Gene editing to improve joint function

Abstract

The present disclosure provides compositions and methods for treating joint disorders characterized by an inflammatory component. In some aspects, the compositions and methods are for preventing the progression of osteoarthritis and other arthritis and for treating osteoarthritis and other arthritis in mammalian joints.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 222,972, filed July 17, 2021, U.S. Provisional Patent Application No. 63 / 300,822, filed January 19, 2022, U.S. Provisional Patent Application No. 63 / 326,571, filed April 1, 2022, and International Application No. 2022 / 016121, filed July 16, 2021, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] Described herein are compositions and methods for treating synovial joint dysfunction. Additionally, disclosed herein are methods for gene editing of synovial cells and / or synovial cells, chondrocytes, synovial macrophages, and synovial fibroblasts, and uses of gene-edited synovial cells and / or synovial cells, chondrocytes, synovial macrophages, and synovial fibroblasts in the treatment of diseases such as osteoarthritis. [Background technology]

[0003] Treatment of osteoarthritis, degenerative joint disease, and other joint dysfunction is complex, and few long-term options exist for symptomatic relief or restoration of joint function. Osteoarthritis (OA) is a leading cause of pain-related disability. Neogi, Osteoarthritis Cartilage 2013;21:1145-53. All mammalian species are affected: working animals, livestock, and their owners all suffer from OA-related discomfort, pain, and disability, depending on the extent of disease progression.

[0004] OA is a complex disease characterized by a progressive and disabling course. Systemic inflammation is associated with OA and OA disease progression. Inflammation is caused by increased levels of pro-inflammatory cytokines. New methods and compositions for treating this disease are urgently needed. Disclosed herein are compositions and methods useful for treating OA and other inflammatory joint disorders. Summary of the Invention

[0005] The present disclosure provides compositions and methods for treating joint disorders characterized by an inflammatory component. In some aspects, the compositions and methods are for preventing the progression of osteoarthritis and other arthritides and for treating osteoarthritis and other arthritides in mammalian joints. According to exemplary embodiments, at least a portion of joint synoviocytes and / or synoviocytes, chondrocytes, synovial macrophages, or synovial fibroblasts are gene-edited to reduce expression of pro-inflammatory cytokines. In some aspects, at least a portion of joint synoviocytes and / or synoviocytes, chondrocytes, synovial macrophages, or synovial fibroblasts are gene-edited to reduce expression of IL-1α, IL-1β, or both IL-1α and IL-1β.

[0006] In some embodiments, the gene editing silences or reduces expression of one or more cytokine and / or growth factor genes in at least a portion of cells comprising a mammalian joint. In some aspects, the cells are synoviocytes. In some aspects, the cells are synoviocytes. In some aspects, the cells are synovial fibroblasts. In some aspects, the cells are synoviocytes. In some aspects, the cells are chondrocytes. In some aspects, the cells are synovial macrophages.

[0007] In some embodiments, the one or more cytokine and / or growth factor genes are selected from the group comprising IL-1α and IL-1β.

[0008] In some embodiments, gene editing involves the use of programmable nucleases to mediate the creation of double- or single-strand breaks in one or more cytokine and / or growth factor genes.

[0009] In some embodiments, the gene editing comprises one or more methods selected from CRISPR, TALE, zinc finger, and combinations thereof.

[0010] In some embodiments, the gene editing comprises CRISPR technology.

[0011] In some embodiments, the CRISPR method is a CRISPR-Cas9 method.

[0012] In some embodiments, the gene editing comprises the TALE method.

[0013] In some embodiments, the gene editing comprises zinc finger technology.

[0014] In some embodiments, the gene editing silences or reduces expression of one or more cytokine and / or growth factor genes in at least a portion of cells comprising the joint. In some embodiments, the edited portion of cells are synovial cells. In one aspect, the edited portion of cells are synovial fibroblasts. In some embodiments, the edited portion of cells are synovial cells. In some embodiments, the edited portion of cells are chondrocytes. In some embodiments, the edited portion of cells are synovial macrophages.

[0015] 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 intra-articularly.

[0016] Some aspects of the present disclosure provide pharmaceutical compositions for treating or preventing a disease or condition of a joint, 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 loci selected from the group consisting of IL-1α, IL-1β, TNF-α, IL-6, IL-8, and IL-18.

[0017] In some embodiments, the gene editing system comprises a composition for the treatment or prevention of a disease or condition of a joint, 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 that targets an IL-1α or IL-1β gene, wherein the guide RNA specifically binds to a target sequence adjacent to the protospacer adjacent motif (PAM) sequence of a Cas9 protein.

[0018] In some embodiments, at least one guide RNA targets human IL-1α and comprises a crRNA sequence having at least 85%, 90%, 95%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 168-187, 298-387, and 681-710.

[0019] In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence having at least 85%, 90%, 95%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 188-201, 388-496, and 711-740.

[0020] In some embodiments, at least one guide RNA targets the canine IL-1α gene and comprises a crRNA sequence having at least 85%, 90%, 95%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 202-216, 552-590, and 741-770.

[0021] In some embodiments, at least one guide RNA targets the canine IL-1β gene and comprises a crRNA sequence having at least 85%, 90%, 95%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 217-235, 497-551, and 771-800.

[0022] 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.

[0023] In some embodiments, the LNPs comprise a first plurality of LNPs comprising a first nucleic acid encapsulating a nucleic acid encoding an RNA-guided nuclease, and a second plurality of LNPs comprising a second nucleic acid encapsulating a nucleic acid encoding at least one guide RNA.

[0024] 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 encapsulating a nucleic acid encoding at least one guide RNA.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] An embodiment provides a method of treating lameness in a dog, the method comprising administering a gene-editing composition that silences or reduces expression of IL-1α and IL-1β in a portion of synoviocytes, chondrocytes, synovial macrophages, or synovial fibroblasts in a lame joint.

[0030] 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.

[0031] In some embodiments, the gene editing composition is formulated for parenteral administration, hi some embodiments, the gene editing composition is formulated for intra-articular injection into a joint of a subject.

[0032] In some embodiments, the above methods further comprise one or more features recited in any of the methods and compositions described herein. [Brief explanation of the drawings]

[0033] Embodiments of the present disclosure will be further described with reference to the accompanying drawings. The drawings shown are not necessarily to scale, emphasis instead generally being placed upon illustrating the principles of embodiments of the present disclosure.

