1 -C 6 )-alkyluracil, 5-methyluracil, 5-(C 2 -C 6)-alkenyluracil, 5-(C 2 -C 6 )-alkynyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5-(C 1 -C 6 )-alkylcytosine, 5-methylcytosine, 5-(C 2 -C 6 )-alkenylcytosine, 5-(C 2 -C 6 )-alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N 2 -dimethylguanine, 7-deazaguanine, 8-azaguanine, 7-deaza-7-substituted guanine, 7-deaza-7-(C2-C6)alkynylguanine, 7-deaza-8-substituted guanine, 8-hydroxyguanine, 6-thioguanine, 8-oxoguanine, 2-aminopurine, 2-amino-6-chloropurine, 2,4-diaminopurine, 2,6-diaminopurine, 8-azapurine, substituted 7-deazapurine, 7-deaza-7-substituted purine, 7-deaza-8-substituted purine, and combinations thereof.

[0238] sgRNA can be synthesized by any method known to those of ordinary skill in the art. In some embodiments, sgRNA is chemically synthesized. Modified sgRNA can be synthesized using 2'-O-thiocarbamate protected nucleoside phosphoramidites. Methods are described in, for example, Dellinger et al., Journal of the American Chemical Society, 133, 11540-11556 (2011); Threlfall et al., Organic & Biomolecular Chemistry, 10, 746-754 (2012); and Dellinger et al., Journal of the American Chemical Society, 125, 940-950 (2003). Modified sgRNA can be commercially available from, for example, TriLink BioTechnology (San Diego, CA).

[0239] Additional detailed descriptions of useful sgRNAs can be found, for example, in Hendel et al., Nat Biotechnol, 2015, 33(9):985-989 and Dever et al., Nature, 2016, 539:384-389, which are incorporated herein by reference in their entirety for all purposes.

[0240] Those skilled in the art will appreciate that guide RNAs as disclosed in the present disclosure can be used in combination with any Cas protein known in the art (e.g., any Cas type from any suitable organism or bacterial species).

[0241] The Cas protein can be a type I, type II, type III, type IV, type V or type VI Cas protein. The Cas protein can include one or more domains. Non-limiting examples of domains include guide nucleic acid recognition and / or binding domains, nuclease domains (e.g., DNA enzyme or RNA enzyme domains, RuvC, HNH), DNA binding domains, RNA binding domains, helicase domains, protein-protein interaction domains and dimerization domains. The guide nucleic acid recognition and / or binding domain can interact with the guide nucleic acid. The nuclease domain can include catalytic activity for nucleic acid cleavage. The nuclease domain can lack catalytic activity that prevents nucleic acid cleavage. The Cas protein can be a chimeric Cas protein fused to other proteins or polypeptides. The Cas protein can be a chimera of various Cas proteins, for example, including domains from different Cas proteins.

[0242] Non-limiting examples of Cas proteins include c2c1, C2c2, c2c3, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cash, Cas6e, Cas6f, Cas7, Cas8a, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csb1, Csb2, Csb3, Csx14, Csx1O, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cul966, and homologs or modified forms thereof.

[0243] Cas proteins can be from any suitable organism. Non-limiting examples include Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus species, Staphylococcus aureus, Nocardiopsis dassonvillei, Streptomyces pristinae spiralis, Streptomyces viridochromo genes, Streptomyces viridochromo genes, Streptomyces roseus, Streptomyces roseus, AlicyclobacHlus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina), Burkholderialesbacterium, Polaromonas naphthalenivorans, Polaromonas species, Crocosphaera watsonii, Cyanobacterium species, Microcystis aeruginosa, Pseudomonas aeruginosa, Synechococcus species, Acetobacter arabinovus, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Pelotomaculum thermopropionicum thermopropionicum), Acidithiobacilluscaldus, Acidithiobacillus ferrooxidansferrooxidans), Allochromatiumvinosum, species of the genus Marinobacter, Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobiumevestigatum, Anabaena variabilis, Nodularia spumigena, species of the genus Nostoc, Arthrospira maxima, Arthrospira platensis, species of the genus Arthrospira, species of the genus Lyngbya, Microcoleus chthonoplastes, species of the genus Oscillatoria sp.), Petrotoga mobilis, Thermosipho africanus, Acaryochloris marina, Leptotrichia shahii, and Francisella novicida. In some aspects, the organism is Streptococcus pyogenes (S. pyogenes). In some aspects, the organism is Staphylococcus aureus (S. aureus). In some aspects, the organism is Streptococcus thermophilus (S. thermophilus).

[0244] Cas proteins can be derived from a variety of bacterial species, including but not limited to, Veillonella atypical, Fusobacterium nucleatum, Filifactor alocis, Solobacterium moorei, Coprococcus catus, Treponema denticola, Peptoniphilus duerdenii, Catenibacterium mitsuokai, Streptococcus mutans, Listeria innocua, Staphylococcus pseudintermedius, Acidaminococcus intestine, Olsenella uli, Oenococcus kitaharae, Bifidobacterium bifidum, Lactobacillus rhamnosus. rhamnosus), Lactobacillus gasseri, Finegoldia magna, Mycoplasma mobile, Mycoplasma gallisepticum, Mycoplasma ovipneumoniae, Mycoplasma canis, Mycoplasma synoviae, Eubacterium rectale, Streptococcus thermophilus, Eubacterium dolichum, Lactobacillus coryniformis subsp.Torquens), Ilyobacterpolytropus, Ruminococcus albus, Akkermansia muciniphila, Acidothermus cellulolyticus, Bifidobacterium longum, Bifidobacterium dentium, Corynebacterium diphtheria, Elusimicrobium minutum, Nitratifractorsalsuginis, Sphaerochaeta globus, Fibrobactersuccinogenes subsp.Succinogenes, Bacteroides fragilis, Capnocytophaga ochracea, Rhodopseudomonas palustris, Prevotella micans, Prevotella ruminicola, Flavobacterium columnare, Aminomonas paucivorans, Rhodospirillum rubrum, Candidatus Puniceispirillum marinum, Verminephrobacter eiseniae, Ralstonia syzygii, Dinoroseobacter shibae, Azospirillum, Nitrobacter hamburgensis, Bradyrhizobium, Wolinella succinogenes, Campylobacter jejuni subsp.Jejuni, Helicobacter mustelae, Bacillus cereus, Acidovorax ebreus, Clostridium perjringens, Parvibaculum lavamentivorans, Roseburia intestinalis, Neisseria meningitidis, Pasteurella multocida subsp. Multocida, Sutterella wadsworthensis, Proteobacterium, Legionella pneumophila, Parasutterella excrementihominis, Wolinella succinogenes, and Francisella novicida. In this context, the term "derived" is defined as modified from a naturally occurring bacterial species to retain a significant portion or significant homology to the naturally occurring bacterial species. A significant portion may be at least 10 consecutive nucleotides, at least 20 consecutive nucleotides, at least 30 consecutive nucleotides, at least 40 consecutive nucleotides, at least 50 consecutive nucleotides, at least 60 consecutive nucleotides, at least 70 consecutive nucleotides, at least 80 consecutive nucleotides, at least 90 consecutive nucleotides, or at least 100 consecutive nucleotides. Significant homology may be at least 50% homology, at least 60% homology, at least 70% homology, at least 80% homology, at least 90% homology, or at least 95% homology. A derived species may be modified while retaining the activity of the naturally occurring species. .

[0245] Gene Editing Methods

[0246] As discussed above, embodiments of the present disclosure provide compositions and methods for treating joint disorders, wherein a portion of joint cells are genetically modified via gene editing to treat joint disorders. Embodiments of the present disclosure include gene editing by nucleotide insertion (RNA or DNA) or recombinant protein insertion into a population of synovial cells, for promoting the expression of one or more proteins and inhibiting the expression of one or more proteins and combinations thereof. Embodiments of the present disclosure also provide methods for delivering gene editing compositions to joint cells, and in particular for delivering gene editing compositions to synovial cells. There are several gene editing techniques that can be used for genetically modifying joint cells, which are suitable for use according to the present invention.

[0247] In some embodiments, the method of genetically modifying joint cells comprises the step of stably incorporating genes for producing one or more proteins. In one embodiment, the method of genetically modifying a portion of joint synovial cells comprises the step of retroviral transduction. In one embodiment, the method of genetically modifying a portion of joint synovial cells comprises the step of lentiviral transduction. Lentiviral transduction systems are known in the art and are described in, for example, Levine et al., Proceedings of the National Academy of Sciences, 2006, 103, 17372-77; Zufferey et al., Nature Biotechnology, 1997, 15, 871-75; Dull et al., Journal of Virology, 1998, 72, 8463-71 and U.S. Patent No. 6,627,442, the disclosure of each of which is incorporated herein by reference. In one embodiment, the method of genetically modifying a portion of joint synovial cells comprises the step of γ-retroviral transduction. Gamma-retroviral transduction systems are known in the art and are described, for example, in Cepko and Pear, Current Protocols in Molecular Biology, 1996, 9.9.1-9.9.16, the disclosure of which is incorporated herein by reference. In one embodiment, the method of genetically modifying a portion of synovial cells of a joint comprises a step of transposon-mediated gene transfer. Transposon-mediated gene transfer systems are known in the art and include systems in which the transposase is provided as a DNA expression vector or as an expressible RNA or protein so that long-term expression of the transposase does not occur in the transgenic cell, for example, the transposase is provided as an mRNA (e.g., an mRNA comprising a cap and a poly-A tail). Suitable transposon-mediated gene transfer systems, including salmonid Tel-like transposases (SB or Sleeping Beauty transposases), such as SB10, SB11, and SB100x, and engineered enzymes with increased enzymatic activity, are described, for example, in Hackett et al., Mol. Therapy, 2010, 18, 674-83, and U.S. Pat. No. 6,489,458, the disclosures of each of which are incorporated herein by reference.

[0248] In some aspects, a viral vector or system is used to introduce a gene editing system into a cell including a joint. In some aspects, the cell is a synovial fibroblast. In some aspects, the viral vector is an AAV vector. In some aspects, the AAV vector includes a serotype selected from the group consisting of: AAV1, AAV1 (Y705 + 731F + T492V), AAV2 (Y444 + 500 + 730F + T491V), AAV3 (Y705 + 731F), AAV4, AAV5, AAV5 (Y436 + 693 + 719F), AAV6, AAV6 (VP3 variant Y705F / Y731F / T492V), AAV-7m8, AAV8, AAV8 (Y733F), AAV9, AAV9 (VP3 variant Y731F), AAV10 (Y733F), AAV-ShH10 and AAV-DJ / 8. In some aspects, the AAV vector comprises a serotype selected from the group consisting of: AAV1, AAV5, AAV6, AAV6(Y705F / Y731F / T492V), AAV8, AAV9, and AAV9(Y731F).

[0249] In some aspects, the viral vector is a lentivirus. In one aspect, the lentivirus is selected from the group consisting of human immunodeficiency virus-1 (HIV-1), human immunodeficiency virus-2 (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Jembrana Disease Virus (JDV), equine infectious anemia virus (EIAV) and caprine arthritis encephalitis virus (CAEV).

[0250] In one embodiment, the method of genetically modifying a portion of synovial cells of a joint comprises the step of stably incorporating a gene for producing or inhibiting (e.g., silencing) one or more proteins. In one embodiment, the method of genetically modifying a portion of synovial cells of a joint comprises the step of liposome transfection. Liposomal transfection methods, such as methods using a 1:1 (w / w) liposome formulation of the cationic lipids N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA) and dioleoylphosphatidylethanolamine (DOPE) in filtered water, are known in the art and are described in Rose et al., Biotechniques, 1991, 10, 520-525 and Felgner et al., Proceedings of the National Academy of Sciences of the United States of America, 1987, 84, 7413-7417; and U.S. Pat. Nos. 5,279,833; 5,908,635; 6,056,938; 6,110,490; 6,534,484; and 7,687,070, the disclosures of each of which are incorporated herein by reference. In one embodiment, a method of genetically modifying a portion of joint synovial cells comprises a step of transfection using the methods described in U.S. Patent Nos. 5,766,902; 6,025,337; 6,410,517; 6,475,994; and 7,189,705; the disclosures of each of which are incorporated herein by reference.

[0251] According to one embodiment, the gene editing process may include using a programmable nuclease that mediates the generation of double-stranded or single-stranded breaks at one or more immune checkpoint genes.Such programmable nucleases can be accurately edited by introducing breaks at a specific genomic locus, i.e., they rely on the recognition of a specific DNA sequence in the genome to target the nuclease domain to the position, and mediate the generation of double-stranded breaks at the target sequence.Double-strand breaks in DNA subsequently recruit endogenous repair mechanisms to the break site, to mediate genome editing by non-homologous end joining (NHEJ) or homology-directed repair (HDR).Therefore, the repair of the break can result in the introduction of insertion / deletion mutations that destroy (e.g., silence, suppress or enhance) the target gene product.

[0252] The main categories of nucleases that have been developed to enable site-specific genome editing include zinc finger nucleases (ZFNs), transcription activator-like nucleases (TALENs), and CRISPR-related nucleases (e.g., CRISPR-Cas9). These nuclease systems can be roughly divided into two categories based on their DNA recognition patterns: ZFNs and TALENs achieve specific DNA binding via protein-DNA interactions, while CRISPR systems such as Cas9 guide molecules through short RNAs that are directly paired with target DNA bases and target specific DNA sequences through protein-DNA interactions. See, for example, Cox et al., Nature Medicine, 2015, Vol. 21, No. 2.

[0253] Non-limiting examples of gene editing methods that can be used according to the methods of the present disclosure include CRISPR methods, TALE methods, and ZFN methods, which are described in more detail below.

[0254] CRISPR Methods

[0255] The pharmaceutical composition for treating or preventing a joint disease or condition includes a gene editing system, wherein the gene editing system targets at least one locus associated with joint function, wherein at least a portion of the synovial cells of the joint are gene edited by a CRISPR method (e.g., CRISPR-Cas9, CRISPR-Cas13a, or CRISPR / Cpf1 (also known as CRISPR-Cas12a)). According to a specific embodiment, gene editing of joint synovial cells using a CRISPR method silences or reduces the expression of one or more immune checkpoint genes in at least a portion of the synovial cells of the joint.

[0256] CRISPR stands for "Clustered Regularly Interspaced Short Palindromic Repeats". Methods of gene editing using CRISPR systems are also referred to herein as CRISPR methods. There are three types of CRISPR systems that incorporate RNA and Cas proteins and can be used in accordance with the present disclosure: Type II, Type V, and Type VI. Type II CRISPR (exemplified by Cas9) is one of the most well-characterized systems.

[0257] CRISPR technology is adapted from the natural defense mechanisms of bacteria and archaea (unicellular microbial domains). These organisms use CRISPR-derived RNA and various Cas proteins (including Cas9) to resist attacks from viruses and other foreign bodies by cutting off and destroying the DNA or RNA of foreign invaders. CRISPR is a specialized region of DNA with two distinct features: the presence of nucleotide repeats and spacers. The repeat sequences of nucleotides are distributed throughout the CRISPR region, with short segments of foreign DNA (spacers) interspersed between the repeated sequences. In the type II CRISPR-Cas system, the spacers are integrated into the CRISPR genomic locus and transcribed and processed into short CRISPR RNAs (crRNAs). These crRNAs anneal with trans-activated crRNAs (tracrRNAs) and guide sequence-specific cutting and silencing of pathogenic DNA by Cas proteins. Targeted recognition by Cas9 proteins requires a "seed" sequence within the crRNA and a conserved dinucleotide-containing protospacer sequence adjacent motif (PAM) sequence upstream of the crRNA binding region. Therefore, the CRISPR-Cas system can be retargeted to cut almost any DNA sequence by redesigning crRNA. The crRNA and tracrRNA in the natural system can be simplified into single-stranded guide RNA (sgRNA) of about 100 nucleotides for genetic engineering. The CRISPR-Cas system can be directly transplanted into human cells by co-delivering a plasmid expressing the Cas9 endonuclease and the necessary crRNA and tracrRNA (or sgRNA) components. Different variants of Cas proteins can be used to reduce targeting restrictions (e.g., orthologs of CasCas9, such as Cpf1).