[0034] [Figure 1A] Figure 1 illustrates in vitro agarose gel electrophoresis analysis of 100 ng of mouse DNA (gBlocks, Integrated DNA Technologies) designed against the Mus musculus Il1a and Il1b genes, and 200 ng of phosphorothioate-modified single guide (sg) RNA targeted against the Il1a gene (#43-46) and the Il1b gene (#47-50), cleaved by 0.5 μg of SpyCas9 (TrueCut™ Cas9 Protein v2, ThermoFisher Scientific). [Figure 1B] Figure 1 illustrates agarose gel electrophoresis analysis of 100 ng of mouse DNA (gBlocks, Integrated DNA Technologies) designed against the Mus musculus Il1a and Il1b genes, and 200 ng of phosphorothioate-modified guide sgRNAs against the Il1a gene (#51-53) and the Il1b gene (#54-56), cleaved by 0.5 μg of SauCas9 (GeneSnipper™ Cas9, BioVision). [Figure 2A] 2A and 2B show in vivo cleavage of Il1a edited with 4x sgRNA (SpyCas9) in two separate pools (Pool 1 and 2) across two cell lines, NIH 3T3 ("N") and J774.2 ("J"). FIG. 2B shows in vivo cleavage of Il1b edited with 4x sgRNA (SpyCas9) in two separate pools (Pool 1 and 2) across two cell lines, NIH 3T3 ("N") and J774.2 ("J"). FIG. 2C shows in vivo cleavage of Il1b edited with 4x sgRNA (SpyCas9) in two separate pools (Pool 1 and 2) across two cell lines, NIH 3T3 ("N") and J774.2 ("J"). FIG. 2C shows in vivo cleavage of Il1b edited with 4x sgRNA (SpyCas9) in two separate pools (Pool 1 and 2) across two cell lines, NIH 3T3 ("N") and J774.2 ("J"). In vivo cleavage of Il1a edited with 3x sgRNA (SaCas9) in two separate pools (Pool 1 and 2) across 3T3 ("N") and J774.2 ("J"). Editing efficiency is determined using deconvolution of Sanger sequencing traces (ICE tool, Synthego) of each pool. [Figure 2B]2A and 2B show in vivo cleavage of Il1a edited with 4x sgRNA (SpyCas9) in two separate pools (Pool 1 and 2) across two cell lines, NIH 3T3 ("N") and J774.2 ("J"). FIG. 2B shows in vivo cleavage of Il1b edited with 4x sgRNA (SpyCas9) in two separate pools (Pool 1 and 2) across two cell lines, NIH 3T3 ("N") and J774.2 ("J"). FIG. 2C shows in vivo cleavage of Il1b edited with 4x sgRNA (SpyCas9) in two separate pools (Pool 1 and 2) across two cell lines, NIH 3T3 ("N") and J774.2 ("J"). FIG. 2C shows in vivo cleavage of Il1b edited with 4x sgRNA (SpyCas9) in two separate pools (Pool 1 and 2) across two cell lines, NIH 3T3 ("N") and J774.2 ("J"). In vivo cleavage of Il1a edited with 3x sgRNA (SaCas9) in two separate pools (Pool 1 and 2) across 3T3 ("N") and J774.2 ("J"). Editing efficiency is determined using deconvolution of Sanger sequencing traces (ICE tool, Synthego) of each pool. [Figure 2C] 2A and 2B show in vivo cleavage of Il1a edited with 4x sgRNA (SpyCas9) in two separate pools (Pool 1 and 2) across two cell lines, NIH 3T3 ("N") and J774.2 ("J"). FIG. 2B shows in vivo cleavage of Il1b edited with 4x sgRNA (SpyCas9) in two separate pools (Pool 1 and 2) across two cell lines, NIH 3T3 ("N") and J774.2 ("J"). FIG. 2C shows in vivo cleavage of Il1b edited with 4x sgRNA (SpyCas9) in two separate pools (Pool 1 and 2) across two cell lines, NIH 3T3 ("N") and J774.2 ("J"). FIG. 2C shows in vivo cleavage of Il1b edited with 4x sgRNA (SpyCas9) in two separate pools (Pool 1 and 2) across two cell lines, NIH 3T3 ("N") and J774.2 ("J"). In vivo cleavage of Il1a edited with 3x sgRNA (SaCas9) in two separate pools (Pool 1 and 2) across 3T3 ("N") and J774.2 ("J"). Editing efficiency is determined using deconvolution of Sanger sequencing traces (ICE tool, Synthego) of each pool. [Figure 2D]2A and 2B show in vivo cleavage of Il1a edited with 4x sgRNA (SpyCas9) in two separate pools (Pool 1 and 2) across two cell lines, NIH 3T3 ("N") and J774.2 ("J"). FIG. 2B shows in vivo cleavage of Il1b edited with 4x sgRNA (SpyCas9) in two separate pools (Pool 1 and 2) across two cell lines, NIH 3T3 ("N") and J774.2 ("J"). FIG. 2C shows in vivo cleavage of Il1b edited with 4x sgRNA (SpyCas9) in two separate pools (Pool 1 and 2) across two cell lines, NIH 3T3 ("N") and J774.2 ("J"). FIG. 2C shows in vivo cleavage of Il1b edited with 4x sgRNA (SpyCas9) in two separate pools (Pool 1 and 2) across two cell lines, NIH 3T3 ("N") and J774.2 ("J"). In vivo cleavage of Il1a edited with 3x sgRNA (SaCas9) in two separate pools (Pool 1 and 2) across 3T3 ("N") and J774.2 ("J"). Editing efficiency is determined using deconvolution of Sanger sequencing traces (ICE tool, Synthego) of each pool. [Figure 3] Figure 1 illustrates GFP expression measured using the IVIS system. Flux values ​​were based on a region of interest centered on the injected knee joint of the animal. Data are presented as the mean (SD) of four specimens per group. [Figure 4] 1 illustrates the design of the study described in Example 5 of the present disclosure. [Figure 5] 1 illustrates survival outcome measures obtained in the study described in Example 5 of the present disclosure. [Figure 6] 1 illustrates the weight change in mice treated with intra-articular (IA) injection 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 the study described in Example 5 of the present disclosure. [Figure 7A]1A and 1B collectively illustrate (A) the change in knee caliper measurements from baseline of mouse joints over time, and (B) the mean difference in ankle caliper measurements by AUC, in mice treated with intra-articular (IA) injection 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 the study described in Example 5 of the present disclosure. [Figure 7B] 1A and 1B collectively illustrate (A) the change in knee caliper measurements from baseline of mouse joints over time, and (B) the mean difference in ankle caliper measurements by AUC, in mice treated with intra-articular (IA) injection 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 the study described in Example 5 of the present disclosure. [Figure 8A] Collectively, (A) the change in von Frey measurements, and (B) the mean absolute threshold of von Frey measurements, obtained from mice treated with intra-articular (IA) injection 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 the study described in Example 5 of the present disclosure. [Figure 8B] Collectively, (A) the change in von Frey measurements, and (B) the mean absolute threshold of von Frey measurements, obtained from mice treated with intra-articular (IA) injection 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 the study described in Example 5 of the present disclosure. [Figure 9]Illustrated are the results of a qPCR assay for IL-1β expression in synovial fluid obtained from mice treated with intra-articular (IA) injection 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 the study described in Example 5 of the present disclosure. [Figure 10A] Figures 10A and 10B collectively illustrate immunohistochemistry for mouse IL-1β in synovial tissue from MSU-injected animals pretreated with PBS (A, B) and treated with CRISPR (C, D). Figures 10B and 10D show isotype controls for Figures 10A and 10C, respectively. [Figure 10B] Figures 10A and 10B collectively illustrate immunohistochemistry for mouse IL-1β in synovial tissue from MSU-injected animals pretreated with PBS (A, B) and treated with CRISPR (C, D). Figures 10B and 10D show isotype controls for Figures 10A and 10C, respectively. [Figure 10C] Figures 10A and 10B collectively illustrate immunohistochemistry for mouse IL-1β in synovial tissue from MSU-injected animals pretreated with PBS (A, B) and treated with CRISPR (C, D). Figures 10B and 10D show isotype controls for Figures 10A and 10C, respectively. [Figure 10D] Figures 10A and 10B collectively illustrate immunohistochemistry for mouse IL-1β in synovial tissue from MSU-injected animals pretreated with PBS (A, B) and treated with CRISPR (C, D). Figures 10B and 10D show isotype controls for Figures 10A and 10C, respectively. [Figure 11A] Collectively, an alignment between the mouse, human, equine, feline, and canine IL-1 alpha genes is illustrated. [Figure 11B] Collectively, an alignment between the mouse, human, equine, feline, and canine IL-1 alpha genes is illustrated. [Figure 11C]Collectively, an alignment between the mouse, human, equine, feline, and canine IL-1 alpha genes is illustrated. [Figure 12A] Collectively, an alignment between the mouse, human, equine, feline, and canine IL-1 beta genes is illustrated. [Figure 12B] Collectively, an alignment between the mouse, human, equine, feline, and canine IL-1 beta genes is illustrated. [Figure 12C] Collectively, an alignment between the mouse, human, equine, feline, and canine IL-1 beta genes is illustrated. [Figure 12D] Collectively, an alignment between the mouse, human, equine, feline, and canine IL-1 beta genes is illustrated. [Figure 13A] Collectively, examples of CRISPR / Cas9 crRNA sequences designed to edit the human IL-1 alpha gene are illustrated. [Figure 13B] Collectively, examples of CRISPR / Cas9 crRNA sequences designed to edit the human IL-1 alpha gene are illustrated. [Figure 13C] Collectively, examples of CRISPR / Cas9 crRNA sequences designed to edit the human IL-1 alpha gene are illustrated. [Figure 13D] Collectively, examples of CRISPR / Cas9 crRNA sequences designed to edit the human IL-1 alpha gene are illustrated. [Figure 14A] Collectively, exemplary CRISPR / Cas9 crRNA sequences designed to edit the human IL-1 beta gene are illustrated. [Figure 14B] Collectively, exemplary CRISPR / Cas9 crRNA sequences designed to edit the human IL-1 beta gene are illustrated. [Figure 14C] Collectively, exemplary CRISPR / Cas9 crRNA sequences designed to edit the human IL-1 beta gene are illustrated. [Figure 14D] Collectively, exemplary CRISPR / Cas9 crRNA sequences designed to edit the human IL-1 beta gene are illustrated. [Figure 14E] Collectively, exemplary CRISPR / Cas9 crRNA sequences designed to edit the human IL-1 beta gene are illustrated. [Figure 15A] 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 , [Figure 15B] 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 , [Figure 15C] 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 , [Figure 16A] 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 , [Figure 16B] 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 , [Figure 17A]Figures 7A and 7B collectively illustrate the results of cell-based and in silico gene editing analyses of crRNA sequences targeting the human IL-1 alpha gene (Figure 7A), human IL-1 beta gene (Figure 7B), canine IL-1 alpha gene (Figure 7C), and canine IL-1 beta gene (Figure 7D), as described in Example 8. ° CRISPR cleavage position within the amino acid (AA) translation frame. *Optimized score from Doench, Fusi et al. (2016). This score is optimized for 20-bp guides containing 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. ***This score is based on experiments in U2OS. A high-precision score (>0.4) suggests that DNA repair outcomes are uniform and concentrated in only a handful of unique genotypes. ****This score is based on experiments in U2OS. High (>80%) frameshift frequencies tend to place protein-coding genes out of frame. Typical genomic frameshift frequencies are above 66%, as 1-bp insertions and 1-2 bp deletions are particularly common repair outcomes. ^Composite score = (off-target score + precision score * 100 + frameshift) / 3. †The pipe symbol "|" indicates the CRISPR cut site. Curly brackets "{}" indicate an insertion. A hyphen "-" indicates a deletion. $Potential off-target site. Scoring according to Hsu et al. (2013). An off-target site has a score of 100. [Figure 17B]Figures 7A and 7B collectively illustrate the results of cell-based and in silico gene editing analyses of crRNA sequences targeting the human IL-1 alpha gene (Figure 7A), human IL-1 beta gene (Figure 7B), canine IL-1 alpha gene (Figure 7C), and canine IL-1 beta gene (Figure 7D), as described in Example 8. ° CRISPR cleavage position within the amino acid (AA) translation frame. *Optimized score from Doench, Fusi et al. (2016). This score is optimized for 20-bp guides containing 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. ***This score is based on experiments in U2OS. A high-precision score (>0.4) suggests that DNA repair outcomes are uniform and concentrated in only a handful of unique genotypes. ****This score is based on experiments in U2OS. High (>80%) frameshift frequencies tend to place protein-coding genes out of frame. Typical genomic frameshift frequencies are above 66%, as 1-bp insertions and 1-2 bp deletions are particularly common repair outcomes. ^Composite score = (off-target score + precision score * 100 + frameshift) / 3. †The pipe symbol "|" indicates the CRISPR cut site. Curly brackets "{}" indicate an insertion. A hyphen "-" indicates a deletion. $Potential off-target site. Scoring according to Hsu et al. (2013). An off-target site has a score of 100. [Figure 17C]Figures 7A and 7B collectively illustrate the results of cell-based and in silico gene editing analyses of crRNA sequences targeting the human IL-1 alpha gene (Figure 7A), human IL-1 beta gene (Figure 7B), canine IL-1 alpha gene (Figure 7C), and canine IL-1 beta gene (Figure 7D), as described in Example 8. ° CRISPR cleavage position within the amino acid (AA) translation frame. *Optimized score from Doench, Fusi et al. (2016). This score is optimized for 20-bp guides containing 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. ***This score is based on experiments in U2OS. A high-precision score (>0.4) suggests that DNA repair outcomes are uniform and concentrated in only a handful of unique genotypes. ****This score is based on experiments in U2OS. High (>80%) frameshift frequencies tend to place protein-coding genes out of frame. Typical genomic frameshift frequencies are above 66%, as 1-bp insertions and 1-2 bp deletions are particularly common repair outcomes. ^Composite score = (off-target score + precision score * 100 + frameshift) / 3. †The pipe symbol "|" indicates the CRISPR cut site. Curly brackets "{}" indicate an insertion. A hyphen "-" indicates a deletion. $Potential off-target site. Scoring according to Hsu et al. (2013). An off-target site has a score of 100. [Figure 17D]Figures 7A and 7B collectively illustrate the results of cell-based and in silico gene editing analyses of crRNA sequences targeting the human IL-1 alpha gene (Figure 7A), human IL-1 beta gene (Figure 7B), canine IL-1 alpha gene (Figure 7C), and canine IL-1 beta gene (Figure 7D), as described in Example 8. ° CRISPR cleavage position within the amino acid (AA) translation frame. *Optimized score from Doench, Fusi et al. (2016). This score is optimized for 20-bp guides containing 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. ***This score is based on experiments in U2OS. A high-precision score (>0.4) suggests that DNA repair outcomes are uniform and concentrated in only a handful of unique genotypes. ****This score is based on experiments in U2OS. High (>80%) frameshift frequencies tend to place protein-coding genes out of frame. Typical genomic frameshift frequencies are above 66%, as 1-bp insertions and 1-2 bp deletions are particularly common repair outcomes. ^Composite score = (off-target score + precision score * 100 + frameshift) / 3. †The pipe symbol "|" indicates the CRISPR cut site. Curly brackets "{}" indicate an insertion. A hyphen "-" indicates a deletion. $Potential off-target site. Scoring according to Hsu et al. (2013). An off-target site has a score of 100. [Figure 18A] Figures 18A and 18B collectively illustrate canine IL-1 alpha (Figures 18A and 18B) and canine IL-1 beta (Figures 18C and 18D) release from non-edited (control) and double IL-1 alpha / IL-1 beta KO (edited) canine chondrocytes 6 hours (Figures 18A and 18C) and 24 hours (Figures 18B and 18D) after exposure to PBS or LPS, as described in Example 9. [Figure 18B]Figures 18A and 18B collectively illustrate canine IL-1 alpha (Figures 18A and 18B) and canine IL-1 beta (Figures 18C and 18D) release from non-edited (control) and double IL-1 alpha / IL-1 beta KO (edited) canine chondrocytes 6 hours (Figures 18A and 18C) and 24 hours (Figures 18B and 18D) after exposure to PBS or LPS, as described in Example 9. [Figure 18C] Figures 18A and 18B collectively illustrate canine IL-1 alpha (Figures 18A and 18B) and canine IL-1 beta (Figures 18C and 18D) release from non-edited (control) and double IL-1 alpha / IL-1 beta KO (edited) canine chondrocytes 6 hours (Figures 18A and 18C) and 24 hours (Figures 18B and 18D) after exposure to PBS or LPS, as described in Example 9. [Figure 18D] Figures 18A and 18B collectively illustrate canine IL-1 alpha (Figures 18A and 18B) and canine IL-1 beta (Figures 18C and 18D) release from non-edited (control) and double IL-1 alpha / IL-1 beta KO (edited) canine chondrocytes 6 hours (Figures 18A and 18C) and 24 hours (Figures 18B and 18D) after exposure to PBS or LPS, as described in Example 9. [Figure 19A] Figures 19A and 19B collectively illustrate human IL-1 alpha (Figures 19A and 19B) and canine IL-1 beta (Figures 19C and 19D) release from non-edited (control) and double IL-1 alpha / IL-1 beta KO (edited) canine chondrocytes 6 hours (Figures 19A and 19C) and 24 hours (Figures 19B and 19D) after exposure to PBS or LPS, as described in Example 9. [Figure 19B] Figures 19A and 19B collectively illustrate human IL-1 alpha (Figures 19A and 19B) and canine IL-1 beta (Figures 19C and 19D) release from non-edited (control) and double IL-1 alpha / IL-1 beta KO (edited) canine chondrocytes 6 hours (Figures 19A and 19C) and 24 hours (Figures 19B and 19D) after exposure to PBS or LPS, as described in Example 9. [Figure 19C]Figures 19A and 19B collectively illustrate human IL-1 alpha (Figures 19A and 19B) and canine IL-1 beta (Figures 19C and 19D) release from non-edited (control) and double IL-1 alpha / IL-1 beta KO (edited) canine chondrocytes 6 hours (Figures 19A and 19C) and 24 hours (Figures 19B and 19D) after exposure to PBS or LPS, as described in Example 9. [Figure 19D] Figures 19A and 19B collectively illustrate human IL-1 alpha (Figures 19A and 19B) and canine IL-1 beta (Figures 19C and 19D) release from non-edited (control) and double IL-1 alpha / IL-1 beta KO (edited) canine chondrocytes 6 hours (Figures 19A and 19C) and 24 hours (Figures 19B and 19D) after exposure to PBS or LPS, as described in Example 9. [Figure 20] 1 illustrates the results of tissue-specific splicing and expression analysis of the human IL1A (IL-1a) gene. [Figure 21] 1 illustrates the results of tissue-specific splicing and expression analysis of the human IL1B (IL-1b) gene. [Figure 22] The results of in silico analysis of human IL-1a and IL-1b-targeting gRNA targeting domains are illustrated. The on-target score (see Doench et al.) is optimized for 20-bp gRNAs with an NGG protospacer adjacent motif (PAM). Scores range from 0 to 1. The precision score is based on experiments in U2OS cells. A high precision score (>0.4) suggests that DNA repair outcomes are uniform and concentrated in only a handful of unique genotypes. The frameshift percentage is based on experiments in U2OS cells. A high (>80%) frameshift frequency tends to shift protein-coding genes out of frame. Because 1-bp insertions and 1-2 bp deletions are particularly common repair outcomes, the typical genomic frameshift frequency is greater than 66%. The off-target score from CRISPOR assesses the number of matches within the genome with a given number of mismatches. Mismatches in the seed sequence have a more detrimental effect. [Figure 23A]The results of splicing and functional analyses of the canine IL1A (IL-1a) and IL1B (IL-1b) genes are collectively illustrated. The reference canine genome assembly (CanFam3.1) was used for these analyses. gRNA designs may be tailored to individual breeds as needed. [Figure 23B] The results of splicing and functional analyses of the canine IL1A (IL-1a) and IL1B (IL-1b) genes are collectively illustrated. The reference canine genome assembly (CanFam3.1) was used for these analyses. gRNA designs may be tailored to individual breeds as needed. [Figure 23C] The results of splicing and functional analyses of the canine IL1A (IL-1a) and IL1B (IL-1b) genes are collectively illustrated. The reference canine genome assembly (CanFam3.1) was used for these analyses. gRNA designs may be tailored to individual breeds as needed. [Figure 23D] The results of splicing and functional analyses of the canine IL1A (IL-1a) and IL1B (IL-1b) genes are collectively illustrated. The reference canine genome assembly (CanFam3.1) was used for these analyses. gRNA designs may be tailored to individual breeds as needed. [Figure 24] Illustrates the results of in silico analysis of canine IL-1a and IL-1b targeting gRNA targeting domains. [Figure 25A] The figures collectively illustrate the knockdown efficacy of selected gRNA targeting domains in human chondrocytes (Figure 25A), canine chondrocytes (Figure 25B), and synoviocytes (Figure 25C). Genomic DNA was collected from pooled cells 8-10 days after administration prior to sequencing analysis. [Figure 25B] The figures collectively illustrate the knockdown efficacy of selected gRNA targeting domains in human chondrocytes (Figure 25A), canine chondrocytes (Figure 25B), and synoviocytes (Figure 25C). Genomic DNA was collected from pooled cells 8-10 days after administration prior to sequencing analysis. [Figure 25C]The figures collectively illustrate the knockdown efficacy of selected gRNA targeting domains in human chondrocytes (Figure 25A), canine chondrocytes (Figure 25B), and synoviocytes (Figure 25C). Genomic DNA was collected from pooled cells 8-10 days after administration prior to sequencing analysis. [Figure 26] 1 illustrates the results of an in silico analysis of off-target effects of lead candidate gRNA targeting domains in canine cells. [Figure 27A] The figures collectively illustrate the effectiveness of enhanced specificity Cas9 (espCas9) in disrupting off-target editing of sgRNA 242 in canine cells. Figure 27A shows the effect of canonical spCas9, resulting in potent on-target editing but also high off-target activity. Figure 27B shows that espCas9 maintains high on-target efficiency without off-target effects. [Figure 27B] The figures collectively illustrate the effectiveness of enhanced specificity Cas9 (espCas9) in disrupting off-target editing of sgRNA 242 in canine cells. Figure 27A shows the effect of canonical spCas9, resulting in potent on-target editing but also high off-target activity. Figure 27B shows that espCas9 maintains high on-target efficiency without off-target effects. [Figure 28A] Collectively, the editing activity of sgRNAs targeting IL-1a and IL-1b in human (A) and canine (B) chondrocytes is illustrated. [Figure 28B] Collectively, the editing activity of sgRNAs targeting IL-1a and IL-1b in human (A) and canine (B) chondrocytes is illustrated. [Figure 29] 1 shows the results of simultaneous or sequential administration of lead candidate sgRNAs in canine cells. [Figure 30A] Illustrates sequence alignment of IL1A(A) and IL1B(B) genomic sequences from human, horse, mouse, and dog for potential cross-species targets using a single sgRNA. [Figure 30B]Illustrates sequence alignment of IL1A(A) and IL1B(B) genomic sequences from human, horse, mouse, and dog for potential cross-species targets using a single sgRNA. [Figure 31A] Figures 31A and 31B collectively illustrate CRISPR-mediated editing of canine IL-1A. Specifically, Figure 31A shows the average KO scores determined by inferring CRISPR editing from Sanger traces in canine chondrocytes (n=3+), fibroblast-like synoviocytes (n=3), and monocyte DH82 (n=2). Stacked bar graphs indicate the top and remaining KO genotypes for wild-type Cas9 and assembled sgRNAs, respectively. Figure 31B shows the amino acid sequences of wild-type and truncated canine IL1A before (top) and after (bottom) CRISPR-mediated genome editing. Wild-type IL1A contains the propeptide, which is cleaved to create the functional (mature) protein (highlighted). Figure 31C illustrates the predicted three-dimensional structures of wild-type and truncated IL1A as predicted by AlphaFold2. N marks the N-terminus, the canonical translation start site. [Figure 31B] Figures 31A and 31B collectively illustrate CRISPR-mediated editing of canine IL-1A. Specifically, Figure 31A shows the average KO scores determined by inferring CRISPR editing from Sanger traces in canine chondrocytes (n=3+), fibroblast-like synoviocytes (n=3), and monocyte DH82 (n=2). Stacked bar graphs indicate the top and remaining KO genotypes for wild-type Cas9 and assembled sgRNAs, respectively. Figure 31B shows the amino acid sequences of wild-type and truncated canine IL1A before (top) and after (bottom) CRISPR-mediated genome editing. Wild-type IL1A contains the propeptide, which is cleaved to create the functional (mature) protein (highlighted). Figure 31C illustrates the predicted three-dimensional structures of wild-type and truncated IL1A as predicted by AlphaFold2. N marks the N-terminus, the canonical translation start site. [Figure 31C]Figures 31A and 31B collectively illustrate CRISPR-mediated editing of canine IL-1A. Specifically, Figure 31A shows the average KO scores determined by inferring CRISPR editing from Sanger traces in canine chondrocytes (n=3+), fibroblast-like synoviocytes (n=3), and monocyte DH82 (n=2). Stacked bar graphs indicate the top and remaining KO genotypes for wild-type Cas9 and assembled sgRNAs, respectively. Figure 31B shows the amino acid sequences of wild-type and truncated canine IL1A before (top) and after (bottom) CRISPR-mediated genome editing. Wild-type IL1A contains the propeptide, which is cleaved to create the functional (mature) protein (highlighted). Figure 31C illustrates the predicted three-dimensional structures of wild-type and truncated IL1A as predicted by AlphaFold2. N marks the N-terminus, the canonical translation start site. [Figure 32A] Figure 32A collectively illustrates 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. Figure 32A shows the target sequence edited by the guide (the guide sequence is identical to the crRNA sequence in the guide, except that it contains uracil instead of thymidine). Figure 32B shows the predicted on-target, off-target, and frameshift effect scores for each of the gRNAs, as described in Example 16. Figure 32C shows the results of an in vitro editing assay analyzed by sequencing the edited targets. [Figure 32B]Figure 32A collectively illustrates 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. Figure 32A shows the target sequence edited by the guide (the guide sequence is identical to the crRNA sequence in the guide, except that it contains uracil instead of thymidine). Figure 32B shows the predicted on-target, off-target, and frameshift effect scores for each of the gRNAs, as described in Example 16. Figure 32C shows the results of an in vitro editing assay analyzed by sequencing the edited targets. [Figure 32C] Figure 32A collectively illustrates 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. Figure 32A shows the target sequence edited by the guide (the guide sequence is identical to the crRNA sequence in the guide, except that it contains uracil instead of thymidine). Figure 32B shows the predicted on-target, off-target, and frameshift effect scores for each of the gRNAs, as described in Example 16. Figure 32C shows the results of an in vitro editing assay analyzed by sequencing the edited targets. [Figure 33A]Figure 33A collectively illustrates 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). Figure 33A shows the target sequence edited by the guide (the guide sequence is identical to the crRNA sequence in the guide, except that it contains uracil instead of thymidine). Figure 32B shows the predicted on-target, off-target, and frameshift effect scores for each of the gRNAs, as described in Example 17. Figure 32C shows the results of an in vitro editing assay analyzed by sequencing the edited targets. Figure 33D shows the results of an anti-IL-1B ELISA assay on supernatants of canine monocytes targeted with editing constructs. [Figure 33B] Figure 33A collectively illustrates 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). Figure 33A shows the target sequence edited by the guide (the guide sequence is identical to the crRNA sequence in the guide, except that it contains uracil instead of thymidine). Figure 32B shows the predicted on-target, off-target, and frameshift effect scores for each of the gRNAs, as described in Example 17. Figure 32C shows the results of an in vitro editing assay analyzed by sequencing the edited targets. Figure 33D shows the results of an anti-IL-1B ELISA assay on supernatants of canine monocytes targeted with editing constructs. [Figure 33C]Figure 33A collectively illustrates 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). Figure 33A shows the target sequence edited by the guide (the guide sequence is identical to the crRNA sequence in the guide, except that it contains uracil instead of thymidine). Figure 32B shows the predicted on-target, off-target, and frameshift effect scores for each of the gRNAs, as described in Example 17. Figure 32C shows the results of an in vitro editing assay analyzed by sequencing the edited targets. Figure 33D shows the results of an anti-IL-1B ELISA assay on supernatants of canine monocytes targeted with editing constructs. [Figure 33D] Figure 33A collectively illustrates 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). Figure 33A shows the target sequence edited by the guide (the guide sequence is identical to the crRNA sequence in the guide, except that it contains uracil instead of thymidine). Figure 32B shows the predicted on-target, off-target, and frameshift effect scores for each of the gRNAs, as described in Example 17. Figure 32C shows the results of an in vitro editing assay analyzed by sequencing the edited targets. Figure 33D shows the results of an anti-IL-1B ELISA assay on supernatants of canine monocytes targeted with editing constructs. [Figure 34A]Collectively illustrated are the performance of IL1B-targeting sgRNAs #241 (OCB01) and #242 (OCB02) as measured by the mean IL1B KO scores inferred from Sanger sequencing CRISPR-mediated genome editing in chondrocytes, synoviocytes, and monocyte DH82 (Figure 34A), as well as ELISA results for IL1B in either control or two IL1A / B double KO monocytes generated from different combinations of sgRNAs (Figure 34B, left, Sanger sequence inferred KO scores for each combination). [Figure 34B] Collectively illustrated are the performance of IL1B-targeting sgRNAs #241 (OCB01) and #242 (OCB02) as measured by the mean IL1B KO scores inferred from Sanger sequencing CRISPR-mediated genome editing in chondrocytes, synoviocytes, and monocyte DH82 (Figure 34A), as well as ELISA results for IL1B in either control or two IL1A / B double KO monocytes generated from different combinations of sgRNAs (Figure 34B, left, Sanger sequence inferred KO scores for each combination). [Figure 35A] 1A-1C collectively illustrate the effect of electroporation on canine monocytes with either OCB02 / sg242 (KO1) or OCB01 / sg241 (KO2) as measured by supernatant ELISA either (A) 6 hours and (B) 24 hours after challenge with lipopolysaccharide (LPS). [Figure 35B] 1A-1C collectively illustrate the effect of electroporation on canine monocytes with either OCB02 / sg242 (KO1) or OCB01 / sg241 (KO2) as measured by supernatant ELISA either (A) 6 hours and (B) 24 hours after challenge with lipopolysaccharide (LPS). [Figure 36] Figure 1 illustrates a comparison of the editing efficiency of five potential gRNAs targeting canine IL-1beta in monocytes as measured by the Abcam IL-1beta ELISA kit. [Figure 37A]Collectively, the effects of different Cas9 variants with enhanced specificity are illustrated, including (A) a comparison of on-target editing efficiency with OCB02 gRNA plus either standard Cas9 or an enhanced specificity / fidelity Cas9 variant, and (B) a comparison of the on-target editing efficiency of OCB01 with the ARCas9 variant or wild-type Cas9. [Figure 37B] Collectively, the effects of different Cas9 variants with enhanced specificity are illustrated, including (A) a comparison of on-target editing efficiency with OCB02 gRNA plus either standard Cas9 or an enhanced specificity / fidelity Cas9 variant, and (B) a comparison of the on-target editing efficiency of OCB01 with the ARCas9 variant or wild-type Cas9. [Figure 38A] These figures collectively illustrate the in vitro performance of Cas9 mutants with enhanced specificity. Figure 38A illustrates on-target and off-target editing efficiencies, with percentages representing KO scores and editing efficiencies, respectively. Figure 38B shows the predicted off-target editing sites of OCB01. [Figure 38B] These figures collectively illustrate the in vitro performance of Cas9 mutants with enhanced specificity. Figure 38A illustrates on-target and off-target editing efficiencies, with percentages representing KO scores and editing efficiencies, respectively. Figure 38B shows the predicted off-target editing sites of OCB01. [Figure 39A] Collectively, exemplary CRISPR / Cas9 crRNA sequences designed to edit the human IL-1 alpha gene are illustrated. [Figure 39B] Collectively, exemplary CRISPR / Cas9 crRNA sequences designed to edit the human IL-1 alpha gene are illustrated. [Figure 40A] Collectively, exemplary CRISPR / Cas9 crRNA sequences designed to edit the human IL-1 beta gene are illustrated. [Figure 40B] Collectively, exemplary CRISPR / Cas9 crRNA sequences designed to edit the human IL-1 beta gene are illustrated. [Figure 41A] 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 , [Figure 41B] 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 , [Figure 42A] 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 , [Figure 42B] 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 , [Figure 43A] collectively illustrate exemplary CRISPR / Cas9 crRNA sequences designed to edit the equine IL-1 alpha gene. [Figure 43B] collectively illustrate exemplary CRISPR / Cas9 crRNA sequences designed to edit the equine IL-1 alpha gene. [Figure 44A] 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 , [Figure 44B] 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 , [Figure 45A] Collectively, exemplary CRISPR / Cas9 crRNA sequences designed to edit the feline IL-1 beta gene are illustrated. [Figure 45B] Collectively, exemplary CRISPR / Cas9 crRNA sequences designed to edit the feline IL-1 beta gene are illustrated. [Figure 46A] Collectively, exemplary CRISPR / Cas9 crRNA sequences designed to edit the feline IL-1 alpha gene are illustrated. [Figure 46B]Collectively, exemplary CRISPR / Cas9 crRNA sequences designed to edit the feline IL-1 alpha gene are illustrated.

[0035] While the above-identified drawings describe the presently disclosed embodiments, other embodiments are also intended to be described in the discussion. The present disclosure presents exemplary embodiments by way of representation, not limitation. Numerous other modifications and embodiments can be devised by those skilled in the art which fall within the scope and spirit of the principles of the disclosed embodiments. DETAILED DESCRIPTION OF THE INVENTION

[0036] As described herein, embodiments of the present disclosure provide compositions and methods for improving joint function and treating joint diseases. In certain embodiments, compositions and methods are provided for gene editing synovial fibroblasts, synoviocytes, chondrocytes, or synovial macrophages to reduce the expression of pro-inflammatory cytokines, e.g., IL-1α, IL-1β, TNF-α, IL-6, IL-8, IL-18, one or more matrix metalloproteinases (MMPs), or one or more components of the NLRP3 inflammasome. Embodiments are used to treat osteoarthritis and other inflammatory joint diseases. Embodiments are further useful for treating canine lameness caused by osteoarthritis. Embodiments are further useful for treating equine lameness caused by joint disease. Embodiments are also useful for treating post-traumatic arthritis, gout, pseudogout, and other inflammatory- or immune-mediated joint diseases.

[0037] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents and publications referenced herein are incorporated by reference in their entirety.

[0038] The term "in vivo" refers to events that take place inside a subject's body.

[0039] The term "in vitro" refers to events that take place outside a subject's body. In vitro assays include cell-based assays, in which live or dead cells are used, and can also include cell-free assays, in which intact cells are used.

[0040] The term "ex vivo" refers to events involving treating or performing treatment on cells, tissues, and / or organs that have been removed from a subject's body. Suitably, the cells, tissues, and / or organs may be returned to the subject's body in a surgical or therapeutic procedure.

[0041] 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 IL-1-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, called the type I IL-1 receptor (IL-1R1). A third ligand for this receptor exists: the interleukin-1 receptor antagonist (IL-1Ra), which acts as an inhibitor of IL-1α and IL-1β signaling by not activating downstream signaling and therefore competing with IL-1α and IL-1β signaling for the receptor's binding site. 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, for example, in Dinarello, Cytokine Growth Factor Rev. 8:253-65 (1997), the disclosure of which is incorporated herein by reference. For example, the term IL-1 includes human recombinant forms of IL-1. [Table 1]

[0042] The term "NLRP3 inflammasome" refers to a multiprotein complex involved in the activation of several 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, ASC (apoptosis-associated speck-like protein containing CARD), and caspase-1, as described by Lee et al., Lipids Health Dis. 16:271 (2017) and Groslambert and Py, J. Inflamm. Res. 11:359-374 (2018).

[0043] The terms "matrix metalloproteinase" and "MMP" are defined as any one of the members of the matrix metalloproteinase family of zinc endopeptidases, as characterized, for example, 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, matrilysins, and / or furin-activatable 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.

[0044] As used herein, the terms "co-administration," "co-administer," "administered in combination," "administering in combination," "simultaneously," and "combination" encompass administration of two or more active pharmaceutical ingredients to a subject such that both active pharmaceutical ingredients and / or their metabolites are present in the subject at the same time (in preferred embodiments of the present disclosure, for example, at least one anti-inflammatory compound is functionally engineered in combination with a viral vector to deliver a gene-edited nucleic acid described herein). 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. Co-administration in separate compositions and administration in a composition in which both agents are present are preferred.

[0045] The term "effective amount" or "therapeutically effective amount" refers to an amount of a composition or combination of compositions described herein sufficient to produce an effect for the intended use, including, but not limited to, disease treatment. A therapeutically effective amount may vary depending on the intended use (in vitro or in vivo), or the subject and disease state being treated (e.g., the subject's weight, age, or sex), the severity of the disease state, or the mode of administration. The term also applies to a dose that will elicit a particular response in target cells (e.g., reduced platelet adhesion and / or cell migration). A particular dose will vary depending on the particular composition selected, the administration regimen to be followed, whether the composition is administered in combination with other compositions or compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system through which the composition is transported.

[0046] The terms "treatment," "treating," "treat," and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic, in terms of partially or completely curing the disease and / or adverse effects resulting from the disease. For example, a composition, method, or system of the present disclosure may be administered as a prophylactic treatment to a subject predisposed to a given condition (e.g., arthritis). As used herein, "treatment" covers any treatment of a disease in a mammal, particularly a human, dog, cat, or horse, and includes (a) preventing the disease from occurring in a subject predisposed to the disease but not yet diagnosed with the disease, (b) inhibiting the disease, i.e., arresting the onset or progression of the disease, and (c) relieving the disease, i.e., causing regression of the disease and / or alleviating one or more disease symptoms. "Treatment" is also meant to encompass the delivery of an agent to provide a pharmacological effect even in the absence of a disease or condition. For example, "treatment" includes the delivery of a composition that can induce an immune response or confer immunity in the absence of a disease state, e.g., in the case of a vaccine. It is understood that the compositions and methods of the present disclosure are applicable to treating all mammals, including, but not limited to, human, canine, feline, equine, and bovine subjects.

[0047] The term "heterologous" when used with reference to portions of a nucleic acid or protein indicates that the nucleic acid or protein comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, nucleic acids are typically produced recombinantly, having two or more sequences from unrelated genes arranged to create a new functional nucleic acid, e.g., 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 comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0048] 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 antagonists, and inhibitory DNA or RNA (e.g., RNAi, e.g., small or short hairpin (sh)RNA, microRNA (miRNA), aptamers, small or short interfering (si)RNA, trans-splicing RNA, or antisense RNA). Polynucleotides also include non-coding RNA, including, but not limited to, RNAi, miRNA, lncRNA, RNA antagonists, aptamers, and any other non-coding RNA known to those skilled in the art. Polynucleotides include naturally occurring, synthetic, and intentionally altered or modified polynucleotides, as well as analogs and derivatives. The term "polynucleotide" also refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof, and is synonymous with nucleic acid sequence. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The term polynucleotide, as used herein, refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment described herein that includes a polynucleotide encompasses both the double-stranded form and two complementary single-stranded forms known or predicted to constitute a double-stranded form. Polynucleotides may be single-, double-, or triplex-stranded, linear or circular, and may be of any length. When discussing polynucleotides, the sequence or structure of a particular polynucleotide may be described herein according to the convention of providing the sequence in the 5' to 3' direction.

[0049] The term "gene" or "nucleotide sequence encoding a polypeptide" refers to a segment of DNA involved in producing a polypeptide chain. The DNA segment can include regions preceding and following the coding region (leader and trailer) involved in the transcription / translation of the gene product and the regulation of transcription / translation, as well as intervening sequences (introns) between individual coding segments (exons). For example, a gene comprises a polynucleotide containing at least one open reading frame that, after being transcribed and translated, is capable of encoding a specific protein or polypeptide.

[0050] The term "homologous" in the context of a nucleotide sequence includes a nucleotide (nucleic acid) sequence that is either identical or substantially similar to a known reference sequence. In one embodiment, the term "homologous nucleotide sequence" is used to characterize a sequence that has 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.

[0051] "Heterologous" means derived from a genotypically distinct entity from the rest of the entity to which it is being compared. For example, a polynucleotide introduced by genetic engineering techniques into a plasmid or vector derived from a different species is a heterologous polynucleotide. A promoter has been removed from its native coding sequence and is operably linked to a non-naturally occurring coding sequence linked to a heterologous promoter. Although the term "heterologous" is not always used herein in reference to polynucleotides, reference to a polynucleotide in the absence of the modifier "heterologous" is intended to include a heterologous polynucleotide despite the omission.

[0052] The terms "sequence identity," "percent identity," and "sequence percent identity" (or synonyms thereof, e.g., "99% identical") in the context of two or more nucleic acids or polypeptides refer to two or more sequences or subsequences that are the same or have a specified percentage of identical nucleotides or amino acid residues when compared and aligned for maximum correspondence (introducing gaps, if necessary), without considering any conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignments of amino acid or nucleotide sequences are known in the art. Suitable programs for determining percent sequence identity include, for example, the BLAST suite of programs available from the U.S. government's National Center for Biotechnology Information BLAST website. Comparisons between two sequences can be performed using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare 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 (Web Server issue):W 597-600 (2013). Those skilled in the art can determine appropriate parameters for maximal alignment depending on the particular alignment software. In certain embodiments, the default parameters of the alignment software are used.

[0053] As used herein, the term "variant" includes, but is not limited to, an antibody or fusion protein 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 specific positions within or adjacent to the amino acid sequence of the reference antibody. A variant may include one or more conservative substitutions in its amino acid sequence compared to the amino acid sequence of the reference antibody. Conservative substitutions may involve, for example, the substitution of similarly charged or uncharged amino acids. A variant retains the ability of the reference antibody to specifically bind to an antigen. The term variant also includes pegylated antibodies or proteins.