[0258] CRISPR-Cas mediated homologous recombination

[0259] The CRISPR-Cas system for homologous recombination (HR) comprises a Cas nuclease (e.g., Cas9 nuclease) or a variant or fragment thereof, a DNA targeting RNA (e.g., single-stranded guide RNA (sgRNA)) containing a guide sequence that targets the Cas nuclease to the target genomic DNA and a scaffold sequence that interacts with the Cas nuclease, and a donor template. The CRISPR-Cas system can be used to generate double-strand breaks at the desired target gene locus in the genome of a cell, and the induced breaks are repaired by homology-directed repair (HDR) using the cell's endogenous machinery.

[0260] CRISPR-Cas9 nuclease can promote the locus-specific chromosomal integration of the exogenous DNA delivered by AAV vector. Generally, the size of the exogenous DNA (e.g., transgenic, expression cassette, etc.) that can be integrated is limited by the DNA packaging capacity of the AAV vector, which is about 4.0kb. By including two homology arms necessary for homologous recombination, a single AAV vector can only deliver an exogenous DNA less than about 3.7kb. The method described herein allows the delivery of 4kb or longer exogenous DNA by splitting the nucleotide sequence between two different AAV vectors. The donor template is designed to be used for the continuous homologous recombination events of the two parts that can integrate and fuse the nucleotide sequence.

[0261] Homologous recombination disclosed herein can be performed using engineered nuclease systems for genome editing, such as, but not limited to, CRISPR-Cas nucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), engineered meganucleases. In one aspect, a CRISPR-Cas-based nuclease system is used. A detailed description of useful nuclease systems can be found in, for example, Gaj et al., Trends Biotechnol, July 2013, 31(7): 397-405.

[0262] Any suitable CRISPR / Cas system can be used for the methods and compositions disclosed herein. The CRISPR / Cas system can be referred to using various naming systems. Exemplary naming systems are provided in Makarova, KS et al., "An updated evolutionary classification of CRISPR-Cas systems", "Nat Rev Microbiol", (2015) 13:722-736 and Shmakov, S. et al., "Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems", "Mol Cell", (2015) 60:1-13. The CRISPR / Cas system can be a type I, type II, type III, type IV, type V, type VI system or any other suitable CRISPR / Cas system. As used herein, the CRISPR / Cas system can be a CRISPR / Cas system of category 1, category 2 or any other appropriate classification. Class 1 CRISPR / Cas systems can use a complex of multiple Cas proteins to achieve regulation. Class 1 CRISPR / Cas systems may include, for example, type I (e.g., I, IA, IB, IC, ID, IE, IF, IU), type III (e.g., III, IIIA, IIIB, IIIC, IIID), and type IV (e.g., IV, IVA, IVB) CRISPR / Cas types. Class 2 CRISPR / Cas systems can use a single large Cas protein to achieve regulation. Class 2 CRISPR / Cas systems may include, for example, type II (e.g., II, IIA, IIB) and type V CRISPR / Cas types. CRISPR systems may complement each other, and / or functional units may be provided in trans to facilitate CRISPR locus targeting.

[0263] In some embodiments, the nucleotide sequence encoding the Cas nuclease is present in a recombinant expression vector. In some cases, the recombinant expression vector is a viral construct, such as a recombinant adeno-associated virus construct, a recombinant adenovirus construct, a recombinant lentivirus construct, etc. For example, a viral vector can be based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, etc. Retroviral vectors can be based on murine leukemia virus, spleen necrosis virus and vectors derived from retroviruses, such as Rous sarcoma virus, Harvey sarcoma virus (HarveySarcoma Virus), avian leukosis virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, mammary tumor virus, etc. Useful expression vectors are known to those skilled in the art, and many are commercially available. The following vectors are provided by, for example, eukaryotic host cells: pXT1, pSG5, pSVK3, pBPV, pMSG and pSVLSV40. However, any other vector can be used if it is compatible with the host cell. For example, useful expression vectors containing a nucleotide sequence encoding a Cas9 enzyme are commercially available from, for example, Addgene, Life Technologies, Sigma-Aldrich, and Origene.

[0264] Host cells are necessary for the production of infectious AAV vectors and for the production of AAV virions based on the disclosed AAV vectors. Various host cells are known in the art and can find application in the methods disclosed herein. Any host cell described herein or known in the art can be used with the compositions and methods described herein.

[0265] In some embodiments, the host cell for producing infectious virions can be selected from any biological organism, including prokaryotic (e.g., bacterial) cells and eukaryotic cells, including insect cells, yeast cells and mammalian cells. A variety of cells can be used, such as mammalian cells, including, for example, murine cells and primate cells (e.g., human cells). Particularly desirable host cells are selected from any mammalian species, including but not limited to, cells such as A549, WEHI, 3T3, 10T1 / 2, BHK, MDCK, COS1, COS 7, BSC 1, BSC 40, BMT 10, VERO, WI38, HeLa, CHO, 293, Vero, NIH 3T3, PC12, Huh-7 Saos, C2C12, RAT1, Sf9, L cells, HT1080, human embryonic kidney (HEK), human embryonic kidney (HEK), human embryonic stem cells, human adult tissue stem cells, pluripotent stem cells, induced pluripotent stem cells, reprogrammed stem cells, organoid stem cells, bone marrow stem cells, HLHepG2, HepG2 and primary fibroblasts, hepatocytes and myoblasts derived from mammals (including humans, monkeys, mice, rats, rabbits and hamsters). The requirement for the cells used is that they can be infected or transfected by AAV vectors. In some embodiments, the host cell is a cell stably transfected with rep and cap.

[0266] In some embodiments, the preparation of host cells according to the present disclosure involves techniques such as assembling a selected DNA sequence. This assembly can be achieved using conventional techniques. Such techniques include cDNA and genomic cloning, the use of overlapping oligonucleotide sequences of adenovirus and AAV genomes, the use of combinations with polymerase chain reactions, synthetic methods, and any other suitable methods for providing the desired nucleotide sequence, which are well known and described in Sambrook et al. cited above.

[0267] In addition to the AAV vector, the host cell may also contain sequences that drive the expression of the AAV capsid polypeptides (in the host cell), and a rep (replication) sequence of the same serotype as the serotype or cross-complementary serotype of the AAV inverted terminal repeats (ITRs) found in the AAV vector. The AAV capsid and rep (replication) sequences can be obtained independently from an AAV source and can be introduced into the host cell in any manner known to those skilled in the art or as described herein. In addition, when the AAV vector is pseudotyped in an AAV8 capsid, for example, the sequence encoding each of the essential rep (replication) proteins can be provided by AAV8, or the sequence encoding the rep (replication) protein can be provided by a different AAV serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and / or AAV9).

[0268] In some embodiments, the host cell stably contains a capsid protein under the control of a suitable promoter. In some embodiments, the capsid protein is supplied to the host cell in trans. When delivered to the host cell in trans, the capsid protein can be delivered via a plasmid containing a sequence necessary for the expression of the selected capsid protein in the host cell. In some embodiments, when delivered to the host cell in trans, the vector encoding the capsid protein also carries other sequences required for packaging AAV, such as a rep (replication) sequence.

[0269] In some embodiments, the host cell stably contains a rep (replication) sequence under the control of a suitable promoter. In another embodiment, the rep (replication) protein is supplied to the host cell in trans. When delivered to the host cell in trans, the rep (replication) protein can be delivered via a plasmid containing a sequence necessary for the expression of the selected rep (replication) protein in the host cell. In some embodiments, when delivered to the host cell in trans, the vector encoding the capsid protein also carries other sequences required for packaging the AAV vector, such as the rep (replication) sequence.

[0270] In some embodiments, rep (replication) and capsid sequences can be transfected into host cells as single nucleic acid molecules and stably present in cells as unintegrated epitopes. In another embodiment, rep (replication) and capsid sequences are stably integrated into the chromosome of the cell. Another embodiment has rep (replication) and capsid sequences transiently expressed in host cells. For example, useful nucleic acid molecules for such transfections include, from 5' to 3', a promoter, an optional spacer present between the start site of the promoter and the rep (replication) gene sequence, an AAV rep (replication) gene sequence, and an AAV capsid gene sequence.

[0271] Although the molecules providing rep (replication) and capsid can be transiently present in the host cell (i.e., by transfection), in some embodiments, one or both of the rep (replication) and capsid proteins and a promoter controlling their expression are stably expressed in the host cell, for example as an epitope or by integration into the chromosome of the host cell. The methods used to construct the embodiments of the present disclosure are conventional genetic engineering or recombinant engineering techniques, such as those described in the references above.

[0272] Various methods for producing AAV virions are known in the art and can be used to produce AAV virions including the AAV vectors described herein. Typically, the method involves inserting or transducing the AAV vector of the present disclosure into a host cell capable of packaging the AAV vector into AAV virions. Exemplary methods are described and referenced below; however, any method known to those skilled in the art can be used to produce AAV virions disclosed herein.

[0273] Methods well known in the art can be used to construct AAV vectors comprising heterologous nucleic acids (e.g., donor templates) and used to generate AAV virions. See, for example, Koerber et al., (2009) Mol. Ther., 17:2088; Koerber et al., (2008) Mol. Ther., 16:1703-1709; and U.S. Pat. Nos. 7,439,065, 6,951,758, and 6,491,907. For example, heterologous sequences can be directly inserted into the AAV genome, with the major AAV open reading frame ("ORF") excised therefrom. Other parts of the AAV genome can also be deleted, as long as enough ITR portions are retained to allow for replication and packaging functions. Such constructs can be designed using techniques well known in the art. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92 / 01070 (published Jan. 23, 1992) and WO 93 / 03769 (published Mar. 4, 1993); Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, BJ (1992) Current Opinion in Biotechnology 3:533-539; Muzyczka, N. (1992) Curr. Topics in Microbiology and Immunology. Microbiol. Immunol., 158:97-129; Kotin, RM (1994) Human Gene Therapy, 5:793-801; Shelling and Smith (1994) Gene Therapy, 1:165-169; and Zhou et al., (1994) Journal of Experimental Medicine (J. Exp. Med.), 179:1867-1875.

[0274] In order to produce AAV virions, known techniques are used, such as by transfection to introduce the AAV vector into a suitable host cell. Various transfection techniques are generally known in the art. See, for example, Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986), Basic Methods in Molecular Biology, Elsevier and Chu et al. (1981), Gene, 13:197. Particularly suitable transfection methods include calcium phosphate coprecipitation (Graham et al. (1973) Virol., 52:456-467), direct microinjection into cultured cells (Capecchi, MR (1980) Cell 22:479-488), electroporation (Shigekawa et al. (1988) BioTechniques, 6:742-751), liposome-mediated gene transfer (Mannino et al. (1988) BioTechniques, 6:682-690), lipid-mediated transduction (Felgner et al., (1987) Proc. Natl. Acad. Sci. USA, 84:7413-7417), and nucleic acid delivery using high-speed microprojectile bombardment (Klein et al., (1987) Nature 327:70-73).

[0275] Depending on the expression system used, any of a number of transcriptional and translational control elements, including promoters, transcription enhancers, transcription terminators, etc., can be used in expression vectors. Useful promoters can be derived from viruses or any organism, such as prokaryotes or eukaryotes. Suitable promoters include but are not limited to SV40 early promoter, mouse mammalian tumor virus long terminal repeat (LTR) promoter, adenovirus major late promoter (Ad MLP), herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoter (such as CMV immediate early promoter region; CMVIE), Rous sarcoma virus (RSV) promoter, human U6 small nuclear promoter (U6), enhanced U6 promoter and human HI promoter (HI), etc.

[0276] In some embodiments, polynucleotides encoding Cas nucleases can be used in the present disclosure. Such polynucleotides (e.g., mRNA) can be obtained commercially from, for example, TriLink BioTechnologies, GE Dharmacon, ThermoFisher, etc.

[0277] In certain embodiments, Cas nucleases (e.g., Cas9 polypeptides) can be used in the present disclosure. Detailed descriptions of useful Cas9 polypeptides can be found in, for example, Hendel et al., Nature Biology, 2015, 33(9): 985-989 and Dever et al., Nature, 2016, 539: 384-389, which are incorporated herein by reference in their entirety for all purposes.

[0278] In some embodiments, the Cas nuclease (e.g., Cas9 polypeptide) is complexed with the sgRNA to form a Cas ribonucleoprotein (e.g., Cas9 ribonucleoprotein). The molar ratio of the Cas nuclease to the sgRNA can be any range that promotes continuous homologous recombination of the targeted AAV vector and the target gene locus. In some embodiments, the molar ratio of the Cas9 polypeptide to the sgRNA is about 1:5; 1:4; 1:3; 1:2.5; 1:2; or 1:1. In other embodiments, the molar ratio of the Cas9 polypeptide to the sgRNA is about 1:2 to about 1:3. In certain embodiments, the molar ratio of the Cas9 polypeptide to the sgRNA is about 1:2.5.

[0279] Cas nucleases and variants or fragments thereof can be introduced into cells (e.g., cells isolated from a subject or cells in vivo, such as in a subject) as Cas polypeptides or variants or fragments thereof, mRNA encoding Cas polypeptides or variants or fragments thereof, recombinant expression vectors comprising nucleotide sequences encoding Cas polypeptides or variants or fragments thereof, or Cas ribonucleoproteins. Those skilled in the art will recognize that any method for delivering exogenous polynucleotides, polypeptides, or ribonucleoproteins can be used. Non-limiting examples of such methods include electroporation, nuclear transfection, transfection, lipofection, transduction, microinjection, electroinjection, electrofusion, nanoparticle bombardment, transformation, conjugation, and the like.

[0280] In one aspect, the present disclosure provides the use of nanoparticles as a means of delivering CRISPR components to a subject in need thereof. In some embodiments, the nanoparticles are selected from lipid nanoparticles (LNPs) or liposomes, hydrogel nanoparticles, metal organic nanoparticles, gold nanoparticles, and magnetic nanoparticles, see, for example, Xu, CF et al., (2021), Advanced Drug Delivery Reviews, 168, 3-29; see also Buschmann et al., (2021), Vaccines 9: 65; Kenjo, E. et al., (2021), Nature Communications, 12 (1), 7101.

[0281] In some embodiments, CRISPR components are delivered via nanoparticles. Without wishing to be bound by any particular theory, in certain embodiments, when present in nanoparticles, the nucleic acid is resistant to degradation by nucleases in aqueous solution. Lipid nanoparticles comprising nucleic acids and methods of making the same are disclosed in at least WO2017 / 019935, WO2017 / 049074, WO2017 / 201346, WO2017 / 218704, WO2018 / 006052, WO2018 / 013525, WO2018 / 089540, WO2018 / 119115, WO2018 / 126084, WO2018 / 157009, WO2018 / 170336, WO2018 / 222890, WO2019 / 046809, WO2019 / 089828, WO2020 / 061284, WO2020 / 061317, WO2020 / 081938, WO2020 / 097511, WO2020 / 097520, WO2020 / 097540, WO2020 / 097548, WO2020 / 2149 46. ​​WO2020 / 219941, WO2020 / 232276, WO2020 / 227615, WO2020 / 061295, WO2021 / 007278, WO2021 / 016430, WO2021 / 021988, EPPatent No.EP 2 972 360, US20200155691, US20200237671, USPatent and 11,246,933, which are incorporated herein by reference in their entirety for all purposes.