[0054] "Joint disease" is defined as a measurable abnormality in the cells or tissues of a joint that may lead to, for example, metabolic and molecular disorders, a condition that triggers anatomical and / or physiological changes in the joint, including, but not limited to, radiological detection of joint space narrowing, subchondral sclerosis, subchondral cysts, and osteophyte formation.

[0055] "Joint disease" is defined in a human subject as a symptom that prompts the subject to seek medical intervention, e.g., pain, stiffness, swelling, or rigidity reported by the subject. For non-human mammals, "joint disease" is defined as, e.g., lameness, observable changes in gait, weight bearing, allodynia, or exploratory behavior.

[0056] As used herein, sgRNA (single guide RNA) is an RNA, preferably a synthetic RNA, composed of a targeting sequence and a scaffold. In genome engineering experiments, it is used to guide Cas9 to a specific locus. The sgRNA can be administered or formulated, for example, as synthetic RNA or as a nucleic acid containing a sequence encoding the gRNA, which is then expressed in the target cell. As will be apparent to those skilled in the art, various tools can be used to design and / or optimize the sequence of the sgRNA, for example, to improve the specificity and / or accuracy of genome editing. Generally, candidate sgRNAs can be designed by identifying sequences within the target region with high predicted on-target efficiency and low off-target efficiency based on any of the available web-based tools. Candidate sgRNAs can be further evaluated by manual inspection and / or experimental screening. Examples of web-based tools include, without limitation, CRISPRSeek, CRISPR Design Tool, Cas-OFFinder, E-CRISP, ChopChop, CasOT, CRISPRDirect, CRISPOR, BREAKING-CAS, CrispRGold, and CCTop. See, e.g., Safari, et al. Current Pharma. Biotechnol. (2017) 18(13), which is incorporated by reference in its entirety for all purposes. Such tools are also described, for example, in PCT Application Publication No. WO2014 / 093701A1 and Liu, et al., "Computational approach for effective CRISPR guide RNA design and evaluation," Comput Struct Biotechnol J., 2020;18:35-44, each of which is incorporated by reference in its entirety for all purposes.

[0057] As used herein, "Cas9" refers to a CRISPR-associated protein, and Cas9 nuclease is the active enzyme of the type II CRISPR system. "nCas9" refers to Cas9 with one of two inactivated nuclease domains, i.e., either the RuvC or HNH domain. nCas9 can cleave only one strand of target DNA ("nickases"). The term "Cas9" refers to an RNA-guided double-stranded DNA-binding nuclease protein or nickase protein, or variants thereof. As used herein, "Cas9" refers to both native and recombinant Cas9s. Wild-type Cas9 nucleases have two functional domains, e.g., RuvC and HNH, that cleave different DNA strands. The Cas9 enzymes described herein can contain an HNH or HNH-like nuclease domain and / or a RuvC or RuvC-like nuclease domain. Cas9 can induce double-stranded breaks in genomic DNA (target loci) when both functional domains are active. The Cas9 enzyme can comprise one or more catalytic domains of Cas9 proteins from bacteria belonging to the group consisting of Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, and Campylobacter. In some embodiments, the two catalytic domains are derived from different bacterial species.

[0058] As used herein, "PAM" refers to a protospacer-adjacent motif, which is required for Cas9 to bind target DNA and immediately follows the target sequence. Cas9 can be administered or formulated, for example, as a protein (e.g., a recombinant protein) or as a nucleic acid containing a sequence encoding the Cas9 protein, which is then expressed in target cells. Native Cas9 molecules recognize specific PAM sequences (e.g., the PAM recognition sequences of S. pyogenes, S. thermophilus, S. mutans, S. aureus, and N. meningitidis). In one embodiment, the Cas9 molecule has the same PAM specificity as a native Cas9 molecule. In other embodiments, the Cas9 molecule has a PAM specificity that is not associated with the native Cas9 molecule to which it has closest sequence homology. For example, native Cas9 molecules can be modified so that PAM sequence recognition is altered to reduce off-target sites, improve specificity, or eliminate the PAM recognition requirement. In one embodiment, the Cas9 molecule can be modified (e.g., to lengthen the PAM recognition sequence, to improve Cas9 specificity to a higher level of identity, to reduce off-target sites, and / or to increase specificity). In one embodiment, the length of 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 modified to ablate PAM recognition.

[0059] An "expression cassette" is a recombinantly or synthetically produced nucleic acid construct having a set of specified nucleic acid elements that allow for transcription of a particular polynucleotide sequence in a host cell. An expression cassette or vector may 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 and operably linked to a promoter.

[0060] The term "promoter" is used herein to refer to an array of nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which may be located as much as several thousand base pairs from the start site of transcription. Other elements that may be present in an expression vector include elements that enhance transcription (e.g., enhancers) and terminate transcription (e.g., terminators), as well as elements that confer specific binding affinity or antigenicity to a recombinant protein produced from the expression vector.

[0061] The term "operably linked" refers to the juxtaposition of genetic elements, wherein the elements are in a relationship permitting them to function in their expected manner. For example, a promoter is operably linked to a coding region if it helps initiate transcription of the coding sequence. Intervening residues between the promoter and coding region can be present so long as this functional relationship is maintained.

[0062] An "isolated" plasmid, nucleic acid, vector, virus, virion, host cell, or other substance refers to a preparation of the substance that lacks at least some of the other components present in the location where the substance or similar substance naturally occurs or where it was originally prepared. Thus, for example, an isolated substance can be prepared by concentrating it from a source mixture using purification techniques. Concentration can be measured in absolute terms, such as weight per volume of solution, or relative to secondary, potentially interfering substances present in the source mixture. The greater the concentration of an embodiment of the present disclosure, the more isolated it becomes. An isolated plasmid, nucleic acid, vector, virus, host cell, or other substance, in some embodiments, is purified, e.g., 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% pure or greater.

[0063] As used herein, "AAV vector" refers to an AAV vector nucleic acid sequence encoding a variety of nucleic acid sequences, and in some embodiments, includes a variant or chimeric capsid polypeptide (i.e., the AAV vector includes a nucleic acid sequence encoding a variant or chimeric capsid polypeptide). An AAV vector may also include a heterologous nucleic acid sequence not of AAV origin as part of the nucleic acid insert. This heterologous nucleic acid sequence typically includes a sequence of interest for genetic transformation of a cell. Generally, the heterologous nucleic acid sequence is flanked by at least one, and generally two, AAV inverted terminal repeats (ITRs). In certain embodiments, the Cas sequence, guide RNA sequence, and any other genetic elements (e.g., promoter sequence, PAM sequence, etc.) may be on the same AAV vector or on two or more different AAV vectors when administered to a subject. In certain embodiments, the Cas sequence, guide RNA sequence, and any other genetic elements (e.g., promoter sequence, PAM sequence, etc.) may be on two or more different AAV vectors when administered to a subject, and the AAV may be of the same serotype, or the AAV may be of two or more different serotypes (e.g., AAV5 and AAV6).

[0064] An "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 encapsidated polynucleotide AAV transfer vector. If the particle contains heterologous nucleic acid (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to a cell), it may be referred to as an "AAV vector particle" or simply an "AAV vector." Thus, production of an AAV virion or AAV particle necessarily includes production of an AAV vector, since the vector is contained within the AAV virion or AAV particle.

[0065] As used herein, "carrier" or "vehicle" refers to a carrier material suitable for administering a pharmaceutical. Carriers and vehicles useful herein include any such material known in the art that is non-toxic and does not interact with other components of the composition in a deleterious manner, such as any liquid, gel, solvent, liquid diluent, solubilizer, surfactant, etc.

[0066] The phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0067] 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 inactive ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Except insofar as 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 drugs, may also be incorporated into the compositions and methods described.

[0068] The term "pharmaceutically acceptable excipient" is intended to include vehicles and carriers that can be co-administered with a compound to facilitate the performance of its intended function. The use of such vehicles for pharmaceutically active substances is well known in the art. Examples of such vehicles and carriers include solutions, solvents, dispersion media, delay agents, emulsions, etc. Any other conventional carriers suitable for use with multibinding compounds also fall within the scope of this disclosure.

[0069] As used herein, the terms "a," "an," or "the" are generally intended to cover both the singular and the plural.

[0070] The terms "about" and "approximately" refer to a statistically significant range of values. Such a range can be within an order of 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 acceptable variation encompassed by the terms "about" or "approximately" depends on the particular system under study and can be readily understood by one of ordinary skill in the art. Furthermore, as used herein, the terms "about" and "approximately" mean that compositions, amounts, formulations, parameters, shapes, and other quantities and characteristics are not, and need not be, exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding, measurement error, etc., and other factors known to those of ordinary skill in the art. Generally, a dimension, size, formulation, parameter, shape, or other quantity or characteristic is "about" or "approximately" whether or not explicitly stated as such. Note that embodiments of widely different sizes, shapes, and dimensions may employ the described configurations.

[0071] As used herein, the term "substantially" can 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.

[0072] When used in the appended claims, in their original and amended forms, the transitional phrases "comprising," "consisting essentially of," and "consisting of" define the claim in terms of which additional unrecited claim elements or steps, if any, are excluded from the claim. 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 element, step, or material other than that specified in the claim, and in the latter case, excludes impurities normally associated with the specified material. The term "consisting essentially of" limits the claim to the specified elements, steps, or materials, and those that do not materially affect the basic and novel characteristics of the claimed methods and compositions. All compositions, methods, and kits described herein that embody the present disclosure may, in alternative embodiments, be more specifically defined by any of the transitional phrases "comprising," "essentially consisting of," and "consisting of."

[0073] A subject treated by any of the methods or compositions described herein can be of any age, and can be an adult, an infant, or a 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, The subject may be 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 of age, or within a range therein (e.g., without limitation, 2-20 years, 20-40 years, or 40-90 years). The subject can be a human or non-human subject. Particular classes of subjects that can benefit from the compositions and methods of the present disclosure include subjects over 40, 50, or 60 years of age. Another class of subjects that can benefit from the compositions and methods of the present disclosure are subjects with arthritis (e.g., osteoarthritis).

[0074] Any of the compositions disclosed herein can be administered to a non-human subject, such as a laboratory or livestock animal. Non-limiting examples of non-human subjects include laboratory or research animals, pets, wild or livestock animals, livestock, etc., such as dogs, goats, guinea pigs, hamsters, mice, pigs, non-human primates (e.g., gorillas, monkeys, orangutans, lemurs, baboons, etc.), rats, sheep, horses, cows, etc.

[0075] CRISPR / Cas Systems - Minimum Requirements In one aspect, the clustered regularly interspaced short palindromic repeats and CRISPR-associated RNA-guided nuclease related methods, components, and compositions (hereinafter CRISPR / Cas systems) of the present disclosure minimally 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 effect the augmentation of a nucleic acid sequence (e.g., genomic DNA).

[0076] In some embodiments, the CRISPR / Cas system modifies a target gene or locus in a eukaryotic cell by effecting a sequence modification at a targeted location (e.g., by creating an insertion or deletion (collectively, indels)), resulting in a loss of function (i.e., knockout) of the affected gene or allele; e.g., a nucleotide substitution, resulting in a truncation, nonsense mutation, or other type of loss of function of the encoded gene product, e.g., the encoded IL1A or IL1B mRNA or protein; a deletion of one or more nucleotides, resulting in a truncation, nonsense mutation, or other type of loss of function of the encoded IL1A or IL1B gene product; a loss of function of the encoded mRNA or protein, e.g., by a single nucleotide, double nucleotide, or other frameshift deletion, or a deletion resulting in a premature stop codon; or an insertion, resulting in a truncation, nonsense mutation, or other type of loss of function of the encoded gene product, e.g., the encoded IL1A or IL1B mRNA or protein; e.g., a single nucleotide, double nucleotide, or other frameshift insertion, or an insertion resulting in a premature stop codon. In some embodiments, the CRISPR / Cas systems of the present disclosure provide for the modification (e.g., knockout) of genes associated with inflammatory joint diseases (e.g., rheumatoid arthritis or osteoarthritis) by modifying sequences at target locations, e.g., generating indels that result in nonsense-mediated decay of the encoded gene product, e.g., the encoded transcript.

[0077] In one aspect, the CRISPR / Cas system alters the sequence of a nucleic acid through nuclease activity. For example, in the case of genomic DNA, a nuclease guided by a protein-associated exogenous nucleic acid located at a target location within a targeted gene or locus by sequence complementarity with the target genomic sequence (e.g., the complementary component of a CRISPR RNA (crRNA) or synthetic single guide RNA (sgRNA)) cleaves the genomic DNA upon recognizing a specific nuclease-specific motif called a protospacer adjacent motif (PAM). See generally, Collias, D., & Beisel, CL (2021). Nature Communications, 12(1), 1-12.

[0078] Nuclease activity (i.e., cleavage) induces double-strand breaks (DSBs) in genomic DNA. Endogenous cellular mechanisms for DSB repair, i.e., nonhomologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and homologous recombination, result in erroneous repair at a given target location with some calculable frequency as a result of interference from relevant components of the CRISPR / Cas system, thereby introducing substitutions or indels into genomic DNA. See generally Scully, R., et al. (2019). Nature Reviews Molecular Cell Biology, 20(11), 698-714. At some frequency, these indels and / or substitutions can result in frameshifts, nonsense mutations (i.e., premature stop codons), or truncations, which affect the availability of gene products such as mRNA and / or proteins. In certain embodiments, the CRISPR / Cas system can induce a homology-directed repair (HDR) mechanism that leads to the insertion of non-random sequences at the target location through the use of a template (e.g., an HDR template) that is provided to the cell as part of the system along with a nuclease and a gRNA. See Bloh, K., & Rivera-Torres, N. (2021). International Journal of Molecular Sciences, 22(8), 3834.

[0079] Generally, the minimum requirements for a CRISPR / Cas system depend on the nuclease (i.e., Cas protein) provided with it. To this extent, these bacterial nucleases have been functionally divided into types I, III, and V, which are all classified into class 1, and types II, IV, and VI, which are classified into class 2.

[0080] Class 1 CRISPR / Cas systems: The exact components, compositions, and methods for effecting alteration of a target nucleic acid sequence using a Class 1 CRISPR / Cas system will vary, but should minimally include a nuclease (selected from at least Type I and Type III), 1) at least one guide RNA selected from sgRNAs, or 2) a combination of 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. To this end, compositions, components, and methods among Class 1 components may be considered functionally interchangeable, and the following details, provided solely for illustrative purposes, do not represent an exhaustive list of class members.

[0081] Cas3 (see Table 1) is a prototypical type I DNA nuclease that functions as an effector protein as part of a larger complex (the Cascade complex, which includes Cse1 and Cse2) capable of genome editing. See generally He, L., et al. (2020). Genes, 11(2), 208. Unlike other CRISPR / Cas systems, type I systems localize to DNA targets without the Cas3 nuclease via the Cascade complex, which then recruits Cas3 to cleave DNA upon binding and localizing the 3' PAM. The Cascade complex is also responsible for processing crRNA so that it can be used to guide crRNA to target locations. This functionality allows Cascade to process multiple arrayed crRNAs from a single molecule. See Luo, M. (2015). Nucleic Acids Research, 43(1), 674-681. Thus, type I systems may be used to edit multiple targeted genes or loci from a single molecule.

[0082] Because the natural Cas3 substrate is ssDNA, its function in genome editing is thought to be as a nicases; however, when targeted in tandem, the resulting edits are the result of blunt-end cleavage on opposite strands to approximate blunt-cutting endonucleases such as Cas9. See Pickar-Oliver, A., & Gersbach, CA (2019). Nature Reviews Molecular Cell Biology, 20(8), 490-507.

[0083] Similar to type I nucleases, type III systems rely on a complex of proteins to effect nucleic acid cleavage. In particular, Cas10 possesses nuclease activity for cleaving ssDNA in prokaryotes. See Tamulaitis, G., Trends in Microbiology, 25(1), 49-61. Interestingly, this archaeal CRISPR / Cas system exhibits dual specificity, targeting both ssDNA and ssRNA. Apart from this change, the system functions very similarly to type I in that the crRNA targets an effector complex (similar to a Cascade) in a sequence-dependent manner. Similarly, the effector complex processes the crRNA before assembly. The dual nature of this nuclease potentially makes its application in genome editing more powerful, as both genomic DNA and, in some cases, mRNA with the same sequence can be targeted to silence specific target genes.