[0282] Lipid nanoparticle compositions

[0283] In some embodiments, the maximum dimension of the nanoparticle composition is 1 micron or shorter (e.g., 1 micron, 900nm, 800nm, 700nm, 600nm, 500nm, 400nm, 300nm, 200nm, 175nm, 150nm, 125nm, 100nm, 75nm, 50nm or shorter), for example when measured by dynamic light scattering (DLS), transmission electron microscopy, scanning electron microscopy or another method. The nanoparticle composition includes, for example, lipid nanoparticles (LNP), liposomes, lipid vesicles and lipid complexes (lipoplexes). In some embodiments, the nanoparticle composition is a vesicle comprising one or more lipid bilayers. In certain embodiments, the nanoparticle composition includes two or more concentric bilayers separated by an aqueous compartment. The lipid bilayers can be functionalized and / or cross-linked to each other. The lipid bilayer can include one or more ligands, proteins or channels.

[0284] In various embodiments, the lipid nanoparticles described herein have an average diameter of about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 nm to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm and are substantially non-toxic.

[0285] In certain embodiments, lipid nanoparticles described herein include one or more components, including lipid components and (optionally) structural components. The lipid component includes a lipid selected from ionizable lipids and / or cationic lipids (i.e., lipids that can have a positive charge or a partially positive charge at physiological pH), neutral lipids (e.g., phospholipids or sphingolipids) and polymer-conjugated lipids (e.g., PEGylated lipids). In some embodiments, the lipid component includes a single ionizable lipid. In other embodiments, the lipid component includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 kinds of ionizable lipids. In some embodiments, the lipid component includes a single neutral lipid. In other embodiments, the lipid component includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 kinds of neutral lipids. In some embodiments, the lipid component includes a single polymer-conjugated lipid. In other embodiments, the lipid component includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 kinds of polymer-conjugated lipids. In some embodiments, the structural component comprises a single structural lipid. In other embodiments, the structural component comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 structural lipids. In some embodiments, the lipid component comprises at least one cationic lipid, at least one neutral lipid and at least one polymer-conjugated lipid. The present disclosure contemplates that the lipid component may comprise any combination of the aforementioned components.

[0286] Ionizable lipids / cationic lipids

[0287] In some embodiments, the lipid component comprises an ionizable lipid. In some embodiments, the ionizable lipid is anionic. In other embodiments, the ionizable lipid is a cationic lipid. In some embodiments, the lipid component comprises a cationic lipid, including but not limited to a cationic lipid selected from the group consisting of: 3-(didodecylamino)-N1,N1,4-triadecyl-1-piperazineethylamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-triadecyl-1,4-piperazinediethylamine (KL22), 14,25-tricosyl-15,18,21,24-tetraaza-triacontane (KL25), 1 ,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptathriacontane-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA) , 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3.β.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA), (2R)-2-({8-[(3.β.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3 -[(9Z-,12Z)-octadec-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA(2R)), (2S)-2-({8-[(3.β.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z-,12Z)-octadec-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA(2S)), lipids including cyclic amine groups, and mixtures thereof.

[0288] Non-limiting examples of cationic lipids include:

[0289]

[0290]

[0291]

[0292]

[0293]

[0294] Neutral lipids / phospholipids

[0295] In some embodiments, the lipid component further comprises a neutral lipid, including but not limited to a phospholipid selected from the group consisting of: 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-heneicosanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0Diether PC), 1-oleoyl-2-cholesterol hemisuccinyl-sn-glyceryl-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glyceryl-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glyceryl-3-phosphocholine, 1,2-diamidonoyl-sn-glyceryl-3-phosphocholine, 1,2-docosahexaenoyl-sn-glyceryl-3-phosphocholine, 1,2-dioleoyl-sn-glyceryl-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glyceryl-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-dialinolenoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-docosahexaenoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-dioleoyl-sn-glyceryl-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin (SM) and mixtures thereof.

[0296] Polymer-conjugated lipids

[0297] In some embodiments, the lipid component further comprises a polymer-conjugated lipid, the polymer-conjugated lipid including but not limited to a PEGylated lipid selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. For example, the PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DMA, or PEG-DSPE lipid.

[0298] Non-exhaustive and non-limiting examples of PEG lipids include:

[0299]

[0300]

[0301]

[0302] Structured lipids / sterols

[0303] In some embodiments, LNP further comprises a structural component. See generally Patel, S. et al., (2020), Nature Communications, 11 (1), 1-13. In some embodiments, the structural component comprises sterols, including but not limited to sterols selected from the group consisting of cholesterol, fecosterol, stigmasterol, stigmasterol, sitosterol, β-sitosterol, lupeol, betulin, ursolic acid, oleanolic acid, campesterol, fucosterol, brassicasterol, ergosterol, 9,11-dehydroergosterol, tomatine, tomato, alpha-tocopherol and mixtures thereof. In other embodiments, the structural lipids include cholesterol and corticosteroids (e.g., prednisolone, dexamethasone, prednisone and hydrocortisone) or a combination thereof.

[0304] Non-exhaustive and non-limiting examples of structured lipids include:

[0305]

[0306]

[0307] formula

[0308] Nanoparticle compositions can include lipid components and one or more other components, such as therapeutic and / or preventive agents.Nanoparticle compositions can be designed to be used for one or more specific applications or targets.The elements of nanoparticle compositions can be selected based on specific applications or targets and / or based on the efficacy, toxicity, expense, ease of use, availability or other characteristics of one or more elements.Similarly, the specific formulation of nanoparticle compositions can be selected for specific applications or targets according to the efficacy and toxicity of the specific combination of elements, for example.

[0309] The lipid component of the nanoparticle composition can include, for example, cationic lipids, phospholipids (eg, unsaturated lipids such as DOPE or DSPC), PEG lipids, and structural lipids. The elements of the lipid component can be provided in specific fractions.

[0310] In some embodiments, the lipid component of the nanoparticle composition includes ionizable lipids, phospholipids, PEG lipids and structural lipids. In certain embodiments, the lipid component of the nanoparticle composition includes about 30mol% to about 60mol% of ionizable lipids, about 0mol% to about 30mol% of phospholipids, about 0mol% to about 10mol% of PEG lipids and about 17.5mol% to about 50mol% of structural lipids, provided that the total mol% does not exceed 100%. In some embodiments, the lipid component of the nanoparticle composition includes about 35mol% to about 55mol% of the compound of ionizable lipids, about 5mol% to about 25mol% of phospholipids, about 0mol% to about 10mol% of PEG lipids and about 30mol% to about 40mol% of structural lipids. In a specific embodiment, the lipid component includes about 50mol% of the compound, about 10mol% of phospholipids, about 38.5mol% of structural lipids and about 1.5mol% of PEG lipids. In another embodiment, the lipid component comprises about 40mol% of the compound, about 20mol% of the phospholipids, about 38.5mol% of the structural lipids and about 1.5mol% of the PEG lipids. In some embodiments, the phospholipids can be DOPE or DSPC. In other embodiments, the PEG lipid can be PEG-DMG and / or the structural lipid can be cholesterol.

[0311] In some embodiments, the ionizable lipids constitute about 20mol% to about 60mol% of the lipid component. In other embodiments, the ionizable lipids constitute about 35mol% to about 55mol% of the lipid component. In various embodiments, the ionizable lipids constitute about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55 or 60mol% of the lipid component.

[0312] In some embodiments, neutral lipids constitute about 0 to about 30 mol% of the lipid component. In other embodiments, neutral lipids constitute about 5 to about 25 mol% of the lipid component. In various embodiments, neutral lipids constitute about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 mol% of the lipid component.

[0313] In some embodiments, the polymer-conjugated lipids constitute about 0 to about 15 mol% of the lipid component. In other embodiments, the polymer-conjugated lipids constitute about 0.5 mol% to about 10 mol% of the lipid component. In various embodiments, the polymer-conjugated lipids constitute about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5 9, 9.5, 10, or 15 mol% of the lipid component.

[0314] In some embodiments, the structural component constitutes about 17.5mol% to about 50mol% of the lipid component. In other embodiments, the structural component constitutes about 30mol% to about 40mol% of the lipid component. In various embodiments, the structural component constitutes about 17.5, 20, 22.5, 25, 27.5, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50mol% of the lipid component.

[0315] The structural components can alternatively be expressed as a ratio relative to the lipid component. In some embodiments, the ratio of the structural components to the lipid component (sterol: lipid) is about 1: 1. In other embodiments, the ratio of the structural components to the lipid component (sterol: lipid) is about 1: 5. In various embodiments, the ratio of the structural components to the lipid component (sterol: lipid) is about 1: 1, 1: 2, 1: 3, 1: 4, 1: 5, 1: 6, 1: 7, 1: 8, 1: 9, 1: 10, 1: 15, 1: 20 or 1: 25.

[0316] Nanoparticle compositions can be designed to be used for one or more specific applications or targets. For example, nanoparticle compositions can be designed to deliver therapeutic and / or preventive agents such as RNA to specific cells, tissues, organs or systems or groups thereof in mammals. The physicochemical properties of nanoparticle compositions can be changed to increase the selectivity of specific body targets. For example, the particle size can be adjusted based on the window size of different organs. The therapeutic and / or preventive agents included in the nanoparticle compositions can also be selected based on one or more desired delivery targets. For example, therapeutic and / or preventive agents can be selected for specific indications, conditions, diseases or illnesses and / or for delivery to specific cells, tissues, organs or systems or groups thereof (for example, local or specific delivery). In certain embodiments, nanoparticle compositions can include mRNA encoding a polypeptide of interest, which can be translated intracellularly to produce a polypeptide of interest. Such compositions can be designed to be specifically delivered to specific organs. In some embodiments, compositions can be designed to be specifically delivered to mammalian joints.

[0317] The amount of therapeutic and / or preventive agent in the nanoparticle composition can depend on the size, composition, desired target and / or application or other properties of the nanoparticle composition and the property of the therapeutic and / or preventive agent. For example, the amount of RNA that can be used in the nanoparticle composition can depend on the size, sequence and other characteristics of the RNA. The relative amount of therapeutic and / or preventive agent and other elements (for example, lipid) in the nanoparticle composition can also change. In some embodiments, the wt / wt ratio of lipid component and therapeutic and / or preventive agent in the nanoparticle composition can be about 5:1 to about 60:1, such as 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1 and 60:1. For example, the wt / wt ratio of the lipid component to the therapeutic and / or prophylactic agent can be about 10: 1 to about 40: 1. In certain embodiments, the wt / wt ratio is about 20: 1. The amount of the therapeutic and / or prophylactic agent in the nanoparticle composition can be measured, for example, using absorption spectroscopy (e.g., UV-visible spectroscopy).

[0318] In some embodiments, the therapeutic and / or preventive agent comprises a nucleic acid component. In some embodiments, the nucleic acid component comprises RNA, including but not limited to RNA selected from the group consisting of messenger RNA (mRNA), CRISPR RNA (crRNA), tracrRNA, single guide RNA (sgRNA), short interfering RNA (siRNA), antisense oligonucleotides (ASOs) and mixtures thereof. In other embodiments, the nucleic acid component comprises DNA, including but not limited to DNA selected from the group consisting of linear DNA, plasmid DNA, antisense oligonucleotides and mixtures thereof.

[0319] In some embodiments, the nanoparticle composition comprises one or more RNA, and one or more RNA, lipid and its amount can be selected to provide a specific N: P ratio. The N: P ratio of the composition refers to the molar ratio of the number of nitrogen atoms in one or more lipids and the phosphate groups in the RNA. In general, a lower N: P ratio is preferred. One or more RNA, lipid and its amount can be selected to provide an N: P ratio of about 2: 1 to about 30: 1, such as 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 12: 1, 14: 1, 16: 1, 18: 1, 20: 1, 22: 1, 24: 1, 26: 1, 28: 1 or 30: 1. In certain embodiments, the N: P ratio can be about 2: 1 to about 8: 1. In other embodiments, the N: P ratio is about 5: 1 to about 8: 1. For example, the N:P ratio can be about 5.0: 1, about 5.5: 1, about 5.67: 1, about 6.0: 1, about 6.5: 1, or about 7.0: 1. For example, the N:P ratio can be about 5.67:1.

[0320] In some embodiments, the nucleic acid component is composed of modified nucleic acids. For example, RNA can be modified RNA. That is, RNA can include one or more nuclear bases, nucleosides, nucleotides or joints that are not naturally occurring. "Modified" species may also be referred to as "altered" species in this article. Species can be modified or changed chemically, structurally or functionally. For example, modified nuclear base species can include one or more substitutions that are not naturally occurring.

[0321] In certain embodiments, the disclosure includes methods for treating joint diseases or disorders. In other embodiments, the disclosure includes methods for treating osteoarthritis. In some embodiments, the disclosure includes methods for treating joint inflammation, the methods comprising administering a therapeutically effective amount of a CRISPR-Cas composition encapsulated in or associated with a lipid nanoparticle (LNP) wherein the composition comprises one or more non-naturally occurring polynucleotides encoding Cas proteins and at least one sgRNA. In some embodiments, LNP is administered intra-articularly.

[0322] In certain embodiments, the present disclosure includes methods for treating fibrosis or scarring. In some embodiments, fibrosis and / or scarring is postoperative and / or postoperative fibrosis and / or scarring. In some embodiments, fibrosis and / or scarring is after ligament reconstruction. In some embodiments, fibrosis and / or scarring is reconstruction after the anterior cruciate ligament (ACL). In some embodiments, fibrosis and / or scarring is reconstruction of the anterior cruciate ligament (ACL) after autologous transplantation. In some embodiments, fibrosis and / or scarring is reconstruction of the anterior cruciate ligament (ACL) after allograft. In some embodiments, fibrosis and / or scarring is due to fibrosis of the knee joint. In some embodiments, fibrosis and / or scarring is due to intra-articular fibrous nodules. In some embodiments, fibrosis and / or scarring is after total knee arthroplasty (TKA). In some embodiments, fibrosis and / or scarring is due to fibrosis of the knee joint after TKA. In some embodiments, fibrosis and / or scarring is after microdiscectomy. In some embodiments, fibrosis and / or scarring is due to epidural fibrosis after microdiscectomy. In some embodiments, the disclosure includes a method for treating fibrosis or scarring, the method comprising administering a therapeutically effective amount of a CRISPR-Cas composition encapsulated in or associated with a lipid nanoparticle (LNP) wherein the composition comprises one or more non-naturally occurring polynucleotides encoding Cas proteins and at least one sgRNA. In some embodiments, LNP is administered topically. In other embodiments, LNP is administered intra-articularly. In some embodiments, the pharmaceutical composition is administered during surgery and / or after surgery.

[0323] In certain embodiments, the disclosure includes methods for treating low back pain. In other embodiments, the disclosure includes methods for treating discogenic disorders. In some embodiments, the disclosure includes methods for treating local nociception, inflammation or morphological changes associated with back or spinal pathology or disorders in a subject in need thereof, the method comprising administering a therapeutically effective amount of a CRISPR-Cas composition encapsulated in or associated with a lipid nanoparticle (LNP) wherein the composition comprises one or more non-naturally occurring polynucleotides encoding a Cas protein and at least one sgRNA. In some embodiments, LNP is administered intradiscally. In other embodiments, LNP is administered epidurally.

[0324] As used herein, a "lipid component" is a component of a nanoparticle composition that contains one or more lipids. For example, the lipid component can include one or more cationic / ionizable, pegylated, structural lipids or other lipids, such as phospholipids.

[0325] As used herein, the term "delivery" means providing an entity to a destination. For example, delivering a therapeutic and / or prophylactic agent to a subject may involve administering to the subject a nanoparticle composition comprising the therapeutic and / or prophylactic agent (e.g., by intravenous, intramuscular, intradermal, subcutaneous, intraarticular, or intradiscal route). Administering a nanoparticle composition to a mammal or mammalian cell may involve contacting one or more cells with the nanoparticle composition.

[0326] As used herein, "naturally occurring" means occurring in nature without the assistance of human intervention.

[0327] As used herein, "PEG lipid" or "PEGylated lipid" refers to a lipid comprising a polyethylene glycol component. These lipids may also be referred to as PEG-modified lipids.

[0328] As used herein, "phospholipid" is a lipid comprising a phosphate moiety and one or more carbon chains (e.g., unsaturated fatty acid chains). Phospholipids can include one or more multiple bonds (e.g., double bonds or triple bonds) (e.g., one or more unsaturated bonds). Specific phospholipids can promote fusion with membranes. For example, cationic phospholipids can interact with one or more negatively charged phospholipids of membranes (e.g., cell membranes or intracellular membranes). The fusion of phospholipids with membranes can allow one or more elements of the composition comprising lipids to pass through the membrane, thereby allowing, for example, one or more elements to be delivered to cells.

[0329] Physical properties

[0330] The properties of a nanoparticle composition can depend on its components. For example, a nanoparticle composition comprising cholesterol as a structural lipid can have different properties than a nanoparticle composition comprising a different structural lipid. Similarly, the properties of a nanoparticle composition can depend on the absolute or relative amounts of its components. For example, a nanoparticle composition comprising a higher mole fraction of phospholipids can have different properties than a nanoparticle composition comprising a lower mole fraction of phospholipids. The properties can also vary depending on the method and conditions of preparation of the nanoparticle composition.

[0331] Nanoparticle compositions can be characterized by a variety of methods. For example, a microscope (e.g., a transmission electron microscope or a scanning electron microscope) can be used to examine the morphology and size distribution of the nanoparticle composition. Dynamic light scattering or potentiometric determination (e.g., potentiometric titration) can be used to measure the zeta potential. Dynamic light scattering can also be used to determine particle size. Instruments such as ZetasizerNano ZS (Malvern Instruments, Malvern, Worcestershire, UK) can also be used to measure the various characteristics of the nanoparticle composition, such as particle size, polydispersity index, and zeta potential.

[0332] The average size of the nanoparticle composition can be between 10nm and 1 micron, for example, measured by dynamic light scattering (DLS). For example, the average size can be from about 40nm to about 150nm, for example, about 40nm, 45nm, 50nm, 55nm, 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 90nm, 95nm, 100nm, 105nm, 110nm, 115nm, 120nm, 125nm, 130nm, 135nm, 140nm, 145nm or 150nm. In some embodiments, the average size of the nanoparticle composition can be from about 50nm to about 100nm, from about 50nm to about 90nm, from about 50nm to about 80nm, from about 50nm to about 70nm, from about 50nm to about 60nm, from about 60nm to about 100nm, from about 60nm to about 90nm, from about 60nm to about 80nm, from about 60nm to about 70nm, from about 70nm to about 100nm, from about 70nm to about 90nm, from about 70nm to about 80nm, from about 80nm to about 100nm, from about 80nm to about 90nm, or from about 90nm to about 100nm. In certain embodiments, the average size of the nanoparticle composition can be from about 70nm to about 100nm. In specific embodiments, the average size can be about 80nm. In other embodiments, the average size can be about 100nm.

[0333] The nanoparticle composition can be relatively uniform. The polydispersity index can be used to indicate the uniformity of the nanoparticle composition, such as the particle size distribution of the nanoparticle composition. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. The nanoparticle composition can have a polydispersity index of about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24 or 0.25. In some embodiments, the polydispersity index of the nanoparticle composition can be about 0.10 to about 0.20.

[0334] The zeta potential of a nanoparticle composition can be used to indicate the electrokinetic potential of the composition. For example, the zeta potential can describe the surface charge of a nanoparticle composition. Nanoparticle compositions with relatively low charge (positive or negative) are generally desirable because higher charged materials may interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of the nanoparticle composition can be from about -10 mV to about +20 mV, from about -10 mV to about +15 mV, from about -10 mV to about +10 mV, from about -10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about -5 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV.

[0335] The encapsulation efficiency of therapeutic and / or prophylactic agents describes the amount of therapeutic and / or prophylactic agents encapsulated in the nanoparticle composition or otherwise associated with the nanoparticle composition after preparation relative to the initial amount provided. The encapsulation efficiency is ideally high (e.g., close to 100%). For example, the encapsulation efficiency can be measured by comparing the amount of therapeutic and / or prophylactic agents in a solution containing the nanoparticle composition before and after decomposing the nanoparticle composition with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free therapeutic and / or prophylactic agents (e.g., RNA) in the solution. For the nanoparticle compositions described herein, the encapsulation efficiency of the therapeutic and / or prophylactic agent can be at least 50%, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In certain embodiments, the encapsulation efficiency may be at least 90%.

[0336] The nanoparticle composition may optionally include one or more coatings. For example, the nanoparticle composition may be formulated in a capsule, film, or tablet having a coating. The capsule, film, or tablet comprising the composition described herein may have any useful size, tensile strength, hardness, or density.

[0337] Non-limiting examples of genes that can be silenced or inhibited by permanent gene editing of synoviocytes via the CRISPR approach include IL-1α, IL-1β, IL-4, IL-9, IL-10, IL-13, and TNF-α.

[0338] Non-limiting examples of genes that can be enhanced by permanent gene editing of synoviocytes via the CRISPR approach include IL-1α, IL-1β, IL-4, IL-9, IL-10, IL-13, and TNF-α.

[0339] Examples of systems, methods, and compositions for altering expression of target gene sequences by CRISPR methods and that can be used according to embodiments of the present disclosure are described in U.S. Pat. Nos. 8,697,359; 8,993,233; 8,795,965; 8,771,945; 8,889,356; 8,865,406; 8,999,641; 8,945,839; 8,932,814; 8,871,445; 8,906,616; and 8,895,308, which are incorporated herein by reference. Resources for performing CRISPR methods, such as plasmids for expressing CRISPR-Cas9 and CRISPR-Cpf1, are commercially available from companies such as GenScript.

[0340] In one embodiment, genetic modification of at least a portion of the joint synovial cells as described herein can be performed using the CRISPR-Cpf1 system as described in U.S. Pat. No. 9,790,490, the disclosure of which is incorporated herein by reference.

[0341] In one embodiment, genetic modification of at least a portion of the joint synovial cells as described herein can be performed using a CRISPR-Cas system comprising a single vector system as described in U.S. Pat. No. 9,907,863, the disclosure of which is incorporated herein by reference.

[0342] The pharmaceutical composition for treating or preventing joint diseases or conditions includes a gene editing system, wherein the gene editing system targets at least one locus associated with joint function, wherein the method further includes gene editing at least a portion of joint synovial cells by a TALE method. According to a specific embodiment, the TALE method is used to target at least one locus associated with joint function, wherein at least a portion of the synovial cells of the joint is gene edited. Alternatively, the TALE method is used during targeting at least one locus associated with joint function, wherein at least a portion of the synovial cells of the joint is gene edited so that the expression of at least one locus associated with joint function genes is enhanced in at least a portion of the synovial cells of the joint.

[0343] TALE stands for "transcription activator-like effector" protein, which includes TALEN ("transcription activator-like effector nuclease"). The method of gene editing using the TALE system may also be referred to as the TALE method herein. TALE is a naturally occurring protein from the plant pathogenic bacteria genus Xanthomonas (Xanthomonas), and contains a DNA binding domain consisting of a series of 33-35 amino acid repeat domains that each recognizes a single base pair. TALE specificity is determined by two hypervariable amino acids referred to as repeat variable diresidues (RVD). Modular TALE repeats are linked together to recognize adjacent DNA sequences. The specific RVD in the DNA binding domain recognizes the bases in the target locus, providing structural features for assembling predictable DNA binding domains. The DNA binding domain of TALE is fused to the catalytic domain of the IIS type FokI nuclease to form a targetable TALE nuclease. In order to induce site-specific mutations, two separate TALEN arms separated by 14-20 base pair spacers bring the FokI monomers very close to dimerize and produce targeted double-strand breaks.

[0344] Several large systematic studies using various assembly methods have shown that TALE repeats can be combined to recognize almost any user-defined sequence. Custom-designed TALE arrays can also be commercially available from Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuticals (Lexington, KY, USA), and Life Technologies (Grand Island, NY, USA). TALE and TALEN methods suitable for use in the present disclosure are described in U.S. Patent Application Publication Nos. US 2011 / 0201118 A1; US ​​2013 / 0117869 A1; US ​​2013 / 0315884 A1; US ​​2015 / 0203871 A1 and US 2016 / 0120906 A1, which are incorporated herein by reference.

[0345] Non-limiting examples of genes that can be silenced or inhibited by permanent gene editing of synoviocytes via the TALE approach include IL-1α, IL-1β, IL-4, IL-9, IL-10, IL-13, and TNF-α.

[0346] Non-limiting examples of genes that can be enhanced via permanent gene editing of synoviocytes via the TALE approach include IL-1α, IL-1β, IL-4, IL-9, IL-10, IL-13, and TNF-α.

[0347] Examples of systems, methods, and compositions for altering the expression of a target gene sequence by the TALE approach and that can be used according to embodiments of the present disclosure are described in U.S. Pat. No. 8,586,526, which is incorporated herein by reference.

[0348] Zinc finger method

[0349] The pharmaceutical composition for treating or preventing a joint disease or condition includes a gene editing system, wherein the gene editing system targets at least one locus associated with joint function, wherein the method further comprises gene editing at least a portion of the joint synovial cells by a zinc finger or zinc finger nuclease method. According to a specific embodiment, a zinc finger method is used to target at least one locus associated with joint function, wherein at least a portion of the synovial cells of the joint are gene edited. Alternatively, a zinc finger method is used during targeting at least one locus associated with joint function, wherein at least a portion of the synovial cells of the joint are gene edited so that the expression of at least one locus associated with a joint function gene is enhanced in at least a portion of the joint synovial cells.

[0350] A single zinc finger contains approximately 30 amino acids in a conserved ββα configuration. Several amino acids on the surface of the α-helix typically contact 3bp in the major groove of DNA, with varying levels of selectivity. Zinc fingers have two protein domains. The first domain is a DNA binding domain, which contains a eukaryotic transcription factor and contains zinc fingers. The second domain is a nuclease domain, which contains the FokI restriction enzyme and is responsible for catalytic cleavage of DNA.

[0351] The DNA binding domain of a single ZFN typically contains three to six individual zinc finger repeats, and each zinc finger repeat can recognize 9 to 18 base pairs. If the zinc finger domain is specific to its intended target site, then in theory even a pair of 3-finger ZFNs that recognize a total of 18 base pairs can target a single locus in the mammalian genome. One way to generate a new zinc finger array is to combine smaller zinc finger "modules" of known specificity. The most common modular assembly process involves combining three individual zinc fingers that can each recognize a 3 base pair DNA sequence to generate a 3-finger array that can recognize a 9 base pair target site. Alternatively, selection-based methods such as oligomer pool engineering (OPEN) can be used to select new zinc finger arrays from a random library that takes into account context-dependent interactions between adjacent fingers. Engineered zinc fingers are commercially available; Sangamo Biosciences (Richmond, CA, USA) has collaborated with Sigma–Aldrich (St. Louis, MO, USA) to develop a proprietary platform for zinc finger construction.

[0352] Non-limiting examples of genes that can be silenced or inhibited by permanent gene editing of synovial cells via a zinc finger approach include IL-1α, IL-1β, IL-4, IL-9, IL-10, IL-13, TNF-α, IL-6, IL-8, IL-18, matrix metalloproteinases (MMPs) or components of the NLRP3 inflammasome. In some embodiments, components of the NLRP3 inflammasome include NLRP3, apoptosis-associated speck-like protein (ASC) containing CARD, caspase-1, and combinations thereof.

[0353] Non-limiting examples of genes that can be enhanced by permanent gene editing of synoviocytes via a zinc finger approach include the group including IL-1Ra, TIMP-1, TIMP-2, TIMP-3, TIMP-4, and combinations thereof. In one aspect, the present disclosure provides compositions for upregulating anti-inflammatory cytokines.

[0354] Examples of systems, methods, and compositions for altering the expression of a target gene sequence by zinc finger methods that can be used in accordance with embodiments of the present disclosure are described in U.S. Pat. Nos. 6,534,261; 6,607,882; 6,746,838; 6,794,136; 6,824,978; 6,866,997; 6,933,113; 6,979,539; 7,013,219; 7,030,215; 7,220,719; 7,241,573; 7,241,574; 7,585,849; 7,595,376; 6,903,185; and 6,479,626, which are incorporated herein by reference.

[0355] In some aspects, the cell can be ex vivo gene-edited, wherein the gene editing targets one or more anti-inflammatory cytokine loci. In some aspects, the cell is a non-synovial cell. In some aspects, the cell is a mesenchymal stem cell. In some aspects, the cell is a macrophage. In some aspects, the present disclosure provides a pharmaceutical composition for treating or preventing a joint disease or condition, comprising a colony of gene-edited cells, wherein the gene-edited cells are edited by a gene editing system targeting at least one locus associated with joint function. On the one hand, a colony of gene-edited cells is injected into a synovial joint.

[0356] Other examples of systems, methods, and compositions for altering the expression of target gene sequences by zinc finger approaches that can be used in accordance with embodiments of the present disclosure are described in Beane et al., Molecular Therapy, 2015, 23 1380-1390, the disclosure of which is incorporated herein by reference.

[0357] Exemplary embodiments

[0358] In some embodiments, the present disclosure provides a pharmaceutical composition for treating or preventing a joint disease or condition, the composition comprising a therapeutically effective amount of one or more nucleic acids encoding a clustered regularly interspaced short palindromic repeats (CRISPR) gene editing system. The system comprises a CRISPR-associated protein 9 (Cas9) protein and at least one guide RNA targeting an IL-1α or IL-1β gene, wherein the target sequence is adjacent to a protospacer sequence adjacent motif (PAM) sequence of the Cas9 protein.

[0359] In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2 of the IL-1α gene. In some embodiments, the crRNA sequence forms no more than 5 mismatches with the target sequence in exon 2 of the human IL-1α gene. In some embodiments, the crRNA sequence forms no more than 4 mismatches with the target sequence in exon 2 of the human IL-1α gene. In some embodiments, the crRNA sequence forms no more than 3 mismatches with the target sequence in exon 2 of the human IL-1α gene. In some embodiments, the crRNA sequence forms no more than 2 mismatches with the target sequence in exon 2 of the human IL-1α gene. In some embodiments, the crRNA sequence forms no more than 1 mismatch with the target sequence in exon 2 of the human IL-1α gene. In some embodiments, the crRNA sequence does not form a mismatch with the target sequence in exon 2 of the human IL-1α gene.