[0084] Class 2 CRISPR / Cas systems: The exact components, compositions, and methods for effecting alteration of a target nucleic acid sequence using a Class 2 CRISPR / Cas system will vary, but should minimally include a nuclease (selected from at least Type II and Type V), 1) at least one guide RNA selected from sgRNAs, or 2) a combination of crRNA and tracrRNA. These CRISPR / Cas systems are categorized together as Class 2 CRISPR / Cas systems due to their similarities in requirements and mode of action within eukaryotic cells. To this end, compositions, components, and methods among Class 2 components may be considered functionally interchangeable, and the following details, provided solely for illustrative purposes, do not represent an exhaustive list of class members.

[0085] Type II nucleases are the best-characterized CRISPR / Cas system, particularly the standard genome editing nuclease, Cas9 (see Table 1). Multiple Cas9 proteins have been isolated from various bacterial species. The main difference between these nucleases is the required recognition site, PAM, within the targeted dsDNA. After association with the gRNA molecule, the crRNA (or the targeting domain of the sgRNA) orients the nuclease to the appropriate location, while protein recognition of the PAM induces a cleavage event near the site, resulting in a blunt DSB.

[0086] In addition to naturally occurring Cas9 proteins, several engineered variants have also been reported. These range from Cas9s with enhanced specificity (i.e., lower off-target activity), such as espCas9. Others have been catalytically modified via point mutations in the RuvC (e.g., D10A) and HNH (e.g., H840A) domains, so that they induce only single-strand breaks (i.e., Cas9 nickase). See Frock, R. et al. (2015). Nature Biotechnology, 33(2), 179-186. These variants, collectively referred to herein as Cas9 variants with enhanced specificity (spCas9), have also been shown to be less error-prone in editing. Such mitigation of off-target effects is paramount when selecting desired insertions (i.e., knock-in mutations in which the desired nucleotide sequence is introduced into the target nucleic acid molecule) rather than deletions. Indeed, fewer off-target effects may support a preferred DNA repair mechanism (most often HDR for knock-in mutations). See generally Naeem, M., et al. (2020). Cells, 9(7), 1608.

[0087] Additional exemplary further engineered variants (e.g., mutants, chimeras, and the like) of canonical Cas proteins include the following (incorporated herein by reference): WO2015 / 035162A2, WO2019 / 126716A1, WO2019 / 126774A1, WO2014 / 093694A1, and WO2014 / 150624A1.

[0088] For the avoidance of doubt, spCas9 collectively refers to any one of the group consisting of espCas9 (also referred to herein as ESCas9 or esCas9), HFCas9, PECas9, and arCas9.

[0089] Similar to the canonical Cas9 system, type V nucleases require only a synthetic sgRNA with a targeting domain complementary to the genomic sequence to perform genome editing. These nucleases contain a RuvC domain but lack the HNH domain of type II nucleases. Furthermore, Cas12 leaves a staggered cut in the dsDNA substrate distal to the PAM, compared to the blunt cut of Cas9, for example, next to 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-associated nucleases that act on dsDNA as a quaternary structure, simultaneously nicking each strand. See Zetsche B, et al. Cell. 2015;163(3):759-771; see also Liu L, Chen P, Wang M, et al. Mol Cell. 2017;65(2):310-322. Furthermore, Cas12b (C2c1) is a highly accurate nuclease with little tolerance for mismatches. See Yang H, et al. Cell. 2016;167(7):1814-1828.e12. [Table 2] See generally Wang, J., Zhang, C., & Feng, B. (2020). Journal of Cellular and Molecular Medicine, 24(6), 3256-3270, where N = any nucleotide, R = any purine (A or G), Y = any pyrimidine (C or T), W = A or T, and V = A, C, or G.

[0090] In one aspect, the CRISPR / Cas system of the present disclosure comprises at least one Cas protein from one or more of the following selected bacterial genera: Corynebacterium, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flavobacterium, Spirochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Nitratifractor, Campylobacter, Pseudomonas, Streptomyces, Staphylococcus, Francisella, Acidaminococcus, Lachnospiraceae, Leptotrichia, and Prevotella. In some embodiments, the Cas protein is from a Deltaproteobacteria or Planctomycetes bacterial species.

[0091] Some aspects of the present disclosure provide strategies, methods, compositions, and therapeutic modalities for modifying targeted sequences within a genetic locus (e.g., modifying wild-type and / or mutant sequences in cells or in patients having or experiencing the effects of rheumatoid arthritis, osteoarthritis, or other inflammatory diseases of joints or cells therein) by insertion or deletion of one or more nucleotides mediated by RNA-guided nucleases and one or more guide RNAs (gRNAs), resulting in loss of function of the targeted gene product. Such modification is alternatively referred to as "knocking out" (i.e., generating a "knockout") the gene of interest.

[0092] In certain embodiments, any region of the IL1A gene (e.g., the 5' untranslated region [UTR], exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, intron region, intron / exon junction, 3' UTR, or polyadenylation signal) is targeted by an RNA-guided nuclease to modify the gene. In certain embodiments, any region of the IL1B gene (e.g., the 5' untranslated region [UTR], exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, intron region, intron / exon junction, 3' UTR, or polyadenylation signal) is targeted by an RNA-guided nuclease to modify the gene. In certain embodiments, the targeted gene is selected from an inflammatory effector (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 modify 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 modify the gene.

[0093] CRISPR guide RNA: 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) that interacts with the Cas protein. In certain embodiments, the gRNA is an sgRNA in which a targeting (i.e., complementary) domain comprising a nucleotide sequence complementary to a 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 a crRNA provided to a eukaryotic cell harboring a tracrRNA, which acts as a scaffold through interactions with both the crRNA and a nuclease. In some embodiments, the system further optionally comprises an oligonucleotide-HDR template with homology on either side of the target position. See Bloh, K., & Rivera-Torres, N, at 3836.

[0094] In one embodiment, the targeting domain of a gRNA molecule is configured to orient an associated nuclease so that a cleavage event (e.g., a double-stranded or single-stranded break) occurs sufficiently close to the target location in the targeted gene or locus, thereby facilitating modification of the nucleic acid sequence. In some embodiments, the targeting domain is 20 nucleotides in length. In some embodiments, the targeting domain is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0095] 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 location. The double-stranded or single-stranded break may be located either upstream or downstream of the target location, and within or upstream of a functional domain cluster within the targeted gene.

[0096] In certain embodiments, a second gRNA molecule comprising a second targeting domain orients a second associated nuclease so that a cleavage event occurs sufficiently close to the target position of the targeted gene or locus, thereby facilitating modification 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 different targeted gene or locus than the first gRNA molecule. In some embodiments, the second targeting domain is 20 nucleotides in length. In some embodiments, the second targeting domain is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0097] 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 location. The double-stranded or single-stranded break may be located either upstream or downstream of the target location, and within or upstream of a functional domain cluster within the targeted gene.

[0098] In one embodiment, the targeting domains of the first and second gRNA molecules are configured such that the cleavage event 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 respective target position, independently for each gRNA molecule. In one embodiment, the first and second gRNA molecules modify the target nucleic acid sequence simultaneously. In one embodiment, the first and second gRNA molecules modify the target nucleic acid sequence sequentially.

[0099] In one embodiment, the single-stranded breaks are accompanied by second single-stranded breaks positioned by the targeting domains of the first and second gRNA molecules, respectively. For example, the targeting domains can direct the associated nuclease so that the cleavage event (e.g., two single-stranded breaks) is 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 target position. In one embodiment, the targeting domains of the first and second gRNA molecules are configured to orient the associated nucleases such that two single-stranded breaks occur on opposite strands of the genomic DNA, e.g., at the same target location or within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides of each other, thereby essentially approximating a double-stranded break.

[0100] In one embodiment, the nucleic acid encodes the targeting domain of a first gRNA molecule and the targeting domain of a second gRNA molecule selected from the sequences of Figure 5. In one embodiment, the nucleic acid encodes the targeting domain of a first gRNA molecule and the targeting domain of a second gRNA molecule selected from the sequences of 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.

[0101] In one embodiment, the nucleic acid encodes a crRNA sequence for a first gRNA molecule and a crRNA sequence for a second gRNA molecule selected from the sequences of Figure 5. In one embodiment, the nucleic acid encodes a crRNA sequence for a first gRNA molecule and a crRNA sequence for a second gRNA molecule selected from the sequences of Figure 7. In one embodiment, the nucleic acid encodes a first gRNA molecule consisting of a crRNA sequence and a tracrRNA. In one embodiment, the nucleic acid encodes a second gRNA molecule consisting of a crRNA sequence and a tracrRNA. In one embodiment, the nucleic acid encodes a third gRNA molecule consisting of a crRNA sequence and a tracrRNA. In one embodiment, the nucleic acid encodes a fourth gRNA molecule consisting of a crRNA sequence and a tracrRNA.

[0102] 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.

[0103] 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 of a targeted gene or locus, (b) a sequence encoding a second gRNA molecule comprising a targeting domain complementary to a target position of a second targeted gene or locus, and (c) a sequence encoding an RNA-guided nuclease (e.g., Cas9 or other Cas protein). Optionally, (d) and (e) are sequences encoding a third and 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 within 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 within the same nucleic acid molecule. In some embodiments, (a), (b), and (d) are encoded within the same nucleic acid molecule. In some embodiments, (a), (b), and (e) are encoded within the same nucleic acid molecule. In some embodiments, (a), (b), (d), and (e) are encoded within the same nucleic acid molecule. In some embodiments, (a), (b), and (c) are encoded within separate nucleic acid molecules. When more than two gRNAs are used, any combination of (a), (b), (c), (d), and (e) can be encoded within a single or separate nucleic acid molecule.

[0104] In some embodiments, 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 vector, AAV2 vector, AAV3 vector, AAV4 vector, AAV5 vector, AAV6 vector, AAV7 vector, AAV8 vector, AAV9 vector, and AAV10 vector.

[0105] In some embodiments, (a), (b), and (c) are encoded within the same vector. In some embodiments, (a) and (b) are encoded within the same vector. In some embodiments, (a), (b), and (d) are encoded within the same vector. In some embodiments, (a), (b), and (e) are encoded within the same vector. In some embodiments, (a), (b), (d), and (e) are encoded within the same vector. In some embodiments, (a), (b), and (c) are encoded within separate vectors. When more than two gRNAs are used, any combination of (a), (b), (c), (d), and (e) can be encoded within a single or separate vectors.

[0106] In one aspect, a nucleic acid molecule (i.e., encoding (a), (b), (c), (d), or (e)) is delivered to a target cell (i.e., any combination of the encoded RNA-guided nuclease of (c) and at least one encoded gRNA molecule of (a), (b), (d), or (e) contacts the target cell). In some embodiments, the nucleic acid molecule is delivered to the target cell in vivo. In other embodiments, the nucleic acid molecule is delivered to the target cell ex vivo. In some embodiments, the nucleic acid molecule is delivered to the target cell in vitro. In certain embodiments, the nucleic acid molecule is delivered to the target cell as DNA. In other embodiments, the nucleic acid molecule is delivered to the target cell as RNA (e.g., mRNA). In some embodiments, the product of the nucleic acid molecule is delivered as an assembled ribonucleoprotein (RNP).

[0107] 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 nucleic acid molecule of interest selected from (a), (b), (d), and (e).

[0108] In one embodiment, contacting the target cell comprises delivering the encoded RNA-guided nuclease of (c) as DNA together with at least one nucleic acid molecule of interest selected from (a), (b), (d), and (e).

[0109] In certain embodiments, CRISPR components are delivered to target cells via nanoparticles. Exemplary nanoparticles that can be used with any of the 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.

[0110] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this 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. Additionally, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0111] 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 and treating osteoarthritis in mammalian joints.

[0112] In some aspects, the pharmaceutical composition comprises a gene editing system, wherein the gene editing system silences or reduces expression of at least one genetic locus associated with joint function in at least a portion of cells that comprise the joint.

[0113] In one aspect, the pharmaceutical composition comprises a gene editing system, wherein the gene editing system targets one or more of IL-1α and / or IL-1β. In some aspects, the pharmaceutical composition comprises a gene editing system, wherein the gene editing system targets one or more of TNF-α, IL-6, IL-8, IL-18, matrix metalloproteinases (MMPs), or components of the NLRP3 inflammasome.

[0114] In some aspects, the pharmaceutical composition comprises a gene editing system, wherein the gene editing comprises the use of a programmable nuclease to mediate the creation of a double-stranded or single-stranded break in at least one genetic locus associated with joint function. In some embodiments, the gene editing system reduces gene expression at the target locus. In some embodiments, at least one genetic locus associated with joint tissue is silenced or reduced in at least a portion of cells comprising the joint.

[0115] 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.

[0116] In one aspect, the pharmaceutical composition targets one or more cytokines and / or growth factor genes selected from the group including IL-1α, IL-1β, 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, ASC (apoptosis-associated speck-like protein containing CARD), caspase-1, and combinations thereof.

[0117] Also provided is a pharmaceutical composition wherein the gene editing increases expression of one or more cytokine and / or growth factor genes in at least a portion of cells comprising the joint, and the cytokine and / or growth factor genes are selected from the group including IL-1Ra, TIMP-1, TIMP-2, TIMP-3, TIMP-4, and combinations thereof.

[0118] In some embodiments, pharmaceutical compositions are provided for gene editing, wherein the gene editing comprises the use of programmable nucleases to mediate the generation of double-stranded or single-stranded breaks in one or more cytokine and / or growth factor genes. In some embodiments, the gene editing comprises one or more methods selected from CRISPR, TALE, zinc finger, and combinations thereof.

[0119] In one embodiment, the gene editing comprises CRISPR technology. In yet another embodiment, the CRISPR technology is CRISPR-Cas9 technology. In some embodiments, Cas9 is mutated to enhance function.

[0120] Animal models of osteoarthritis Several animal models of osteoarthritis are known in the art.Although exemplary non-limiting animal models are summarized, it is understood that various models can be used.Many different species of animals are used to simulate OA, for example, research has been carried out on mice, rats, rabbits, guinea pigs, dogs, pigs, horses and other animals.For example, see Kuyinu et al., J Orthop Surg Res.11:19(2016) (hereinafter referred to as "Kuyinu,2016").

[0121] It is understood that various methods for inducing OA can be used in any mammal. In mice, spontaneous generation, chemical induction, surgical induction, and non-invasive induction 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 models, chemical induction, traumatic induction, and overuse induction are commonly used. In sheep, surgical induction is the most common, while in guinea pigs, surgical induction, chemical induction, and spontaneous generation (Durkin-Hartley) methods are often used. For example, Bapat, 2018.

[0122] The destabilized medial meniscus (DMM) is often used in mice to model post-traumatic osteoarthritis (e.g., Culley et al., Methods Mol Biol. 1226:143-73 (2015)). The DMM model mimics clinical meniscus injury, a known predisposing factor for the development of human OA, and allows for the study of structural and biological changes over the course of the disease. Mice are attractive model organisms because mouse strains with defined genetic backgrounds can be used. In addition, knockout or other genetically engineered mouse strains can be used to assess the importance of various molecular pathways in the response to various OA treatment modalities and regimens. For example, STR / ort mice possess characteristics that make them particularly susceptible to developing OA, including increased levels of the proinflammatory cytokine IL1β (Bapat et al., Clin Transl Med. 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 those skilled in the art, such as Col9a1(- / -) mice (Allen et al., Arthritis Rheum, 60:2684-2693 (2009)).

[0123] 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 target ACL is surgically severed, causing joint instability. An anterior drawer test with the joint flexed is used to confirm that ligament transection has occurred. In some cases, other ligaments, such as the posterior cruciate ligament, medial collateral ligament, lateral collateral ligament, and / or any of the menisci, may also be severed. Similar to the DMM model, various mouse strains can be used to investigate various molecular pathways.

[0124] Depending on the technical objective, animals of various sizes can be selected for use. Rodents are useful because they require less time for skeletal maturation, resulting in a shorter time for OA to develop following surgery or other techniques that induce OA. Larger animals are particularly useful for evaluating therapeutic interventions. The anatomical structure of larger animals is very similar to that of humans; for example, in dogs, the thickness of cartilage is approximately half that of humans. This striking similarity is an example of why the study of cartilage degeneration and osteochondral defects is much more useful in large animal models. See, for example, McCoy, Vet. Pathol., 52:803-18 (2015), and Pelletier et al., Therapy, 7:621-34 (2010).

[0125] The gene editing process Summary: Compositions for gene editing of synovial cells

[0003] Embodiments of the present disclosure are directed to methods for gene editing synovial cells (synovial cells), including one or more steps of gene editing at least a portion of synovial cells within a joint to treat osteoarthritis or other joint disorders. As used herein, "gene editing," "gene editing," and "genome editing" refer to types of genetic modification in which DNA is permanently modified within a cell's genome, e.g., DNA is inserted, deleted, modified, or replaced within a cell's genome. In some embodiments, gene editing silences (sometimes referred to as gene knockout) or inhibits / reduces (sometimes referred to as gene knockdown) expression of a DNA sequence. In other embodiments, gene editing enhances expression of a DNA sequence (e.g., by causing overexpression). According to embodiments of the present disclosure, gene editing techniques are used to reduce or silence pro-inflammatory genes and / or enhance the expression of regenerative genes.

[0126] Interleukin According to additional embodiments, the gene editing methods 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 demonstrated to enhance inflammatory responses in joint tissues and are associated with disease progression.

[0127] Expression constructs Expression constructs encoding one or both of the guide RNA and / or Cas9 editing enzyme can be administered in any effective carrier, e.g., any formulation or composition capable of effectively delivering the component genes to cells in vivo. Approaches include electroporation and / or insertion in viral vectors, including, for example, recombinant retroviruses, adenoviruses, adeno-associated viruses, lentiviruses, and herpes simplex virus-1, or recombinant bacterial or eukaryotic plasmids. Viral vectors directly transfect cells, and plasmid DNA can be delivered naked or with the aid of, for example, cationic liposomes (lipofectamine), or derivatized (e.g., antibody-conjugated), polylysine conjugates, gramacidin S, artificial viral envelopes, or other such intracellular carriers, as well as direct injection of the gene construct or CaPO4 precipitates carried out in vivo.

[0128] The preferred approach for in vivo introduction of nucleic acid into cells is to use viral vectors containing nucleic acid such as cDNA.The infection of cells with viral vectors has the advantage that most target cells can accept nucleic acid.In addition, the molecule coded in viral vectors, for example, by the cDNA contained in viral vectors, is efficiently expressed in the cells that take up viral vector nucleic acid.

[0129] Retroviral vectors and adeno-associated viral vectors can be used as recombinant gene delivery systems for the transfer of exogenous genes in vivo, especially to humans. These vectors provide efficient delivery of genes into cells. In some cases, the transferred nucleic acid is stably integrated into the host's chromosomal DNA. In other cases, particularly for adeno-associated viral vectors, stable integration into the host's DNA can be a rare event, resulting in episomal expression of the transgene and transient expression of the transgene.

[0130] The development of specialized cell lines (so-called "packaging cells") that produce only replication-defective retroviruses has increased the utility of retroviruses for gene therapy, and defective retroviruses are being characterized for use in gene transfer for gene therapy purposes (for a review, see Miller, Blood 76:271 (1990)). Replication-defective retroviruses can be packaged into virions that can be used to infect target cells through the use of 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, which are known to those skilled in the art. Examples of suitable packaging virus lines for preparing both anisotropic and amphotropic retroviral systems include ΨCrip, ΨCre, Ψ2, and ΨAm.Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, in vitro, and / or in vivo (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 85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al. (1992) Proc. Natl. Acad. Sci. USA 89:1011-1012; each of which is incorporated by reference in its entirety for all purposes). al. (1991) Proc. Natl. Acad. Sci. USA 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. al. (1992) Human Gene Therapy 3:641-647, Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895, Hwu et al. (See, e.g., U.S. Pat. No. 4,868,116, U.S. Pat. No. 4,980,286, PCT Application No. WO 89 / 07136, PCT Application No. WO 89 / 02468, PCT Application No. WO 89 / 05345, and PCT Application No. WO 92 / 07573).

[0131] Another viral gene delivery system useful in the present method utilizes adenovirus-derived vectors. The adenovirus genome can be engineered to encode and express a gene product of interest, but is inactivated with respect to its ability to replicate in the normal lytic viral 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 adenovirus vectors can be derived from any strain of adenovirus (e.g., Ad2, Ad3, Ad5, or Ad7), including adenovirus serotypes from other species (e.g., murine, canine, human, etc.) known to those skilled in the art. Viral particles are relatively stable, amenable to purification and concentration, and can be modified to affect the spectrum of infectivity, as described above. In addition, the introduced adenoviral DNA (and the foreign DNA contained therein) does not integrate into the host cell genome but remains episomal, thereby avoiding potential problems that can arise as a result of in situ insertional mutagenesis, where the introduced DNA becomes integrated into the host genome (e.g., retroviral DNA). Furthermore, the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) compared to other gene delivery vectors (Berkner et al., supra; Haj-Ahmand and Graham, J. Virol. 57:267 (1986)).

[0132] Helper-dependent (HDAd) vectors can also be produced with all adenoviral sequences deleted except for origins of DNA replication at each end of the viral DNA, along with a packaging signal at the 5' 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 necessary for replication.

[0133] Yet another viral vector system useful for delivery of nucleic acids is the adeno-associated virus (AAV), a naturally defective virus that requires another virus, such as an adenovirus or herpesvirus, as a helper virus for efficient replication and a productive life cycle. (For a review, see Muzyczka et al., Curr. Topics in Micro. and Immunol. 158:97-129 (1992). Adeno-associated virus also integrates its DNA into non-dividing cells and is one of the few viruses that exhibits a high frequency of stable integration (see, e.g., Flotte et al., Am. J. Respir. Cell. Mol. Biol. 7:349-356 (1992); Samulski et al., J. Virol. 63:3822-3828 (1989); and McLaughlin et al., J. Virol. 62:1963-1973 (1989)). Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate. Space for exogenous DNA is limited to approximately 4.5 kb. Tratschin et al. AAV vectors, such as those described in (1985) can be used to introduce DNA into cells. A variety of nucleic acids have been introduced into different cell types using AAV vectors (see, e.g., 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 that are more highly stable and effective in vivo and can be easily produced and packaged into adeno-associated virus (AAV) vectors that have been approved by the FDA and tested in multiple clinical trials opens new avenues for therapeutic gene editing.

[0134] In some embodiments, nucleic acids encoding CRISPR IL-1α or IL-1β gene editing complexes (e.g., Cas9 or gRNA) are entrapped in positively charged liposomes (e.g., lipofectin) that can be tagged with antibodies against cell surface antigens of target cells. These delivery vehicles can also be used to deliver the Cas9 protein / gRNA complex.

[0135] In a clinical setting, a gene delivery system for a nucleic acid encoding a CRISPR IL-1α or IL-1β gene editing complex can be introduced into a subject by any of several methods, each of which is well known in the art. For example, a pharmaceutical preparation of the gene delivery system can be introduced systemically, e.g., by intravenous injection, with specific transduction of the protein in target cells resulting primarily from transfection specificity provided by the gene delivery vehicle, cell-type or tissue-type expression due to transcriptional regulatory sequences controlling expression of the receptor gene, or a combination thereof. In other embodiments, the initial delivery of the nucleic acid encoding a CRISPR IL-1α or IL-1β gene editing complex is more limited, resulting in highly localized introduction into the subject. For example, a nucleic acid encoding a CRISPR IL-1α or IL-1β gene editing complex can be introduced by intra-articular injection into a joint exhibiting a joint disease (e.g., osteoarthritis). In some embodiments, the nucleic acid encoding a CRISPR IL-1α or IL-1β gene editing complex is administered during or after surgery, and in some embodiments, a sustained-release hydrogel comprising a nucleic acid encoding a 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 a CRISPR IL-1α or IL-1β gene editing complex over time.

[0136] Pharmaceutical preparations of nucleic acids encoding CRISPR IL-1α or IL-1β gene editing complexes can consist essentially of the gene delivery system (e.g., a viral vector) in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery system can be produced intact from recombinant cells, e.g., adeno-associated viral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.

[0137] Preferably, the CRISPR IL-1α or IL-1β editing complex is specific, i.e., it induces genomic modifications preferentially at the target site (IL-1α or IL-1β) and does not induce modifications at other sites, or only rarely induces modifications 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%.

[0138] An sgRNA for use in a CRISPR / Cas system for HR typically comprises a guide sequence (e.g., crRNA) that is complementary to a target nucleic acid sequence (target locus) and a scaffold sequence (e.g., tracrRNA) that interacts with a Cas nuclease (e.g., Cas9 polypeptide) or a variant or fragment thereof. The single guide RNA (sgRNA) may comprise a crRNA and a tracrRNA.

[0139] Exemplary target sequences for directing genomic modifications in the IL-1α or IL-1β gene 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. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5]

[0140] In certain embodiments, the sequence of the guide RNA (e.g., single guide RNA or sgRNA) may be modified to increase editing efficiency and / or reduce off-target effects. In certain embodiments, the sequence of the guide RNA may vary 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 the guide RNA may vary 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, changes forming a target sequence may refer to the degree of complementarity.

[0141] In certain embodiments, the guide RNA used with a composition, method, or system of the present disclosure is identical to the sequence set forth in any one of SEQ ID NOs: 21-34 and 168-297. In certain embodiments, the guide RNA used with a composition, method, or system of the present disclosure is at least about 95% identical to the sequence set forth in any one of SEQ ID NOs: 21-34 and 168-297. In certain embodiments, the guide RNA used with a composition, method, or system of the present disclosure is at least about 90% identical to the sequence set forth in any one of SEQ ID NOs: 21-34 and 168-297. In certain embodiments, the guide RNA used with a composition, method, or system of the present disclosure is at least about 85% identical to the sequence set forth in any one of SEQ ID NOs: 21-34 and 168-297. In certain embodiments, the guide RNA used with a composition, method, or system of the present disclosure is at least about 80% identical to the sequence set forth in any one of SEQ ID NOs: 21-34 and 168-297. In certain embodiments, a guide RNA used with a composition, method, or system of the present disclosure is at least about 75% identical to the sequence set forth in any one of SEQ ID NOs: 21-34 and 168-297. In certain embodiments, a guide RNA used with a composition, method, or system of the present disclosure is at least about 70% identical to the sequence set forth in any one of SEQ ID NOs: 21-34 and 168-297. In certain embodiments, a guide RNA used with a composition, method, or system of the present disclosure is at least about 65% identical to the sequence set forth in any one of SEQ ID NOs: 21-34 and 168-297. In certain embodiments, a guide RNA used with a composition, method, or system of the present disclosure is at least about 60% identical to the sequence set forth in any one of SEQ ID NOs: 21-34 and 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 set forth in any one of SEQ ID NOs: 21-34 and 168-297.In certain embodiments, a guide RNA used with a composition, method, or system of the present disclosure is at least about 50% identical to the sequence set forth in any one of SEQ ID NOs: 21-34 and 168-297. In certain embodiments, a guide RNA used with a composition, method, or system of the present disclosure is at least about 45% identical to the sequence set forth in any one of SEQ ID NOs: 21-34 and 168-297. In certain embodiments, a guide RNA used with a composition, method, or system of the present disclosure is at least about 40% identical to the sequence set forth in any one of SEQ ID NOs: 21-34 and 168-297. In certain embodiments, a guide RNA used with a composition, method, or system of the present disclosure is at least about 35% identical to the sequence set forth in any one of SEQ ID NOs: 21-34 and 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 set forth in any one of SEQ ID NOs: 21-34 and 168-297.

[0142] In certain embodiments, the guide RNA used with the composition, method, or system of the present disclosure has a single base substitution in the sequence set forth in any one of SEQ ID NOs: 21-34 and 168-297. In certain embodiments, the guide RNA used with the composition, method, or system of the present disclosure has a double base substitution in the sequence set forth in any one of SEQ ID NOs: 21-34 and 168-297. In certain embodiments, the guide RNA used with the composition, method, or system of the present disclosure has a triple base substitution in the sequence set forth in any one of SEQ ID NOs: 21-34 and 168-297. In certain embodiments, the guide RNA used with the composition, method, or system of the present disclosure has a quadruple base substitution in the sequence set forth in any one of SEQ ID NOs: 21-34 and 168-297. In certain embodiments, the guide RNA used with the composition, method, or system of the present disclosure has a quadruple base substitution in the sequence set forth in any one of SEQ ID NOs: 21-34 and 168-297. In certain embodiments, the guide RNA used with the compositions, methods, or systems of the present disclosure has a 6-base substitution in the sequence set forth in any one of SEQ ID NOs: 21-34 and 168-297. In certain embodiments, the guide RNA used with the compositions, methods, or systems of the present disclosure has a 7-base substitution in the sequence set forth in any one of SEQ ID NOs: 21-34 and 168-297. In certain embodiments, the guide RNA used with the compositions, methods, or systems of the present disclosure has an 8-base substitution in the sequence set forth in any one of SEQ ID NOs: 21-34 and 168-297. In certain embodiments, the guide RNA used with the compositions, methods, or systems of the present disclosure has a 9-base substitution in the sequence set forth in any one of SEQ ID NOs: 21-34 and 168-297. In certain embodiments, the guide RNA used with the compositions, methods, or systems of the present disclosure has a 10-base substitution in the sequence set forth in any one of SEQ ID NOs: 21-34 and 168-297.In certain embodiments, the guide RNA used with the compositions, methods, or systems of the present disclosure has an 11-base substitution in the sequence set forth in any one of SEQ ID NOs: 21-34 and 168-297. In certain embodiments, the guide RNA used with the compositions, methods, or systems of the present disclosure has a 12-base substitution in the sequence set forth in any one of SEQ ID NOs: 21-34 and 168-297. In certain embodiments, the guide RNA used with the compositions, methods, or systems of the present disclosure has a 13-base substitution in the sequence set forth in any one of SEQ ID NOs: 21-34 and 168-297. In certain embodiments, the guide RNA used with the compositions, methods, or systems of the present disclosure has a 14-base substitution in the sequence set forth in any one of SEQ ID NOs: 21-34 and 168-297. In certain embodiments, the guide RNA used with the compositions, methods, or systems of the present disclosure has a 15-base substitution in the sequence set forth in any one of SEQ ID NOs: 21-34 and 168-297.

[0143] In certain embodiments, a guide RNA of the present disclosure is designed to and / or is capable of knocking down the expression of a target gene set forth in any one of SEQ ID NOs: 7-20 and 37-167. In certain embodiments, a 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, a 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, a 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, a 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, a 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, a 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, a 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, a 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.

[0144] In certain embodiments, a guide RNA of the present disclosure is designed to or is capable of knocking down the human IL-1β gene by binding to at least a portion of exon 1 of the human IL-1β gene. In certain embodiments, a guide RNA of the present disclosure is designed to or is capable of knocking down the human IL-1β gene by binding to at least a portion of exon 2 of the human IL-1β gene. In certain embodiments, a guide RNA of the present disclosure is designed to or is capable of knocking down the human IL-1β gene by binding to at least a portion of exon 3 of the human IL-1β gene. In certain embodiments, a guide RNA of the present disclosure is designed to or is capable of knocking down the human IL-1β gene by binding to at least a portion of exon 4 of the human IL-1β gene. In certain embodiments, a guide RNA of the present disclosure is designed to or is capable of knocking down the human IL-1β gene by binding to at least a portion of exon 5 of the human IL-1β gene. In certain embodiments, a guide RNA of the present disclosure is designed to or is capable of knocking down the human IL-1β gene by binding to at least a portion of exon 6 of the human IL-1β gene. In certain embodiments, a guide RNA of the present disclosure is designed to or is capable of knocking down the human IL-1β gene by binding to at least a portion of exon 7 of the human IL-1β gene.

[0145] In certain embodiments, a guide RNA of the present disclosure is designed to and / or is capable of knocking down expression of a target gene set forth in any one of SEQ ID NOs: 7-20 and 37-167. In certain embodiments, a guide RNA of the present disclosure is designed to or is capable of knocking down the expression of the canine IL-1α gene by binding to at least a portion of exon 1 of the canine IL-1α gene. In certain embodiments, a 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, a 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, a 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, a 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, a 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, a 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.

[0146] In certain embodiments, a guide RNA of the present disclosure 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, a guide RNA of the present disclosure 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, a guide RNA of the present disclosure 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, a guide RNA of the present disclosure 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, a guide RNA of the present disclosure 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, a guide RNA of the present disclosure is designed to or is 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, a guide RNA of the present disclosure is designed to or is 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, a guide RNA of the present disclosure is designed to or is 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.