[0360] In some embodiments, at least one guide RNA targets a human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 3 of the IL-1α gene. In some embodiments, the crRNA sequence forms no more than 5 mismatches with the target sequence in exon 3 of the human IL-1α gene. In some embodiments, the crRNA sequence forms no more than 4 mismatches with the target sequence in exon 3 of the human IL-1α gene. In some embodiments, the crRNA sequence forms no more than 3 mismatches with the target sequence in exon 3 of the human IL-1α gene. In some embodiments, the crRNA sequence forms no more than 2 mismatches with the target sequence in exon 3 of the human IL-1α gene. In some embodiments, the crRNA sequence forms no more than 1 mismatch with the target sequence in exon 3 of the human IL-1α gene. In some embodiments, the crRNA sequence does not form a mismatch with the target sequence in exon 3 of the human IL-1α gene.

[0361] In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 4 of the IL-1α gene. In some embodiments, the crRNA sequence forms no more than 5 mismatches with the target sequence in exon 4 of the human IL-1α gene. In some embodiments, the crRNA sequence forms no more than 4 mismatches with the target sequence in exon 4 of the human IL-1α gene. In some embodiments, the crRNA sequence forms no more than 3 mismatches with the target sequence in exon 4 of the human IL-1α gene. In some embodiments, the crRNA sequence forms no more than 2 mismatches with the target sequence in exon 4 of the human IL-1α gene. In some embodiments, the crRNA sequence forms no more than 1 mismatch with the target sequence in exon 4 of the human IL-1α gene. In some embodiments, the crRNA sequence does not form a mismatch with the target sequence in exon 4 of the human IL-1α gene.

[0362] In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 5 of the IL-1α gene. In some embodiments, the crRNA sequence forms no more than 5 mismatches with the target sequence in exon 5 of the human IL-1α gene. In some embodiments, the crRNA sequence forms no more than 4 mismatches with the target sequence in exon 5 of the human IL-1α gene. In some embodiments, the crRNA sequence forms no more than 3 mismatches with the target sequence in exon 5 of the human IL-1α gene. In some embodiments, the crRNA sequence forms no more than 2 mismatches with the target sequence in exon 5 of the human IL-1α gene. In some embodiments, the crRNA sequence forms no more than 1 mismatch with the target sequence in exon 5 of the human IL-1α gene. In some embodiments, the crRNA sequence does not form a mismatch with the target sequence in exon 5 of the human IL-1α gene.

[0363] In some embodiments, at least one guide RNA targets a human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 6 of the IL-1α gene. In some embodiments, the crRNA sequence forms no more than 5 mismatches with a target sequence in exon 6 of a human IL-1α gene. In some embodiments, the crRNA sequence forms no more than 4 mismatches with a target sequence in exon 6 of a human IL-1α gene. In some embodiments, the crRNA sequence forms no more than 3 mismatches with a target sequence in exon 6 of a human IL-1α gene. In some embodiments, the crRNA sequence forms no more than 2 mismatches with a target sequence in exon 6 of a human IL-1α gene. In some embodiments, the crRNA sequence forms no more than 1 mismatch with a target sequence in exon 6 of a human IL-1α gene. In some embodiments, the crRNA sequence does not form a mismatch with a target sequence in exon 6 of a human IL-1α gene.

[0364] In some embodiments, at least one guide RNA targets a human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 7 of the IL-1α gene. In some embodiments, the crRNA sequence forms no more than 5 mismatches with a target sequence in exon 7 of a human IL-1α gene. In some embodiments, the crRNA sequence forms no more than 4 mismatches with a target sequence in exon 7 of a human IL-1α gene. In some embodiments, the crRNA sequence forms no more than 3 mismatches with a target sequence in exon 7 of a human IL-1α gene. In some embodiments, the crRNA sequence forms no more than 2 mismatches with a target sequence in exon 7 of a human IL-1α gene. In some embodiments, the crRNA sequence forms no more than 1 mismatch with a target sequence in exon 7 of a human IL-1α gene. In some embodiments, the crRNA sequence does not form a mismatch with a target sequence in exon 7 of a human IL-1 gene.

[0365] In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2 or exon 3 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2 or exon 4 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2 or exon 5 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2 or exon 6 of the IL-1α gene. In some embodiments, at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2 or exon 7 of the IL-1α gene.

[0366] In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 3 or exon 4 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 3 or exon 5 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 3 or exon 6 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 3 or exon 7 of the IL-1α gene.

[0367] In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 4 or exon 5 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 4 or exon 6 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 4 or exon 7 of the IL-1α gene.

[0368] In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 5 or exon 6 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 5 or exon 7 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 6 or exon 7 of the IL-1α gene.

[0369] In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 3, or exon 4 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 3, or exon 5 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 3, or exon 6 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 3, or exon 7 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 4, or exon 5 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 4, or exon 6 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 4, or exon 7 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 5, or exon 6 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 5, or exon 7 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 6, or exon 7 of the IL-1α gene.

[0370] In some embodiments, at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 3, exon 4, or exon 5 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 3, exon 4, or exon 6 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 3, exon 4, or exon 7 of the IL-1α gene. In some embodiments, at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 3, exon 5, or exon 6 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 3, exon 5, or exon 7 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 3, exon 6, or exon 7 of the IL-1α gene.

[0371] In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 4, exon 5, or exon 6 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 4, exon 5, or exon 7 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 4, exon 6, or exon 7 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 5, exon 6, or exon 7 of the IL-1α gene.

[0372] In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 3, exon 4, or exon 5 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 3, exon 4, or exon 6 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 3, exon 4, or exon 7 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 4, exon 5, or exon 6 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 4, exon 5, or exon 7 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 4, exon 6, or exon 7 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 5, exon 6, or exon 7 of the IL-1α gene.

[0373] In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 3, exon 4, exon 5, or exon 6 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 3, exon 4, exon 5, or exon 7 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 3, exon 4, exon 6, or exon 7 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 3, exon 5, exon 6, or exon 7 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 4, exon 5, exon 6, or exon 7 of the IL-1α gene.

[0374] In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 3, exon 4, exon 5, or exon 6 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 3, exon 4, exon 5, or exon 7 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 3, exon 4, exon 5, exon 6, or exon 7 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 4, exon 5, exon 6, or exon 7 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 3, exon 5, exon 6, or exon 6 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 3, exon 4, exon 6, or exon 7 of the IL-1α gene. In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 3, exon 4, exon 5, exon 6, or exon 7 of the IL-1α gene.

[0375] In some embodiments, at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence having at least 75% identity to a sequence selected from the group consisting of SEQ ID NOs: 168-187, 298-387, and 681-710. In some embodiments, the crRNA sequence has at least 80% identity to a sequence selected from the group consisting of SEQ ID NOs: 168-187, 298-387, and 681-710. In some embodiments, the crRNA sequence has at least 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 168-187, 298-387, and 681-710. In some embodiments, the crRNA sequence has at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 168-187, 298-387, and 681-710. In some embodiments, the crRNA sequence is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 168-187, 298-387, and 681-710. In some embodiments, the crRNA sequence is selected from the group consisting of SEQ ID NOs: 168-187, 298-387, and 681-710.

[0376] In some embodiments, at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence having at least 75% identity to SEQ ID NO: 301. In some embodiments, the crRNA sequence has at least 80% identity to SEQ ID NO: 301. In some embodiments, the crRNA sequence has at least 85% identity to SEQ ID NO: 301. In some embodiments, the crRNA sequence has at least 90% identity to SEQ ID NO: 301. In some embodiments, the crRNA sequence has at least 95% identity to SEQ ID NO: 301. In some embodiments, the crRNA sequence is SEQ ID NO: 301.

[0377] In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence having at least 75% identity to SEQ ID NO: 309. In some embodiments, the crRNA sequence has at least 80% identity to SEQ ID NO: 309. In some embodiments, the crRNA sequence has at least 85% identity to SEQ ID NO: 309. In some embodiments, the crRNA sequence has at least 90% identity to SEQ ID NO: 309. In some embodiments, the crRNA sequence has at least 95% identity to SEQ ID NO: 309. In some embodiments, the crRNA sequence is SEQ ID NO: 309.

[0378] In some embodiments, at least one guide RNA targets human IL-1β gene and comprises a crRNA sequence having at least 75% identity with a sequence selected from the group consisting of SEQ ID NO: 188-201, 388-496 and 711-740. In some embodiments, the crRNA sequence has at least 80% identity with a sequence selected from the group consisting of SEQ ID NO: 188-201, 388-496 and 711-740. In some embodiments, the crRNA sequence has at least 85% identity with a sequence selected from the group consisting of SEQ ID NO: 188-201, 388-496 and 711-740. In some embodiments, the crRNA sequence has at least 90% identity with a sequence selected from the group consisting of SEQ ID NO: 188-201, 388-496 and 711-740. In some embodiments, the crRNA sequence is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 188-201, 388-496, and 711-740. In some embodiments, the crRNA sequence is selected from the group consisting of SEQ ID NOs: 188-201, 388-496, and 711-740.

[0379] In some embodiments, the at least one guide RNA targets a human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 2 of the IL-1β gene. In some embodiments, the crRNA sequence forms no more than 5 mismatches with the target sequence in exon 2 of the human IL-1β gene. In some embodiments, the crRNA sequence forms no more than 4 mismatches with the target sequence in exon 2 of the human IL-1β gene. In some embodiments, the crRNA sequence forms no more than 3 mismatches with the target sequence in exon 2 of the human IL-1β gene. In some embodiments, the crRNA sequence forms no more than 2 mismatches with the target sequence in exon 2 of the human IL-1β gene. In some embodiments, the crRNA sequence forms no more than 1 mismatch with the target sequence in exon 2 of the human IL-1β gene. In some embodiments, the crRNA sequence does not form a mismatch with the target sequence in exon 2 of the human IL-1β gene.

[0380] In some embodiments, the at least one guide RNA targets a human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 3 of the IL-1β gene. In some embodiments, the crRNA sequence forms no more than 5 mismatches with the target sequence in exon 3 of the human IL-1β gene. In some embodiments, the crRNA sequence forms no more than 4 mismatches with the target sequence in exon 3 of the human IL-1β gene. In some embodiments, the crRNA sequence forms no more than 3 mismatches with the target sequence in exon 3 of the human IL-1β gene. In some embodiments, the crRNA sequence forms no more than 2 mismatches with the target sequence in exon 3 of the human IL-1β gene. In some embodiments, the crRNA sequence forms no more than 1 mismatch with the target sequence in exon 3 of the human IL-1β gene. In some embodiments, the crRNA sequence does not form a mismatch with the target sequence in exon 3 of the human IL-1β gene.

[0381] In some embodiments, at least one guide RNA targets a human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 4 of the IL-1β gene. In some embodiments, the crRNA sequence forms no more than 5 mismatches with a target sequence in exon 4 of a human IL-1β gene. In some embodiments, the crRNA sequence forms no more than 4 mismatches with a target sequence in exon 4 of a human IL-1β gene. In some embodiments, the crRNA sequence forms no more than 3 mismatches with a target sequence in exon 4 of a human IL-1β gene. In some embodiments, the crRNA sequence forms no more than 2 mismatches with a target sequence in exon 4 of a human IL-1β gene. In some embodiments, the crRNA sequence forms no more than 1 mismatch with a target sequence in exon 4 of a human IL-1β gene. In some embodiments, the crRNA sequence does not form a mismatch with a target sequence in exon 4 of a human IL-1β gene.

[0382] In some embodiments, the at least one guide RNA targets a human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 5 of the IL-1β gene. In some embodiments, the crRNA sequence forms no more than 5 mismatches with a target sequence in exon 5 of a human IL-1β gene. In some embodiments, the crRNA sequence forms no more than 4 mismatches with a target sequence in exon 5 of a human IL-1β gene. In some embodiments, the crRNA sequence forms no more than 3 mismatches with a target sequence in exon 5 of a human IL-1β gene. In some embodiments, the crRNA sequence forms no more than 2 mismatches with a target sequence in exon 5 of a human IL-1β gene. In some embodiments, the crRNA sequence forms no more than 1 mismatch with a target sequence in exon 5 of a human IL-1β gene. In some embodiments, the crRNA sequence does not form a mismatch with a target sequence in exon 5 of a human IL-1β gene.

[0383] In some embodiments, the at least one guide RNA targets a human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 6 of the IL-1β gene. In some embodiments, the crRNA sequence forms no more than 5 mismatches with a target sequence in exon 6 of a human IL-1β gene. In some embodiments, the crRNA sequence forms no more than 4 mismatches with a target sequence in exon 6 of a human IL-1β gene. In some embodiments, the crRNA sequence forms no more than 3 mismatches with a target sequence in exon 6 of a human IL-1β gene. In some embodiments, the crRNA sequence forms no more than 2 mismatches with a target sequence in exon 6 of a human IL-1β gene. In some embodiments, the crRNA sequence forms no more than 1 mismatch with a target sequence in exon 6 of a human IL-1β gene. In some embodiments, the crRNA sequence does not form a mismatch with a target sequence in exon 6 of a human IL-1β gene.

[0384] In some embodiments, the at least one guide RNA targets a human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 7 of the IL-1β gene. In some embodiments, the crRNA sequence forms no more than 5 mismatches with a target sequence in exon 7 of a human IL-1β gene. In some embodiments, the crRNA sequence forms no more than 4 mismatches with a target sequence in exon 7 of a human IL-1β gene. In some embodiments, the crRNA sequence forms no more than 3 mismatches with a target sequence in exon 7 of a human IL-1β gene. In some embodiments, the crRNA sequence forms no more than 2 mismatches with a target sequence in exon 7 of a human IL-1β gene. In some embodiments, the crRNA sequence forms no more than 1 mismatch with a target sequence in exon 7 of a human IL-1β gene. In some embodiments, the crRNA sequence does not form a mismatch with a target sequence in exon 7 of a human IL-1β gene.

[0385] In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 2 or exon 3 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 2 or exon 4 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 2 or exon 5 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 2 or exon 6 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 2 or exon 7 of the IL-1β gene.

[0386] In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 3 or exon 4 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 3 or exon 5 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 3 or exon 6 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 3 or exon 7 of the IL-1β gene.

[0387] In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 4 or exon 5 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 4 or exon 6 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 4 or exon 7 of the IL-1β gene.

[0388] In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 5 or exon 6 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 5 or exon 7 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 6 or exon 7 of the IL-1β gene.

[0389] In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 3, or exon 4 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 3, or exon 5 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 3, or exon 6 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 3, or exon 7 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 4, or exon 5 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 4, or exon 6 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 4, or exon 7 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 5, or exon 6 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 5, or exon 7 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 6, or exon 7 of the IL-1β gene.

[0390] In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 3, exon 4, or exon 5 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 3, exon 4, or exon 6 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 3, exon 4, or exon 7 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 3, exon 5, or exon 6 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 3, exon 5, or exon 7 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 3, exon 6, or exon 7 of the IL-1β gene.

[0391] In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 4, exon 5, or exon 6 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 4, exon 5, or exon 7 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 4, exon 6, or exon 7 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 5, exon 6, or exon 7 of the IL-1β gene.

[0392] In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 3, exon 4, or exon 5 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 3, exon 4, or exon 6 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 3, exon 4, or exon 7 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 4, exon 5, or exon 6 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 4, exon 5, or exon 7 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 4, exon 6, or exon 7 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 5, exon 6, or exon 7 of the IL-1β gene.

[0393] In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 3, exon 4, exon 5, or exon 6 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 3, exon 4, exon 5, or exon 7 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 3, exon 4, exon 6, or exon 7 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 3, exon 5, exon 6, or exon 7 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 4, exon 5, exon 6, or exon 7 of the IL-1β gene.

[0394] In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 3, exon 4, exon 5, or exon 6 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 3, exon 4, exon 5, or exon 7 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 3, exon 4, exon 5, exon 6, or exon 7 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 4, exon 5, exon 6, or exon 7 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 3, exon 5, exon 6, or exon 6 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 3, exon 4, exon 6, or exon 7 of the IL-1β gene. In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 3, exon 4, exon 5, exon 6, or exon 7 of the IL-1β gene.

[0395] In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence having at least 75% identity to SEQ ID NO: 462. In some embodiments, the crRNA sequence has at least 80% identity to SEQ ID NO: 462. In some embodiments, the crRNA sequence has at least 85% identity to SEQ ID NO: 462. In some embodiments, the crRNA sequence has at least 90% identity to SEQ ID NO: 462. In some embodiments, the crRNA sequence has at least 95% identity to SEQ ID NO: 462. In some embodiments, the crRNA sequence is SEQ ID NO: 462.

[0396] In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence having at least 75% identity to SEQ ID NO: 391. In some embodiments, the crRNA sequence has at least 80% identity to SEQ ID NO: 391. In some embodiments, the crRNA sequence has at least 85% identity to SEQ ID NO: 391. In some embodiments, the crRNA sequence has at least 90% identity to SEQ ID NO: 391. In some embodiments, the crRNA sequence has at least 95% identity to SEQ ID NO: 391. In some embodiments, the crRNA sequence is SEQ ID NO: 391.

[0397] In some embodiments, the at least one guide RNA targets a human IL-1β gene and comprises a crRNA sequence having at least 75% identity to SEQ ID NO: 393. In some embodiments, the crRNA sequence has at least 80% identity to SEQ ID NO: 393. In some embodiments, the crRNA sequence has at least 85% identity to SEQ ID NO: 393. In some embodiments, the crRNA sequence has at least 90% identity to SEQ ID NO: 393. In some embodiments, the crRNA sequence has at least 95% identity to SEQ ID NO: 393. In some embodiments, the crRNA sequence is SEQ ID NO: 393.

[0398] In some embodiments, the at least one guide RNA targets a human IL-1β gene and comprises a crRNA sequence having at least 75% identity to SEQ ID NO: 388. In some embodiments, the crRNA sequence has at least 80% identity to SEQ ID NO: 388. In some embodiments, the crRNA sequence has at least 85% identity to SEQ ID NO: 388. In some embodiments, the crRNA sequence has at least 90% identity to SEQ ID NO: 388. In some embodiments, the crRNA sequence has at least 95% identity to SEQ ID NO: 388. In some embodiments, the crRNA sequence is SEQ ID NO: 388.

[0399] In some embodiments, the at least one guide RNA targets a human IL-1β gene and comprises a crRNA sequence having at least 75% identity to SEQ ID NO: 389. In some embodiments, the crRNA sequence has at least 80% identity to SEQ ID NO: 389. In some embodiments, the crRNA sequence has at least 85% identity to SEQ ID NO: 389. In some embodiments, the crRNA sequence has at least 90% identity to SEQ ID NO: 389. In some embodiments, the crRNA sequence has at least 95% identity to SEQ ID NO: 389. In some embodiments, the crRNA sequence is SEQ ID NO: 389.

[0400] In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence having at least 75% identity to a sequence selected from the group consisting of SEQ ID NOs: 202-216, 552-590, and 741-770. In some embodiments, the crRNA sequence has at least 80% identity to a sequence selected from the group consisting of SEQ ID NOs: 202-216, 552-590, and 741-770. In some embodiments, the crRNA sequence has at least 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 202-216, 552-590, and 741-770. In some embodiments, the crRNA sequence has at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 202-216, 552-590, and 741-770. In some embodiments, the crRNA sequence is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 202-216, 552-590, and 741-770. In some embodiments, the crRNA sequence is selected from the group consisting of SEQ ID NOs: 202-216, 552-590, and 741-770.

[0401] In some embodiments, the at least one guide RNA targets a canine IL-1 alpha gene and comprises a crRNA sequence that is at least 75% identical to SEQ ID NO: 552. In some embodiments, the crRNA sequence is at least 80% identical to SEQ ID NO: 552. In some embodiments, the crRNA sequence is at least 85% identical to SEQ ID NO: 552. In some embodiments, the crRNA sequence is at least 90% identical to SEQ ID NO: 552. In some embodiments, the crRNA sequence is at least 95% identical to SEQ ID NO: 552. In some embodiments, the crRNA sequence is SEQ ID NO: 552.

[0402] In some embodiments, the at least one guide RNA targets a canine IL-1 alpha gene and comprises a crRNA sequence that is at least 75% identical to SEQ ID NO: 554. In some embodiments, the crRNA sequence is at least 80% identical to SEQ ID NO: 554. In some embodiments, the crRNA sequence is at least 85% identical to SEQ ID NO: 554. In some embodiments, the crRNA sequence is at least 90% identical to SEQ ID NO: 554. In some embodiments, the crRNA sequence is at least 95% identical to SEQ ID NO: 554. In some embodiments, the crRNA sequence is SEQ ID NO: 554.

[0403] In some embodiments, the at least one guide RNA targets a canine IL-1 alpha gene and comprises a crRNA sequence that is at least 75% identical to SEQ ID NO: 578. In some embodiments, the crRNA sequence is at least 80% identical to SEQ ID NO: 578. In some embodiments, the crRNA sequence is at least 85% identical to SEQ ID NO: 578. In some embodiments, the crRNA sequence is at least 90% identical to SEQ ID NO: 578. In some embodiments, the crRNA sequence is at least 95% identical to SEQ ID NO: 578. In some embodiments, the crRNA sequence is SEQ ID NO: 578.

[0404] In some embodiments, the at least one guide RNA targets a canine IL-1 alpha gene and comprises a crRNA sequence that is at least 75% identical to SEQ ID NO: 579. In some embodiments, the crRNA sequence is at least 80% identical to SEQ ID NO: 579. In some embodiments, the crRNA sequence is at least 85% identical to SEQ ID NO: 579. In some embodiments, the crRNA sequence is at least 90% identical to SEQ ID NO: 579. In some embodiments, the crRNA sequence is at least 95% identical to SEQ ID NO: 579. In some embodiments, the crRNA sequence is SEQ ID NO: 579.

[0405] In some embodiments, the at least one guide RNA targets a canine IL-1β gene and comprises a crRNA sequence having at least 75% identity to a sequence selected from the group consisting of SEQ ID NOs: 217-235, 497-551, and 771-800. In some embodiments, the crRNA sequence has at least 80% identity to a sequence selected from the group consisting of SEQ ID NOs: 217-235, 497-551, and 771-800. In some embodiments, the crRNA sequence has at least 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 217-235, 497-551, and 771-800. In some embodiments, the crRNA sequence has at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 217-235, 497-551, and 771-800. In some embodiments, the crRNA sequence is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 217-235, 497-551, and 771-800. In some embodiments, the crRNA sequence is selected from the group consisting of SEQ ID NOs: 217-235, 497-551, and 771-800.

[0406] In some embodiments, the at least one guide RNA targets a canine IL-1β gene and comprises a crRNA sequence that is at least 75% identical to SEQ ID NO: 498. In some embodiments, the crRNA sequence is at least 80% identical to SEQ ID NO: 498. In some embodiments, the crRNA sequence is at least 85% identical to SEQ ID NO: 498. In some embodiments, the crRNA sequence is at least 90% identical to SEQ ID NO: 498. In some embodiments, the crRNA sequence is at least 95% identical to SEQ ID NO: 498. In some embodiments, the crRNA sequence is SEQ ID NO: 498.

[0407] In some embodiments, the at least one guide RNA targets a canine IL-1β gene and comprises a crRNA sequence that is at least 75% identical to SEQ ID NO: 506. In some embodiments, the crRNA sequence is at least 80% identical to SEQ ID NO: 506. In some embodiments, the crRNA sequence is at least 85% identical to SEQ ID NO: 506. In some embodiments, the crRNA sequence is at least 90% identical to SEQ ID NO: 506. In some embodiments, the crRNA sequence is at least 95% identical to SEQ ID NO: 506. In some embodiments, the crRNA sequence is SEQ ID NO: 506.

[0408] In some embodiments, the pharmaceutical composition comprises one or more viral vectors that collectively include one or more nucleic acids as described herein. In some embodiments, the one or more viral vectors comprise a recombinant virus selected from a retrovirus, an adenovirus, an adeno-associated virus, a lentivirus, and herpes simplex virus-1. In some embodiments, the one or more viral vectors comprise a recombinant adeno-associated virus (AAV). In some embodiments, the recombinant AAV is of serotype 5 (AAV5). In some embodiments, the recombinant AAV is of serotype 6 (AAV6).

[0409] In some embodiments, the one or more viral vectors include: a first viral vector, the first viral vector comprising a first nucleic acid in the one or more nucleic acids encoding the Cas9 protein; and a second viral vector, the second viral vector comprising a second nucleic acid in the one or more nucleic acids encoding the at least one guide RNA. In some embodiments, the one or more viral vectors include a viral vector comprising a single nucleic acid, wherein the single nucleic acid encodes the Cas9 protein and at least one guide RNA.

[0410] In some embodiments, the composition comprises one or more liposomes that together include one or more nucleic acids. In some embodiments, the one or more nucleic acids are present in a naked state.

[0411] In some embodiments, the Cas9 protein is a Streptococcus pyogenes Cas9 polypeptide. In some embodiments, the Cas9 protein is a Staphylococcus aureus Cas9 polypeptide.

[0412] In some embodiments, the composition is formulated for parenteral administration.In some embodiments, the composition is formulated for intra-articular injection into a joint of a subject.

[0413] On the other hand, the present disclosure provides a method for treating or preventing a joint disease or condition in a subject in need thereof. The method comprises administering a pharmaceutical composition to a subject's joint, the pharmaceutical composition comprising a pharmaceutically effective amount of a composition comprising one or more nucleic acids encoding a clustered regularly interspaced short palindromic repeat (CRISPR) gene editing system. The system comprises a CRISPR-associated protein 9 (Cas9) protein and at least one guide RNA targeting an IL-1α or IL-1β gene, wherein the target sequence is adjacent to a protospacer sequence adjacent to a motif (PAM) sequence of the Cas9 protein.

[0414] In some embodiments, at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence having at least 75% identity to a sequence selected from the group consisting of SEQ ID NOs: 168-187, 298-387, and 681-710. In some embodiments, the crRNA sequence has at least 80% identity to a sequence selected from the group consisting of SEQ ID NOs: 168-187, 298-387, and 681-710. In some embodiments, the crRNA sequence has at least 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 168-187, 298-387, and 681-710. In some embodiments, the crRNA sequence has at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 168-187, 298-387, and 681-710. In some embodiments, the crRNA sequence is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 168-187, 298-387, and 681-710. In some embodiments, the crRNA sequence is selected from the group consisting of SEQ ID NOs: 168-187, 298-387, and 681-710.

[0415] In some embodiments, at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence having at least 75% identity to SEQ ID NO: 301. In some embodiments, the crRNA sequence has at least 80% identity to SEQ ID NO: 301. In some embodiments, the crRNA sequence has at least 85% identity to SEQ ID NO: 301. In some embodiments, the crRNA sequence has at least 90% identity to SEQ ID NO: 301. In some embodiments, the crRNA sequence has at least 95% identity to SEQ ID NO: 301. In some embodiments, the crRNA sequence is SEQ ID NO: 301.

[0416] In some embodiments, the at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence having at least 75% identity to SEQ ID NO: 309. In some embodiments, the crRNA sequence has at least 80% identity to SEQ ID NO: 309. In some embodiments, the crRNA sequence has at least 85% identity to SEQ ID NO: 309. In some embodiments, the crRNA sequence has at least 90% identity to SEQ ID NO: 309. In some embodiments, the crRNA sequence has at least 95% identity to SEQ ID NO: 309. In some embodiments, the crRNA sequence is SEQ ID NO: 309.

[0417] In some embodiments, at least one guide RNA targets human IL-1β gene and comprises a crRNA sequence having at least 75% identity with a sequence selected from the group consisting of SEQ ID NO: 188-201, 388-496 and 711-740. In some embodiments, the crRNA sequence has at least 80% identity with a sequence selected from the group consisting of SEQ ID NO: 188-201, 388-496 and 711-740. In some embodiments, the crRNA sequence has at least 85% identity with a sequence selected from the group consisting of SEQ ID NO: 188-201, 388-496 and 711-740. In some embodiments, the crRNA sequence has at least 90% identity with a sequence selected from the group consisting of SEQ ID NO: 188-201, 388-496 and 711-740. In some embodiments, the crRNA sequence is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 188-201, 388-496, and 711-740. In some embodiments, the crRNA sequence is selected from the group consisting of SEQ ID NOs: 188-201, 388-496, and 711-740.

[0418] In some embodiments, the at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence having at least 75% identity to SEQ ID NO: 462. In some embodiments, the crRNA sequence has at least 80% identity to SEQ ID NO: 462. In some embodiments, the crRNA sequence has at least 85% identity to SEQ ID NO: 462. In some embodiments, the crRNA sequence has at least 90% identity to SEQ ID NO: 462. In some embodiments, the crRNA sequence has at least 95% identity to SEQ ID NO: 462. In some embodiments, the crRNA sequence is SEQ ID NO: 462.

[0419] In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence having at least 75% identity to SEQ ID NO: 391. In some embodiments, the crRNA sequence has at least 80% identity to SEQ ID NO: 391. In some embodiments, the crRNA sequence has at least 85% identity to SEQ ID NO: 391. In some embodiments, the crRNA sequence has at least 90% identity to SEQ ID NO: 391. In some embodiments, the crRNA sequence has at least 95% identity to SEQ ID NO: 391. In some embodiments, the crRNA sequence is SEQ ID NO: 391.

[0420] In some embodiments, the at least one guide RNA targets a human IL-1β gene and comprises a crRNA sequence having at least 75% identity to SEQ ID NO: 393. In some embodiments, the crRNA sequence has at least 80% identity to SEQ ID NO: 393. In some embodiments, the crRNA sequence has at least 85% identity to SEQ ID NO: 393. In some embodiments, the crRNA sequence has at least 90% identity to SEQ ID NO: 393. In some embodiments, the crRNA sequence has at least 95% identity to SEQ ID NO: 393. In some embodiments, the crRNA sequence is SEQ ID NO: 393.

[0421] In some embodiments, the at least one guide RNA targets a human IL-1β gene and comprises a crRNA sequence having at least 75% identity to SEQ ID NO: 388. In some embodiments, the crRNA sequence has at least 80% identity to SEQ ID NO: 388. In some embodiments, the crRNA sequence has at least 85% identity to SEQ ID NO: 388. In some embodiments, the crRNA sequence has at least 90% identity to SEQ ID NO: 388. In some embodiments, the crRNA sequence has at least 95% identity to SEQ ID NO: 388. In some embodiments, the crRNA sequence is SEQ ID NO: 388.

[0422] In some embodiments, the at least one guide RNA targets a human IL-1β gene and comprises a crRNA sequence having at least 75% identity to SEQ ID NO: 389. In some embodiments, the crRNA sequence has at least 80% identity to SEQ ID NO: 389. In some embodiments, the crRNA sequence has at least 85% identity to SEQ ID NO: 389. In some embodiments, the crRNA sequence has at least 90% identity to SEQ ID NO: 389. In some embodiments, the crRNA sequence has at least 95% identity to SEQ ID NO: 389. In some embodiments, the crRNA sequence is SEQ ID NO: 389.