[0147] In certain embodiments, a guide RNA of the present disclosure is designed to and / or is capable of knocking down expression of a target gene set forth in any one of SEQ ID NOs: 7-20 and 37-167. In certain embodiments, a guide RNA of the present disclosure is designed to or is capable of knocking down the expression of the equine IL-1α gene by binding to at least a portion of exon 1 of the equine IL-1α gene. In certain embodiments, a guide RNA of the present disclosure is designed to or is capable of knocking down the equine IL-1α gene by binding to at least a portion of exon 2 of the equine IL-1α gene. In certain embodiments, a guide RNA of the present disclosure is designed to or is capable of knocking down the equine IL-1α gene by binding to at least a portion of exon 3 of the equine IL-1α gene. In certain embodiments, a guide RNA of the present disclosure is designed to or is capable of knocking down the equine IL-1α gene by binding to at least a portion of exon 4 of the equine IL-1α gene. In certain embodiments, a guide RNA of the present disclosure is designed to or is capable of knocking down the equine IL-1α gene by binding to at least a portion of exon 5 of the equine IL-1α gene. In certain embodiments, a guide RNA of the present disclosure is designed to or is capable of knocking down the equine IL-1α gene by binding to at least a portion of exon 6 of the equine IL-1α gene. In certain embodiments, a guide RNA of the present disclosure is designed to or is capable of knocking down the equine IL-1α gene by binding to at least a portion of exon 7 of the equine IL-1α gene.

[0148] In certain embodiments, a guide RNA of the present disclosure is designed to or is capable of knocking down the equine IL-1β gene by binding to at least a portion of exon 1 of the equine IL-1β gene. In certain embodiments, a guide RNA of the present disclosure is designed to or is capable of knocking down the equine IL-1β gene by binding to at least a portion of exon 2 of the equine IL-1β gene. In certain embodiments, a guide RNA of the present disclosure is designed to or is capable of knocking down the equine IL-1β gene by binding to at least a portion of exon 3 of the equine IL-1β gene. In certain embodiments, a guide RNA of the present disclosure is designed to or is capable of knocking down the equine IL-1β gene by binding to at least a portion of exon 4 of the equine IL-1β gene. In certain embodiments, a guide RNA of the present disclosure is designed to or is capable of knocking down the equine IL-1β gene by binding to at least a portion of exon 5 of the equine IL-1β gene. In certain embodiments, a guide RNA of the present disclosure is designed to or is capable of knocking down the equine IL-1β gene by binding to at least a portion of exon 6 of the equine IL-1β gene. In certain embodiments, a guide RNA of the present disclosure is designed to or is capable of knocking down the equine IL-1β gene by binding to at least a portion of exon 7 of the equine IL-1β gene.

[0149] In certain embodiments, a guide RNA of the present disclosure is designed to and / or is capable of knocking down expression of a target gene set forth in any one of SEQ ID NOs: 7-20 and 37-167. In certain embodiments, a guide RNA of the present disclosure is designed to or is capable of knocking down the mouse IL-1α gene by binding to at least a portion of exon 1 of the mouse IL-1α gene. In certain embodiments, a guide RNA of the present disclosure is designed to or is capable of knocking down the mouse IL-1α gene by binding to at least a portion of exon 2 of the mouse IL-1α gene. In certain embodiments, a guide RNA of the present disclosure is designed to or is capable of knocking down the mouse IL-1α gene by binding to at least a portion of exon 3 of the mouse IL-1α gene. In certain embodiments, a guide RNA of the present disclosure is designed to or is capable of knocking down the murine IL-1α gene by binding to at least a portion of exon 4 of the murine IL-1α gene. In certain embodiments, a guide RNA of the present disclosure is designed to or is capable of knocking down the murine IL-1α gene by binding to at least a portion of exon 5 of the murine IL-1α gene. In certain embodiments, a guide RNA of the present disclosure is designed to or is capable of knocking down the murine IL-1α gene by binding to at least a portion of exon 6 of the murine IL-1α gene. In certain embodiments, a guide RNA of the present disclosure is designed to or is capable of knocking down the murine IL-1α gene by binding to at least a portion of exon 7 of the murine IL-1α gene.In certain embodiments, the guide RNAs of the present disclosure are designed to or are capable of knocking down the mouse IL-1α gene by binding to at least a portion of exon 8 of the mouse IL-1α gene.

[0150] In certain embodiments, a guide RNA of the present disclosure is designed to or is capable of knocking down the murine IL-1β gene by binding to at least a portion of exon 1 of the murine IL-1β gene. In certain embodiments, a guide RNA of the present disclosure is designed to or is capable of knocking down the murine IL-1β gene by binding to at least a portion of exon 2 of the murine IL-1β gene. In certain embodiments, a guide RNA of the present disclosure is designed to or is capable of knocking down the murine IL-1β gene by binding to at least a portion of exon 3 of the murine IL-1β gene. In certain embodiments, a guide RNA of the present disclosure is designed to or is capable of knocking down the murine IL-1β gene by binding to at least a portion of exon 4 of the murine IL-1β gene. In certain embodiments, a guide RNA of the present disclosure is designed to or is capable of knocking down the murine IL-1β gene by binding to at least a portion of exon 5 of the murine IL-1β gene. In certain embodiments, a guide RNA of the present disclosure is designed to or is capable of knocking down the murine IL-1β gene by binding to at least a portion of exon 6 of the murine IL-1β gene. In certain embodiments, a guide RNA of the present disclosure is designed to or is capable of knocking down the murine IL-1β gene by binding to at least a portion of exon 7 of the murine IL-1β gene.

[0151] In some cases, the sgRNA is introduced into cells (e.g., in vitro cells, such as primary cells for ex vivo therapy, or in vivo cells, such as in a patient) using a recombinant expression vector comprising a nucleotide sequence encoding a Cas nuclease (e.g., a Cas9 polypeptide) or a variant or fragment thereof. In some embodiments, the 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 in a patient). In other cases, the sgRNA is introduced into cells (e.g., in vitro cells, such as primary cells for ex vivo therapy, or in vivo cells, such as in a patient) using an mRNA encoding the Cas nuclease (e.g., a Cas9 polypeptide) or a variant or fragment thereof.

[0152] Any heterologous or exogenous nucleic acid (e.g., a polynucleotide encoding a target locus-specific sgRNA and / or a Cas9 polynucleotide) can be introduced into a cell using any method known to those of skill in the art, including, but not limited to, electroporation, nucleofection, transfection, lipofection, transduction, microinjection, electroinjection, electrofusion, nanoparticle bombardment, transformation, conjugation, etc.

[0153] The nucleic acid sequence of the sgRNA can be any polynucleotide sequence that has sufficient complementarity with a target polynucleotide sequence (e.g., a target DNA sequence) to hybridize with the target sequence and direct sequence-specific binding of a 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 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or greater when optimally aligned using a suitable alignment algorithm. Optimal alignment can be determined using any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies), ELAND (Illumina, San Diego, CA). Diego, Calif), SOAP (available at soap.genomics.org.cn), and Maq (available at 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, 35 nucleotides, 40 nucleotides, 45 nucleotides, 50 nucleotides, 75 nucleotides or more in length. In some cases, the guide sequence is about 20 nucleotides in length. In other cases, the guide sequence is about 15 nucleotides in length. In other cases, the guide sequence is about 25 nucleotides in length.The ability of a guide sequence to induce sequence-specific binding of a CRISPR complex to a target sequence can be evaluated by any suitable assay. For example, sufficient components of a CRISPR system to form a CRISPR complex containing a test guide sequence can be provided to a host cell having the corresponding target sequence, such as by transfection with a vector encoding the components of the CRISPR sequence, followed by evaluation of preferential cleavage within the target sequence. Similarly, cleavage of a target polynucleotide sequence can be evaluated in vitro by providing components of a CRISPR complex containing the target sequence, the test guide sequence, and a control guide sequence different from the test guide sequence, and comparing the binding or cleavage rate at the target sequence between the test guide sequence reaction and the control guide sequence reaction.

[0154] The nucleic acid sequence of the sgRNA can be selected using any of the web-based software mentioned above. Considerations for selecting a DNA-targeting RNA include the PAM sequence for the Cas nuclease (e.g., Cas9 polypeptide) to be used and a strategy for minimizing off-target modification. Tools such as CRISPR design tools can provide sequences for preparing sgRNAs, evaluating target modification efficiency, and / or evaluating cleavage at off-target sites. Another consideration for selecting the sequence of the 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), the UNAFold package (Markham et al., Methods Mol Biol, 2008, 453:3-31), and the RNAfold forming Vienna RNa Package.

[0155] The sgRNA may 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, or 200 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, 260 nucleotides, 265 nucleotides, 270 nucleotides, 275 nucleotides, 280 nucleotides, 285 nucleotides, 290 nucleotides, 295 nucleotides, 300 nucleotides, 305 nucleotides 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 500 nucleotides.In certain embodiments, the sgRNA comprises between about 20 and 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, 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.

[0156] The scaffold sequence may be from 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, It can be 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 from 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, 260 nucleotides, 265 nucleotides, 270 nucleotides, 275 nucleotides, 280 nucleotides, 285 nucleotides, 290 nucleotides, 295 nucleotides, 300 nucleotides, 305 nucleotides 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 500 nucleotides.In certain 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, 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.

[0157] The nucleotides of the sgRNA can include modifications in the ribose (e.g., sugar) group, the phosphate group, the nucleobase, or any combination thereof. In some embodiments, the modification in the ribose group includes a modification at the 2' position of the ribose.

[0158] In some embodiments, the nucleotide comprises a 2'fluoro-arabino nucleic acid, a tricyclic DNA (tc-DNA), a peptide nucleic acid, a cyclohexene nucleic acid (CeNA), a locked nucleic acid (LNA), an ethylene-bridged nucleic acid (ENA), a phosphodiamidate morpholino, or a combination thereof.

[0159] Modified nucleotides or nucleotide analogs may include sugar and / or backbone ribonucleotides (i.e., containing modifications to the phosphate-sugar backbone). For example, the phosphodiester linkages of natural or native RNA may include at least one of a nitrogen or sulfur heteroatom. In some backbone-ribonucleotides, the phosphoester group connecting adjacent ribonucleotides may be replaced by a group, such as a phosphothioate group. In some sugar-ribonucleotides, the 2' moiety is selected from H, OR, R, halo, SH, SR, H2, HR, R2, or ON, where R is C1-C6 alkyl, alkenyl, or alkynyl, and halo is F, CI, Br, or I.

[0160] In some embodiments, the nucleotide comprises a sugar modification. Non-limiting examples of sugar modifications include 2'-deoxy-2'-fluoro-oligobonucleotides (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- Examples include 2'-C-alkyl oligoribonucleotides (2'-O-methylcytidine-5'-triphosphate, 2'-methyluridine-5'-triphosphate), 2'-C-alkyl oligoribonucleotides and their isomers (2'-aracytidine-5'-triphosphate, 2'-aruridine-5'-triphosphate), azidotriphosphates (2'-azido-2'-deoxycytidine-5'-triphosphate, 2'-azido-2'-deoxyuridine-5'-triphosphate), and combinations thereof.

[0161] In some embodiments, the sgRNA comprises one or more 2'-fluoro, 2'-amino, and / or 2'-thio modifications. In some cases, the modifications are 2'-fluoro-cytidine, 2'-fluoro-uridine, 2'-fluoro-adenosine, 2'-fluoro-guanosine, 2'-amino-cytidine, 2'-amino-uridine, 2'-amino-adenosine, 2'-amino-guanosine, 2,6-diaminopurine, 4-thio-uridine, 5-amino-allyl-uridine, 5-bromo-uridine, 5-iodo-uridine, 5-methyl-cytidine, ribo-thymidine, 2-aminopurine, 2'-amino-butyryl-pyrene-uridine, 5-fluoro-cytidine, and / or 5-fluoro-uridine.

[0162] There are more than 96 naturally occurring nucleoside modifications found in mammalian RNA. See, for example, Limbach et al., Nucleic Acids Research, 22(12):2183-2196 (1994). Nucleotides and the preparation of nucleotides and nucleosides are well known in the art and are described, for example, in 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. The nucleoside can be an analog of a natural nucleoside. In some cases, the analog is dihydrouridine, methyladenosine, methylcytidine, methyluridine, methylpseudouridine, thiouridine, deoxycytidine, and deoxyuridine.

[0163] In some cases, the sgRNAs described herein comprise nucleobase ribonucleotides, i.e., ribonucleotides that contain at least one unnatural nucleobase in place 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-N6isopentenyladenosine), io6A (N6-(cis-hydroxyisopentenyl)adenosine), and cis-hydroxyisopentenyladenosine. methylthio-N6-(cis-hydroxyisopentenyl)adenosine), g6A (N6-glycinylcarbamoyl adenosine), t6A (N6-threonylcarbamoyl adenosine), ms2t6A (2-methylthio-N6-threonylcarbamoyl adenosine), m6t6A (N6-methyl-N6-threonylcarbamoyl adenosine), hn6A (N6-hydroxynorvalylcarbamoyl adenosine), ms2hn6A (2-methylthio-N6-hydroxynorvalylcarbamoyl adenosine), Ar(p) (2'-O-ribosyladenosine (phosphate)), I (inosine), mi l (1-methylinosine), m'lm (l,2'-O-dimethylinosine), m3C (3-methylcytidine), Cm (2T-o-methylcytidine), s2C (2-thiocytidine), ac4C (N4-acetylcytidine), f5C (5-phonylcytidine), m5Cm (5,2-O-dimethylcytidine), ac4Cm (N4-acetyl 2T-o-methylcytidine), k2C (lysidine), 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-ribosylguanosine (phosphate)), yW(wybutosine), o2yW(peroxywybutosine), OHyW(hydroxywybutosine), OHyW*(hydroxywybutosine), imG(wybutosine), mimG(methylguanosine), Q(queosine), oQ(epoxyqueosine), galQ(galactosyl-queosine), manQ(mannosyl-queosine), preQo(7-cyano-7-deazaguanosine), preQi(7-aminomethyl-7-deazaguanosine), azaguanosine), 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-(carbo (5-(carboxyhydroxymethyl)uridine), mchm5U (5-(carboxyhydroxymethyl)uridine methyl ester), mcm5U (5-methoxycarbonylmethyluridine), 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-methyl ncm5U (5-carbamoylmethyluridine), ncm5Um (5-carbamoylmethyl-2'-O-methyluridine), cmnm5U (5-carboxymethylaminomethyluridine), cnmm5Um (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 (N6,0-dimethyladenosine), rn62Am (N6,N6,0-2-trimethyladenosine), m2'7G (N2,7-dimethylguanosine), m2'2'7G (N2,N2,7-trimethylguanosine), m3Um (3,2T-O-dimethyluridine), m5D (5-methyldihydrouridine), f5Cm (5-formyl-2'-O-methylcytidine), mlGm (1,2'-O-dimethylguanosine), m'Am (1,2-O-dimethyladenosine), tm5s2U (S-taurinomethyl-2-thiouridine), imG-14 (4-demethylguanosine), im G2 (isoguanosine), or ac6A (N6-acetyladenosine), hypoxanthine, inosine, 8-oxo-adenine, their 7-substituted derivatives, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5-(C1-C6)-alkyluracil, 5-methyluracil, 5-(C2-C6)-alkenyluracil, 5-(C2-C6) -Alkynyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5-(C1-C6)-alkylcytosine, 5-methylcytosine, 5-(C2-C6)-alkenylcytosine, 5-(C2-C6)-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-deazapurines, 7-deaza-7-substituted purines, 7-deaza-8-substituted purines, and combinations thereof.

[0164] sgRNAs can be synthesized by any method known to those skilled in the art. In some embodiments, sgRNAs are chemically synthesized. Modified sgRNAs can be synthesized using 2'-O-thionocarbamate-protected nucleoside phosphoramidites. Methods are described, for example, in Dellinger et al., J. American Chemical Society, 133, 11540-11556 (2011); Threlfall et al., Organic & Biomolecular Chemistry, 10, 746-754 (2012); and Dellinger et al., J. American Chemical Society, 125, 940-950 (2003). Modified sgRNAs are commercially available, for example, from TriLink BioTechnologies (San Diego, CA).