[0423] In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence having at least 75% identity to a sequence selected from the group consisting of SEQ ID NOs: 202-216, 552-590, and 741-770. In some embodiments, the crRNA sequence has at least 80% identity to a sequence selected from the group consisting of SEQ ID NOs: 202-216, 552-590, and 741-770. In some embodiments, the crRNA sequence has at least 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 202-216, 552-590, and 741-770. In some embodiments, the crRNA sequence has at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 202-216, 552-590, and 741-770. In some embodiments, the crRNA sequence is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 202-216, 552-590, and 741-770. In some embodiments, the crRNA sequence is selected from the group consisting of SEQ ID NOs: 202-216, 552-590, and 741-770.

[0424] In some embodiments, the at least one guide RNA targets a canine IL-1 alpha gene and comprises a crRNA sequence that is at least 75% identical to SEQ ID NO: 552. In some embodiments, the crRNA sequence is at least 80% identical to SEQ ID NO: 552. In some embodiments, the crRNA sequence is at least 85% identical to SEQ ID NO: 552. In some embodiments, the crRNA sequence is at least 90% identical to SEQ ID NO: 552. In some embodiments, the crRNA sequence is at least 95% identical to SEQ ID NO: 552. In some embodiments, the crRNA sequence is SEQ ID NO: 552.

[0425] In some embodiments, the at least one guide RNA targets a canine IL-1 alpha gene and comprises a crRNA sequence that is at least 75% identical to SEQ ID NO: 554. In some embodiments, the crRNA sequence is at least 80% identical to SEQ ID NO: 554. In some embodiments, the crRNA sequence is at least 85% identical to SEQ ID NO: 554. In some embodiments, the crRNA sequence is at least 90% identical to SEQ ID NO: 554. In some embodiments, the crRNA sequence is at least 95% identical to SEQ ID NO: 554. In some embodiments, the crRNA sequence is SEQ ID NO: 554.

[0426] In some embodiments, the at least one guide RNA targets a canine IL-1 alpha gene and comprises a crRNA sequence that is at least 75% identical to SEQ ID NO: 578. In some embodiments, the crRNA sequence is at least 80% identical to SEQ ID NO: 578. In some embodiments, the crRNA sequence is at least 85% identical to SEQ ID NO: 578. In some embodiments, the crRNA sequence is at least 90% identical to SEQ ID NO: 578. In some embodiments, the crRNA sequence is at least 95% identical to SEQ ID NO: 578. In some embodiments, the crRNA sequence is SEQ ID NO: 578.

[0427] In some embodiments, the at least one guide RNA targets a canine IL-1 alpha gene and comprises a crRNA sequence that is at least 75% identical to SEQ ID NO: 579. In some embodiments, the crRNA sequence is at least 80% identical to SEQ ID NO: 579. In some embodiments, the crRNA sequence is at least 85% identical to SEQ ID NO: 579. In some embodiments, the crRNA sequence is at least 90% identical to SEQ ID NO: 579. In some embodiments, the crRNA sequence is at least 95% identical to SEQ ID NO: 579. In some embodiments, the crRNA sequence is SEQ ID NO: 579.

[0428] In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2 of the IL-1α gene. In some embodiments, the crRNA sequence forms no more than 5 mismatches with the target sequence in exon 2 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms no more than 4 mismatches with the target sequence in exon 2 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms no more than 3 mismatches with the target sequence in exon 2 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms no more than 2 mismatches with the target sequence in exon 2 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms no more than 1 mismatch with the target sequence in exon 2 of the canine IL-1α gene. In some embodiments, the crRNA sequence does not form a mismatch with the target sequence in exon 2 of the canine IL-1α gene.

[0429] In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 3 of the IL-1α gene. In some embodiments, the crRNA sequence forms no more than 5 mismatches with the target sequence in exon 3 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms no more than 4 mismatches with the target sequence in exon 3 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms no more than 3 mismatches with the target sequence in exon 3 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms no more than 2 mismatches with the target sequence in exon 3 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms no more than 1 mismatch with the target sequence in exon 3 of the canine IL-1α gene. In some embodiments, the crRNA sequence does not form a mismatch with the target sequence in exon 3 of the canine IL-1α gene.

[0430] In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 4 of the IL-1α gene. In some embodiments, the crRNA sequence forms no more than 5 mismatches with the target sequence in exon 4 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms no more than 4 mismatches with the target sequence in exon 4 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms no more than 3 mismatches with the target sequence in exon 4 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms no more than 2 mismatches with the target sequence in exon 4 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms no more than 1 mismatch with the target sequence in exon 4 of the canine IL-1α gene. In some embodiments, the crRNA sequence does not form a mismatch with the target sequence in exon 4 of the canine IL-1α gene.

[0431] In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 5 of the IL-1α gene. In some embodiments, the crRNA sequence forms no more than 5 mismatches with the target sequence in exon 5 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms no more than 4 mismatches with the target sequence in exon 5 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms no more than 3 mismatches with the target sequence in exon 5 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms no more than 2 mismatches with the target sequence in exon 5 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms no more than 1 mismatch with the target sequence in exon 5 of the canine IL-1α gene. In some embodiments, the crRNA sequence does not form a mismatch with the target sequence in exon 5 of the canine IL-1α gene.

[0432] In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 6 of the IL-1α gene. In some embodiments, the crRNA sequence forms no more than 5 mismatches with the target sequence in exon 6 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms no more than 4 mismatches with the target sequence in exon 6 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms no more than 3 mismatches with the target sequence in exon 6 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms no more than 2 mismatches with the target sequence in exon 6 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms no more than 1 mismatch with the target sequence in exon 6 of the canine IL-1α gene. In some embodiments, the crRNA sequence does not form a mismatch with the target sequence in exon 6 of the canine IL-1α gene.

[0433] In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 7 of the IL-1α gene. In some embodiments, the crRNA sequence forms no more than 5 mismatches with the target sequence in exon 7 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms no more than 4 mismatches with the target sequence in exon 7 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms no more than 3 mismatches with the target sequence in exon 7 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms no more than 2 mismatches with the target sequence in exon 7 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms no more than 1 mismatch with the target sequence in exon 7 of the canine IL-1α gene. In some embodiments, the crRNA sequence does not form a mismatch with the target sequence in exon 7 of the canine IL-1α gene.

[0434] In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2 or exon 3 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2 or exon 4 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2 or exon 5 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2 or exon 6 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2 or exon 7 of the IL-1α gene.

[0435] In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 3 or exon 4 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 3 or exon 5 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 3 or exon 6 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 3 or exon 7 of the IL-1α gene.

[0436] In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 4 or exon 5 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 4 or exon 6 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 4 or exon 7 of the IL-1α gene.

[0437] In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 5 or exon 6 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 5 or exon 7 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 6 or exon 7 of the IL-1α gene.

[0438] In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 3, or exon 4 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 3, or exon 5 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 3, or exon 6 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 3, or exon 7 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 4, or exon 5 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 4, or exon 6 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 4, or exon 7 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 5, or exon 6 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 5, or exon 7 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 6, or exon 7 of the IL-1α gene.

[0439] In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 3, exon 4, or exon 5 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 3, exon 4, or exon 6 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 3, exon 4, or exon 7 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 3, exon 5, or exon 6 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 3, exon 5, or exon 7 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence that is complementary to a target sequence in exon 3, exon 6, or exon 7 of the IL-1α gene.

[0440] In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 4, exon 5, or exon 6 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 4, exon 5, or exon 7 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 4, exon 6, or exon 7 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 5, exon 6, or exon 7 of the IL-1α gene.

[0441] In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 3, exon 4, or exon 5 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 3, exon 4, or exon 6 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 3, exon 4, or exon 7 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 4, exon 5, or exon 6 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 4, exon 5, or exon 7 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 4, exon 6, or exon 7 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 5, exon 6, or exon 7 of the IL-1α gene.

[0442] In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 3, exon 4, exon 5, or exon 6 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 3, exon 4, exon 5, or exon 7 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 3, exon 4, exon 6, or exon 7 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 3, exon 5, exon 6, or exon 7 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence that is complementary to a target sequence in exon 4, exon 5, exon 6, or exon 7 of the IL-1α gene.

[0443] In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 3, exon 4, exon 5, or exon 6 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 3, exon 4, exon 5, or exon 7 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 3, exon 4, exon 5, exon 6, or exon 7 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 4, exon 5, exon 6, or exon 7 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 3, exon 5, exon 6, or exon 6 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 3, exon 4, exon 6, or exon 7 of the IL-1α gene. In some embodiments, the at least one guide RNA targets a canine IL-1α gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 3, exon 4, exon 5, exon 6, or exon 7 of the IL-1α gene.

[0444] In some embodiments, the at least one guide RNA targets a canine IL-1β gene and comprises a crRNA sequence having at least 75% identity to a sequence selected from the group consisting of SEQ ID NOs: 217-235, 497-551, and 771-800. In some embodiments, the crRNA sequence has at least 80% identity to a sequence selected from the group consisting of SEQ ID NOs: 217-235, 497-551, and 771-800. In some embodiments, the crRNA sequence has at least 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 217-235, 497-551, and 771-800. In some embodiments, the crRNA sequence has at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 217-235, 497-551, and 771-800. In some embodiments, the crRNA sequence is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 217-235, 497-551, and 771-800. In some embodiments, the crRNA sequence is selected from the group consisting of SEQ ID NOs: 217-235, 497-551, and 771-800.

[0445] In some embodiments, the at least one guide RNA targets a canine IL-1β gene and comprises a crRNA sequence that is at least 75% identical to SEQ ID NO: 498. In some embodiments, the crRNA sequence is at least 80% identical to SEQ ID NO: 498. In some embodiments, the crRNA sequence is at least 85% identical to SEQ ID NO: 498. In some embodiments, the crRNA sequence is at least 90% identical to SEQ ID NO: 498. In some embodiments, the crRNA sequence is at least 95% identical to SEQ ID NO: 498. In some embodiments, the crRNA sequence is SEQ ID NO: 498.

[0446] In some embodiments, the at least one guide RNA targets a canine IL-1β gene and comprises a crRNA sequence that is at least 75% identical to SEQ ID NO: 506. In some embodiments, the crRNA sequence is at least 80% identical to SEQ ID NO: 506. In some embodiments, the crRNA sequence is at least 85% identical to SEQ ID NO: 506. In some embodiments, the crRNA sequence is at least 90% identical to SEQ ID NO: 506. In some embodiments, the crRNA sequence is at least 95% identical to SEQ ID NO: 506. In some embodiments, the crRNA sequence is SEQ ID NO: 506.

[0447] In some embodiments, the at least one guide RNA targets a canine IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 2 of the IL-1β gene. In some embodiments, the crRNA sequence forms no more than 5 mismatches with the target sequence in exon 2 of the canine IL-1β gene. In some embodiments, the crRNA sequence forms no more than 4 mismatches with the target sequence in exon 2 of the canine IL-1β gene. In some embodiments, the crRNA sequence forms no more than 3 mismatches with the target sequence in exon 2 of the canine IL-1β gene. In some embodiments, the crRNA sequence forms no more than 2 mismatches with the target sequence in exon 2 of the canine IL-1β gene. In some embodiments, the crRNA sequence forms no more than 1 mismatch with the target sequence in exon 2 of the canine IL-1β gene. In some embodiments, the crRNA sequence does not form a mismatch with the target sequence in exon 2 of the canine IL-1β gene.

[0448] In some embodiments, the at least one guide RNA targets a canine IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 3 of the IL-1β gene. In some embodiments, the crRNA sequence forms no more than 5 mismatches with the target sequence in exon 3 of the canine IL-1β gene. In some embodiments, the crRNA sequence forms no more than 4 mismatches with the target sequence in exon 3 of the canine IL-1β gene. In some embodiments, the crRNA sequence forms no more than 3 mismatches with the target sequence in exon 3 of the canine IL-1β gene. In some embodiments, the crRNA sequence forms no more than 2 mismatches with the target sequence in exon 3 of the canine IL-1β gene. In some embodiments, the crRNA sequence forms no more than 1 mismatch with the target sequence in exon 3 of the canine IL-1β gene. In some embodiments, the crRNA sequence does not form a mismatch with the target sequence in exon 3 of the canine IL-1β gene.

[0449] In some embodiments, the at least one guide RNA targets a canine IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 4 of the IL-1β gene. In some embodiments, the crRNA sequence forms no more than 5 mismatches with the target sequence in exon 4 of the canine IL-1β gene. In some embodiments, the crRNA sequence forms no more than 4 mismatches with the target sequence in exon 4 of the canine IL-1β gene. In some embodiments, the crRNA sequence forms no more than 3 mismatches with the target sequence in exon 4 of the canine IL-1β gene. In some embodiments, the crRNA sequence forms no more than 2 mismatches with the target sequence in exon 4 of the canine IL-1β gene. In some embodiments, the crRNA sequence forms no more than 1 mismatch with the target sequence in exon 4 of the canine IL-1β gene. In some embodiments, the crRNA sequence does not form a mismatch with the target sequence in exon 4 of the canine IL-1β gene.

[0450] In some embodiments, the at least one guide RNA targets a canine IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 5 of the IL-1β gene. In some embodiments, the crRNA sequence forms no more than 5 mismatches with the target sequence in exon 5 of the canine IL-1β gene. In some embodiments, the crRNA sequence forms no more than 4 mismatches with the target sequence in exon 5 of the canine IL-1β gene. In some embodiments, the crRNA sequence forms no more than 3 mismatches with the target sequence in exon 5 of the canine IL-1β gene. In some embodiments, the crRNA sequence forms no more than 2 mismatches with the target sequence in exon 5 of the canine IL-1β gene. In some embodiments, the crRNA sequence forms no more than 1 mismatch with the target sequence in exon 5 of the canine IL-1β gene. In some embodiments, the crRNA sequence does not form a mismatch with the target sequence in exon 5 of the canine IL-1β gene.

[0451] In some embodiments, the at least one guide RNA targets a canine IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 6 of the IL-1β gene. In some embodiments, the crRNA sequence forms no more than 5 mismatches with the target sequence in exon 6 of the canine IL-1β gene. In some embodiments, the crRNA sequence forms no more than 4 mismatches with the target sequence in exon 6 of the canine IL-1β gene. In some embodiments, the crRNA sequence forms no more than 3 mismatches with the target sequence in exon 6 of the canine IL-1β gene. In some embodiments, the crRNA sequence forms no more than 2 mismatches with the target sequence in exon 6 of the canine IL-1β gene. In some embodiments, the crRNA sequence forms no more than 1 mismatch with the target sequence in exon 6 of the canine IL-1β gene. In some embodiments, the crRNA sequence does not form a mismatch with the target sequence in exon 6 of the canine IL-1β gene.

[0452] In some embodiments, the at least one guide RNA targets a canine IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 7 of the IL-1β gene. In some embodiments, the crRNA sequence forms no more than 5 mismatches with the target sequence in exon 7 of the canine IL-1β gene. In some embodiments, the crRNA sequence forms no more than 4 mismatches with the target sequence in exon 7 of the canine IL-1β gene. In some embodiments, the crRNA sequence forms no more than 3 mismatches with the target sequence in exon 7 of the canine IL-1β gene. In some embodiments, the crRNA sequence forms no more than 2 mismatches with the target sequence in exon 7 of the canine IL-1β gene. In some embodiments, the crRNA sequence forms no more than 1 mismatch with the target sequence in exon 7 of the canine IL-1β gene. In some embodiments, the crRNA sequence does not form a mismatch with the target sequence in exon 7 of the canine IL-1β gene.

[0453] In some embodiments, the at least one guide RNA targets a canine IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 2 or exon 3 of the IL-1β gene. In some embodiments, the at least one guide RNA targets a canine IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 2 or exon 4 of the IL-1β gene. In some embodiments, the at least one guide RNA targets a canine IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 2 or exon 5 of the IL-1β gene. In some embodiments, the at least one guide RNA targets a canine IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 2 or exon 6 of the IL-1β gene. In some embodiments, the at least one guide RNA targets a canine IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 2 or exon 7 of the IL-1β gene.

[0454] In some embodiments, the at least one guide RNA targets a canine IL-1 β gene and comprises a crRNA sequence complementary to a target sequence in exon 3 or exon 4 of the IL-1 β gene. In some embodiments, the at least one guide RNA targets a canine IL-1 β gene and comprises a crRNA sequence complementary to a target sequence in exon 3 or exon 5 of the IL-1 β gene. In some embodiments, the at least one guide RNA targets a canine IL-1 β gene and comprises a crRNA sequence complementary to a target sequence in exon 3 or exon 6 of the IL-1 β gene. In some embodiments, the at least one guide RNA targets a canine IL-1 β gene and comprises a crRNA sequence complementary to a target sequence in exon 3 or exon 7 of the IL-1 β gene.

[0455] In some embodiments, the at least one guide RNA targets a canine IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 4 or exon 5 of the IL-1β gene. In some embodiments, the at least one guide RNA targets a canine IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 4 or exon 6 of the IL-1β gene. In some embodiments, the at least one guide RNA targets a canine IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 4 or exon 7 of the IL-1β gene.

[0456] In some embodiments, the at least one guide RNA targets a canine IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 5 or exon 6 of the IL-1β gene. In some embodiments, the at least one guide RNA targets a canine IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 5 or exon 7 of the IL-1β gene. In some embodiments, the at least one guide RNA targets a canine IL-1β gene and comprises a crRNA sequence complementary to a target sequence in exon 6 or exon 7 of the IL-1β gene.

[0457] In some embodiments, the at least one guide RNA targets a canine IL-1 β gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 3, or exon 4 of the IL-1 β gene. In some embodiments, the at least one guide RNA targets a canine IL-1 β gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 3, or exon 5 of the IL-1 β gene. In some embodiments, the at least one guide RNA targets a canine IL-1 β gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 3, or exon 6 of the IL-1 β gene. In some embodiments, the at least one guide RNA targets a canine IL-1 β gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 3, or exon 7 of the IL-1 β gene. In some embodiments, the at least one guide RNA targets a canine IL-1 β gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 4, or exon 5 of the IL-1 β gene. In some embodiments, the at least one guide RNA targets a canine IL-1 β gene and comprises a crRNA sequence complementary to a target sequence in exon 2, exon 4, or exon 6 of the IL-1 β gene. In some embodiments, the at least one guide RNA targets a canine IL-1 β gene and comprises a crRNA sequence complementary to a target sequen...

Claims

1. A composition for treating or preventing a joint disease or condition, comprising: (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease; and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA, wherein the at least one guide RNA targets the IL-1α or IL-1β gene, wherein the guide RNA specifically binds to a target sequence adjacent to a protospacer adjacent motif (PAM) sequence of a Cas9 protein.

2. The composition according to claim 1, wherein the at least one guide RNA targets the human IL-1α gene. 3 . The composition of claim 2 , wherein the at least one guide targets exon 2, exon 3, exon 4, exon 5 or exon 6 of the human IL-1α gene.

4. The composition of claim 2, wherein the at least one guide RNA comprises a crRNA sequence having at least 85% identity, at least 90% identity, at least 95% identity, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 168-187, 298-387, and 681-710. The composition according to claim 1 , wherein the at least one guide RNA targets the human IL-1β gene. 6 . The composition of claim 5 , wherein the at least one guide targets exon 2, exon 3, exon 4, exon 5 or exon 6 of the human IL-1β gene.

7. The composition of claim 5, wherein the at least one guide RNA comprises a crRNA sequence having at least 85% identity, at least 90% identity, at least 95% identity, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 188-201, 388-496, and 711-740.

8. The composition of claim 1, wherein the at least one guide RNA targets a canine IL-1α gene.

9. The composition of claim 8, wherein the at least one guide targets exon 2, exon 3, exon 4, exon 5, or exon 6 of the canine IL-1α gene.

10. The composition of claim 8, wherein the at least one guide RNA comprises a crRNA sequence having at least 85% identity, at least 90% identity, at least 95% identity, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 202-216, 552-590, and 741-770.

11. The composition of claim 1, wherein the at least one guide RNA targets a canine IL-1β gene.

12. The composition of claim 11, wherein the at least one guide targets exon 2, exon 3, exon 4, exon 5, or exon 6 of the canine IL-1β gene.

13. The composition of claim 11, wherein the at least one guide RNA comprises a crRNA sequence having at least 85% identity, at least 90% identity, at least 95% identity, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 217-235, 497-551, and 771-800.

14. The composition of claim 1, wherein the at least one guide RNA targets an equine IL-1 alpha gene.

15. The composition of claim 14, wherein the at least one guide targets exon 2, exon 3, exon 4, exon 5, or exon 6 of the equine IL-1α gene.

16. The pharmaceutical composition of claim 14, wherein the at least one guide RNA comprises a crRNA sequence having at least 85% identity, at least 90% identity, at least 95% identity, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 236-255 and 801-830.

17. The composition of claim 1, wherein the at least one guide RNA targets an equine IL-1β gene.

18. The composition of claim 17, wherein the at least one guide targets exon 2, exon 3, exon 4, exon 5, or exon 6 of the equine IL-1β gene.

19. The pharmaceutical composition of claim 17, wherein the at least one guide RNA comprises a crRNA sequence having at least 85% identity, at least 90% identity, at least 95% identity, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 256-262 and 831-860.

20. The composition of claim 1, wherein the at least one guide RNA targets a feline IL-1 alpha gene.

21. The composition of claim 20, wherein the at least one guide targets exon 2, exon 3, exon 4, exon 5, or exon 6 of the feline IL-1α gene.

22. The pharmaceutical composition of claim 20, wherein the at least one guide RNA comprises a crRNA sequence that is at least 85% identical, at least 90% identical, at least 95% identical, or 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 861-890.

23. The composition of claim 1, wherein the at least one guide RNA targets a feline IL-1β gene.

24. The composition of claim 23, wherein the at least one guide targets exon 2, exon 3, exon 4, exon 5, or exon 6 of the feline IL-1 β gene.

25. The composition of claim 23, wherein the at least one guide RNA comprises a crRNA sequence that is at least 85% identical, at least 90% identical, at least 95% identical, or 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 891-920.

26. The composition of any one of claims 1 to 25, wherein the RNA-guided nuclease is a CRISPR-associated protein 9 (Cas9) protein.

27. The composition of claim 26, wherein the Cas9 protein is a Streptococcus pyogenes Cas9 protein.

28. The composition of claim 26, wherein the Cas9 protein is a Staphylococcus aureus Cas9 protein.

29. The composition of any one of claims 26 to 28, wherein the Cas9 protein is an enhanced specificity Cas9 variant protein.

30. The composition of any one of claims 1 to 29, wherein the RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease is the RNA-guided nuclease.

31. The composition of any one of claims 1 to 29, wherein the RNA-guided nuclease or nucleic acid encoding the RNA-guided nuclease is a DNA encoding the RNA-guided nuclease.

32. The composition of any one of claims 1 to 29, wherein the RNA-guided nuclease or the nucleic acid encoding the RNA-guided nuclease is an mRNA encoding the RNA-guided nuclease.

33. The composition of any one of claims 1 to 32, wherein the at least one guide RNA or a nucleic acid encoding at least one guide RNA is the at least one guide RNA.

34. The composition of any one of claims 1 to 32, wherein the at least one guide RNA or nucleic acid encoding at least one guide RNA is a DNA encoding the at least one guide RNA.

35. The composition of any one of claims 1 to 34, comprising a nucleic acid encoding both the RNA-guided nuclease and the at least one guide RNA.

36. The composition of any one of claims 1 to 35, wherein the at least one guide RNA is a single guide RNA (sgRNA).

37. The composition of any one of claims 1 to 36, wherein the composition comprises one or more lipid nanoparticles (LNPs) co-encapsulating: (i) the RNA-guided nuclease or the nucleic acid encoding the RNA-guided nuclease; and (ii) the at least one guide RNA or the nucleic acid encoding the at least one guide RNA.

38. The composition of claim 37, wherein the one or more LNPs comprise: a first plurality of LNPs encapsulating the RNA-guided nuclease or a nucleic acid encoding the RNA-guided nuclease; and A second plurality of LNPs, wherein the second plurality of LNPs encapsulates the at least one guide RNA or a nucleic acid encoding the at least one guide RNA.

39. The composition of claim 37 or 38, wherein the one or more LNPs comprise a component selected from the group consisting of: 3-(didodecylamino)-N1,N1,4-triadecyl-1-piperazineethylamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-triadecyl-1,4-piperazinediethylamine (KL22), 14,25-tricosyl-15,18,21,24-tetraaza-triacontane (KL2 5), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptathriacontane-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA A), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3.β.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA), (2R)-2-({8-[(3.β.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl- 3-[(9Z-,12Z)-octadec-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA(2R)), (2S)-2-({8-[(3.β.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z-,12Z)-octadec-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA(2S)), lipids including cyclic amine groups, and mixtures thereof.

40. The composition of any one of claims 37 to 39, wherein the LNP comprises a component selected from the group consisting of 1,2-dilinoleoyl-sn-glyceryl-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glyceryl-phosphocholine (DMPC), 1,2-dioleoyl-sn-glyceryl-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glyceryl-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glyceryl-3-phosphocholine (DSPC), 1,2-heneicosanoyl-sn-glyceryl-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glyceryl-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glyceryl-3-phosphocholine (18:0Diether PC), 1-oleoyl-2-cholesterol hemisuccinyl-sn-glyceryl-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glyceryl-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glyceryl-3-phosphocholine, 1,2-diamidonoyl-sn-glyceryl-3-phosphocholine, 1,2-docosahexaenoyl-sn-glyceryl-3-phosphocholine, 1,2-dioleoyl-sn-glyceryl-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glyceryl-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-dialinolenoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-docosahexaenoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-dioleoyl-sn-glyceryl-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin (SM) and mixtures thereof.

41. A composition according to any one of claims 37 to 40, wherein the LNP comprises a component selected from the group consisting of: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol and mixtures thereof, for example, the PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DMA, PEG-DSPE lipid and mixtures thereof.

42. The composition of any one of claims 37 to 41, wherein the LNP comprises a component selected from the group consisting of cholesterol, fecosterol, stigmasterol, stigmasterol, sitosterol, β-sitosterol, lupeol, betulin, ursolic acid, oleanolic acid, campesterol, fuccasterol, brassicasterol, ergosterol, 9,11-dehydroergosterol, tomatine, tomatine, alpha-tocopherol, and mixtures thereof.

43. A composition according to any one of claims 1 to 36, wherein the composition comprises one or more liposomes that co-encapsulate: (i) the RNA-guided nuclease or the nucleic acid encoding the RNA-guided nuclease; and (ii) the at least one guide RNA or the nucleic acid encoding the at least one guide RNA.

44. The composition of claim 43, wherein the one or more liposomes comprise: a first plurality of liposomes, said first plurality of liposomes encapsulating said RNA-guided nuclease or a nucleic acid encoding said RNA-guided nuclease; and A second plurality of liposomes, wherein the second plurality of liposomes encapsulates the at least one guide RNA or a nucleic acid encoding the at least one guide RNA.

45. The composition of any one of claims 1 to 36, wherein the composition comprises one or more recombinant viruses co-encapsulating: (i) the RNA-guided nuclease or the nucleic acid encoding the RNA-guided nuclease; and (ii) the at least one guide RNA or the nucleic acid encoding the at least one guide RNA.

46. ​​The composition of claim 45, wherein the one or more recombinant viruses comprise: a first plurality of recombinant viruses, said first plurality of recombinant viruses encapsulating said RNA-guided nuclease or a nucleic acid encoding said RNA-guided nuclease; and A second plurality of recombinant viruses, wherein the second plurality of recombinant viruses encapsulate the at least one guide RNA or a nucleic acid encoding the at least one guide RNA.

47. The composition of claim 45 or 46, wherein the one or more recombinant viruses are selected from the group consisting of retroviruses, adenoviruses, adeno-associated viruses, lentiviruses, and herpes simplex virus-1.

48. The composition of claim 45 or 46, wherein the one or more recombinant viruses comprise a recombinant adeno-associated virus (AAV).

49. The composition of claim 48, wherein the recombinant AAV is of serotype 5 (AAV5).

50. The composition of claim 48, wherein the recombinant AAV is of serotype 6 (AAV6).

51. The composition of any one of claims 1 to 36, wherein the RNA-guided nuclease or the nucleic acid encoding the RNA-guided nuclease and the at least one guide RNA or the nucleic acid encoding the at least one guide RNA are not encapsulated.

52. A composition according to any one of claims 1 to 51, wherein the composition is formulated for parenteral administration.

53. A composition according to any one of claims 1 to 51, wherein the composition is formulated for intra-articular injection in a joint of a subject.

54. A method for treating a joint disease or condition, the method include: The composition of any one of claims 1 to 53 is administered to a subject in need thereof.

55. The method of claim 54, wherein the joint disease or condition is arthritis.

56. The method of claim 55, wherein the arthritis is osteoarthritis.

57. The method of claim 54, wherein the joint disease or disorder is post-traumatic arthritis, gout, or pseudogout.

58. The method of any one of claims 54 to 57, wherein the subject is a human.

59. The method of any one of claims 54 to 57, wherein the subject is a canine, an equine, or a feline.

60. The method of claim 59, wherein the joint disease or disorder is canine, equine, or feline lameness.

61. The method of any one of claims 54 to 60, wherein the administering comprises intra-articular injection of the composition into the joint of the subject.

62. The method of any one of claims 54 to 61, wherein the composition is administered during surgery.

63. The method of any one of claims 54 to 62, wherein the composition is administered post-surgery.

64. The method of any one of claims 54 to 63, wherein the composition is a controlled release pharmaceutical composition.

65. A composition according to any one of claims 1 to 53 for use in treating a joint disease or disorder.

66. The composition of claim 65, wherein the joint disease or disorder is arthritis.

67. The composition of claim 66, wherein the arthritis is osteoarthritis.

68. The composition of claim 65, wherein the joint disease or disorder is post-traumatic arthritis, gout, or pseudogout.

69. The composition of claim 65, wherein the joint disease or disorder is canine, equine or feline lameness.

70. Use of a composition according to any one of claims 1 to 53 in the manufacture of a medicament for treating a joint disease or disorder.

71. The use according to claim 70, wherein the joint disease or disorder is arthritis.

72. The use according to claim 71, wherein the arthritis is osteoarthritis.

73. The use according to claim 70, wherein the joint disease or disorder is post-traumatic arthritis, gout or pseudogout.

74. The use of claim 70, wherein the joint disease or disorder is canine, equine or feline lameness.

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