[0165] 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, the disclosures of which are incorporated herein by reference in their entirety and for all purposes.

[0166] One of skill in the art will appreciate that the guide RNAs disclosed in this disclosure can be used in combination with any Cas protein known in the art (e.g., from any suitable organism or bacterial species).

[0167] The Cas protein may be a type I, II, III, IV, V, or VI Cas protein. The Cas protein may comprise one or more domains. Non-limiting examples of domains include a guide nucleic acid recognition and / or binding domain, a nuclease domain (e.g., DNase or RNase domain, RuvC, HNH), a DNA-binding domain, an RNA-binding domain, a helicase domain, a protein-protein interaction domain, and a dimerization domain. The guide nucleic acid recognition and / or binding domain may interact with the guide nucleic acid. The nuclease domain may comprise catalytic activity for nucleic acid cleavage. The nuclease domain may lack catalytic activity to prevent nucleic acid cleavage. The Cas protein may also be a chimeric Cas protein fused to another protein or polypeptide. For example, the Cas protein may be a chimera of various Cas proteins, comprising domains from different Cas proteins.

[0168] 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 Csx12), Cas10, Cas10d, Cas1O, Cas1Od, CasF, CasG, CasH, Cpf1, Csy1, Csy2, Csy3, and Cse1 (CasA). , Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx1O, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cul966, and homologs or modified versions thereof.

[0169] Cas proteins can be derived from any suitable organism. Non-limiting examples include Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Nocardiopsis dassonvillei, Streptomyces pristinae spiralis, Streptomyces viridochromo genes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Pseudomonas aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelluliruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Acaryochloris marina, Leptotrichia shahii, and Francisella novicida. In some embodiments, the organism is Streptococcus pyogenes (S. pyogenes). In some embodiments, the organism is Staphylococcus aureus (S. aureus). In some embodiments, the organism is Streptococcus thermophilus (S. thermophilus).

[0170] Cas proteins include, but are 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, Lactobacillus gasseri, Finegoldia magna, Mycoplasma mobile, Mycoplasma gallisepticum, Mycoplasma ovipneumoniae, Mycoplasma canis, Mycoplasma synoviae, Eubacterium rectale, Streptococcus thermophilus, Eubacterium dolichum, Lactobacillus coryniformis subsp.Torquens, Ilyobacter polytropus, Ruminococcus albus, Akkermansia muciniphila, Acidothermus cellulolyticus, Bifidobacterium longum, Bifidobacterium dentium, Corynebacterium diphtheria, Elusimicrobium minutum, Nitratifractor salsuginis, Sphaerochaeta globus, Fibrobacter succinogenes 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 perfringens, Parvibaculum lavamentivorans, Roseburia intestinalis, Neisseria meningitidis, Pasteurella multocida subsp.The derivatives may be derived from various bacterial species, including Multocida, Sutterella wadsworthensis, proteobacterium, Legionella pneumophila, Parasutterella excrementihominis, Wolinella succinogenes, and Francisella novicida. The term "derived" is defined in this case as modified from a naturally occurring variety of bacterial species to maintain a significant portion or significant homology to the naturally occurring variety of bacterial species. A significant portion may be at least 10 contiguous nucleotides, at least 20 contiguous nucleotides, at least 30 contiguous nucleotides, at least 40 contiguous nucleotides, at least 50 contiguous nucleotides, at least 60 contiguous nucleotides, at least 70 contiguous nucleotides, at least 80 contiguous nucleotides, at least 90 contiguous nucleotides, or at least 100 contiguous nucleotides. Significant homology may be at least 50% homologous, at least 60% homologous, at least 70% homologous, at least 80% homologous, at least 90% homologous, or at least 95% homologous. Derived species may be modified while retaining the activity of the naturally occurring variety. .

[0171] Gene editing methods 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 encompass gene editing into a population of synovial cells through nucleotide insertion (RNA or DNA) or recombinant protein insertion, both to promote expression of one or more proteins and to inhibit 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, particularly for delivering gene-editing compositions to synovial cells. There are several gene editing techniques that can be used to genetically modify joint cells, which are suitable for use with the present disclosure.

[0172] In some embodiments, the method for genetically modifying articular cells comprises the step of stable integration of a gene for the production of one or more proteins. In one embodiment, the method for genetically modifying a portion of synoviocytes of a joint comprises the step of retroviral transduction. In one embodiment, the method for genetically modifying a portion of synoviocytes of a joint comprises the step of retroviral transduction. Lentiviral transduction systems are known in the art and are described, for example, in Levine, et al., Proc. Nat'l Acad. Sci. 2006, 103, 17372-77; Zufferey, et al., Nat. Biotechnol. 1997, 15, 871-75; Dull, et al., J. Virology 1998, 72, 8463-71; and U.S. Patent No. 6,627,442, the disclosures of each of which are incorporated herein by reference. In one embodiment, the method for genetically modifying a portion of synoviocytes of a joint comprises the step of gamma retroviral transduction. Gamma-retroviral transduction systems are known in the art and are described, for example, in Cepko and Pear, Cur. Prot. Mol. Biol. 1996, 9.9.1-9.9.16, the disclosure of which is incorporated herein by reference. In one embodiment, a method for genetically modifying a portion of synovial cells of a joint includes 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, whereby long-term expression of the transposase does not occur in transgenic cells, for example, with the transposase provided as mRNA (e.g., mRNA comprising a cap and polyA tail).Suitable transposon-mediated gene transfer systems, including salmonid Tel-like transposases (SB or Sleeping Beauty transposases), such as SB10, SB11, and SB100x, as well as 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.

[0173] In some embodiments, a viral vector or system is used to introduce the gene editing system into cells comprising the joint. In some embodiments, the cells are synovial fibroblasts. In some embodiments, the viral vector is an AAV vector. In some aspects, the AAV vector comprises 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).

[0174] In some embodiments, the viral vector is a lentivirus. In one embodiment, the lentivirus is selected from the group consisting of human immunodeficiency-1 (HIV-1), human immunodeficiency-2 (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Jamburana disease virus (JDV), equine infectious anemia virus (EIAV), and caprine arthritis-encephalitis virus (CAEV).

[0175] In one embodiment, the method for genetically modifying a portion of synovial cells of a joint comprises a step of stable integration of genes for the production or inhibition (e.g., silencing) of one or more proteins, hi one embodiment, the method for genetically modifying a portion of synovial cells of a joint comprises a step of liposomal transduction. Liposome transfection methods, such as those using a 1:1 (w / w) liposome formulation of the cationic lipid 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., Proc. Natl. Acad. Sci. USA, 1987, 84, 7413-7417, as well as U.S. Pat. Nos. 5,279,833, 5,908,635, 6,056,938, 6,110,490, 6,534,484, and 7,687,070. In one embodiment, a method for genetically modifying a portion of synovial cells of a joint comprises a transfection step 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.

[0176] According to embodiments, the gene editing process may involve the use of programmable nucleases that mediate the generation of double- or single-strand breaks in one or more immune checkpoint genes. Such programmable nucleases enable precise genome editing by introducing breaks at specific genomic loci; that is, they rely on the recognition of specific DNA sequences within the genome to target the nuclease domain to this location and mediate the generation of double-strand breaks in the target sequence. The double-strand break in the DNA then triggers endogenous repair mechanisms at the break site to mediate genome editing via either non-homologous end joining (NHEJ) or homology-directed repair (HDR). Thus, repair of the break may result in the introduction of an insertion / deletion mutation that disrupts (e.g., silences, suppresses, or enhances) the target gene product.

[0177] The major classes of nucleases developed to enable site-specific genome editing include zinc finger nucleases (ZFNs), transcription activator-like nucleases (TALENs), and CRISPR-associated nucleases (e.g., CRISPR-Cas9). These nuclease systems can be broadly classified into two categories based on their DNA recognition mode. ZFNs and TALENs achieve specific DNA binding through protein-DNA interactions, whereas CRISPR systems such as Cas9 are targeted to specific DNA sequences by short RNA guide molecules that directly base pair with the target DNA and through protein-DNA interactions. See, for example, Cox et al., Nature Medicine, 2015, Vol. 21, No. 2.

[0178] Non-limiting examples of gene editing methods that can be used in accordance with the methods of the present disclosure include CRISPR, TALE, and ZFN methods, which are described in more detail below.

[0179] CRISPR method 1. A pharmaceutical composition for the treatment or prevention of a disease or condition of a joint, comprising a gene editing system, wherein the gene editing system targets at least one genetic locus associated with joint function and gene edits at least a portion of synovial cells of the joint by CRISPR technology (e.g., CRISPR-Cas9, CRISPR-Cas13a, or CRISPR / Cpf1 (also known as CRISPR-Cas12a). According to certain embodiments, the use of CRISPR technology to gene edit synovial cells of the joint silences or reduces expression of one or more immune checkpoint genes in at least a portion of synovial cells of the joint.

[0180] CRISPR stands for "Clustered Regularly Interspaced Short Palindromic Repeats." Methods using CRISPR systems for gene editing are also referred to herein as CRISPR methods. There are three types of CRISPR systems, type II, type V, and type VI, which incorporate RNA and Cas proteins and can be used in accordance with the present disclosure. Type II CRISPR (exemplified by Cas9) is one of the most well-characterized systems.

[0181] CRISPR technology was adapted from the natural defense mechanisms of bacteria and archaea (domains of single-celled microorganisms). These organisms thwart attacks by viruses and other foreign substances by using CRISPR-derived RNA and various Cas proteins, including Cas9, to shred and destroy the DNA or RNA of foreign invaders. CRISPRs are specialized regions of DNA with two distinct features: the presence of nucleotide repeats and spacers. Repeated sequences of nucleotides are distributed throughout the CRISPR region, and short segments of foreign DNA (spacers) are interspersed among the repeats. In type II CRISPR-Cas systems, the spacers are integrated into the CRISPR genomic locus, transcribed, and processed into short CRISPR RNAs (crRNAs). These crRNAs anneal to trans-activating crRNAs (tracrRNAs), which direct the Cas proteins to perform sequence-specific cleavage and silencing of pathogenic DNA. Target recognition by the Cas9 protein requires a "seed" sequence within the crRNA and a conserved dinucleotide-containing protospacer adjacent motif (PAM) sequence upstream of the crRNA-binding region. The CRISPR-Cas system can thereby be retargeted to cleave virtually any DNA sequence by redesigning the crRNA. The crRNA and tracrRNA in natural systems can be simplified to approximately 100-nucleotide single guide RNAs (sgRNAs) for use in genetic engineering. The CRISPR-Cas system can be directly transplanted into human cells by co-delivery of a plasmid expressing the Cas9 endonuclease and the necessary crRNA and tracrRNA (or sgRNA) components. Different variants of the Cas protein can be used to reduce targeting limitations (e.g., orthologs of Cas9, such as Cpf1).

[0182] CRSIPR-Cas-mediated homologous recombination A CRISPR-Cas system for homologous recombination (HR) includes a Cas nuclease (e.g., Cas9 nuclease) or a variant or fragment thereof, a DNA-targeting RNA (e.g., a single guide RNA (sgRNA)) containing a guide sequence that targets the Cas nuclease to target genomic DNA and a scaffold sequence that interacts with the Cas nuclease, and a donor template. The CRISPR-Cas system generates a double-strand break at a desired target locus in a cell's genome and can be used to repair the break induced by homology-directed repair (HDR) using the cell's endogenous machinery.

[0183] CRISPR-Cas9 nuclease can facilitate the locus-specific chromosomal integration of exogenous DNA delivered by AAV vector. Typically, the size of exogenous DNA (such as transgene, expression cassette, etc.) that can be integrated is limited by the DNA packaging capacity of AAV vector, which is about 4.0 kb. Including the two homologous arms required for homologous recombination, a single AAV vector can only deliver exogenous DNA of less than about 3.7 kb. The method described herein allows the delivery of exogenous DNA of 4 kb or more by dividing the nucleotide sequence between two different AAV vectors. The donor template is designed for successive homologous recombination events, which can integrate and fuse two parts of the nucleotide sequence.

[0184] The homologous recombination of the present disclosure can be carried out using engineered nuclease systems for genome editing, such as but not limited to CRISPR-Cas nuclease, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), engineered meganuclease.In one embodiment, CRISPR-Cas based nuclease system is used.The detailed description of useful nuclease systems can be found, for example, in Gaj et al., Trends Biotechnol, 2013, Jul:31(7):397-405.

[0185] Any suitable CRISPR / Cas system can be used in the methods and compositions disclosed herein. CRISPR / Cas systems can be referred to using various nomenclature systems. Exemplary nomenclature systems are provided in Makarova, K. Set 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. A CRISPR / Cas system can be a Type I, Type II, Type III, Type IV, Type V, Type VI, or any other suitable CRISPR / Cas system. As used herein, a CRISPR / Cas system can be a Class 1, Class 2, or any other suitable classified CRISPR / Cas system. Class 1 CRISPR / Cas systems can use a complex of multiple Cas proteins to produce a regulatory effect. Class 1 CRISPR / Cas systems can 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 provide a regulatory effect. Class 2 CRISPR / Cas systems can include, for example, Type II (e.g., II, IIA, IIB) and Type V CRISPR / Cas types. CRISPR systems can complement each other and / or provide functional units in trans to facilitate targeting of the CRISPR locus.

[0186] In some embodiments, the nucleotide sequence encoding the Cas nuclease is present in a recombinant expression vector. In certain cases, the recombinant expression vector is a viral construct, such as a recombinant adeno-associated virus construct, a recombinant adenovirus construct, or a recombinant lentivirus construct. For example, viral vectors 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, avian leukosis virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus. Useful expression vectors are known to those of skill in the art, and many are commercially available. The following vectors are provided as examples for eukaryotic host cells: pXT1, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40. However, any other vector may be used provided it is compatible with the host cell. For example, useful expression vectors containing nucleotide sequences encoding the Cas9 enzyme are commercially available from, for example, Addgene, Life Technologies, Sigma-Aldrich, and Origene.

[0187] Host cells are necessary for producing infectious AAV vectors and for producing AAV virions based on the disclosed AAV vectors. A variety of host cells are known in the art and find use in the methods of the present disclosure. Any host cell described herein or known in the art can be used with the compositions and methods described herein.

[0188] In some embodiments, host cells for use in 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, for example, mammalian cells, including, for example, mouse cells, and primate cells (e.g., human cells). Particularly desirable host cells include, but are not limited to, cells from any mammalian species, including A549, WEHI, 3T3, 10T1 / 2, BHK, MDCK, COS 1, 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 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 from mammals, including humans, monkeys, mice, rats, rabbits, and hamsters. The cells used must be capable of infection or transfection with an AAV vector. In some embodiments, the host cell is one that has rep and has cap stably transfected within the cell.

[0189] In some embodiments, preparation of host cells according to the present disclosure involves techniques such as assembly of selected DNA sequences. This assembly can be accomplished using conventional techniques. Such techniques are well known and include cDNA and genomic cloning as described in Sambrook et al., cited above, the use of overlapping oligonucleotide sequences of adenovirus and AAV genomes in combination with the polymerase chain reaction, synthetic methods, and any other suitable method for providing a desired nucleotide sequence.

[0190] In addition to the AAV vector, the host cell may contain sequences that drive expression of AAV capsid polypeptides (in the host cell, and rep (replication) sequences of the same serotype as, or a cross-complementary serotype of, the AAV inverted terminal repeats (ITRs) found in the AAV vector. The AAV capsid and rep (replication) sequences may be obtained independently from an AAV source and introduced into the host cell in any manner known to those of skill in the art or as described herein. Additionally, when pseudotyping an AAV vector in an AAV8 capsid, for example, the sequences encoding each of the essential rep (replication) proteins may be provided by AAV8, or the sequences encoding the rep (replication) proteins may be provided by a different AAV serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and / or AAV9).

[0191] 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 the necessary sequences to direct 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 necessary for packaging the AAV, such as a rep (replication) sequence.

[0192] 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 the sequences necessary to induce 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 necessary for packaging the AAV vector, such as the rep (replication) sequence.

[0193] In some embodiments, the rep (replication) sequence and the capsid sequence are transfected into the host cell on a single nucleic acid molecule, which may stably exist as a non-integrated episome within the cell. In another embodiment, the rep (replication) and capsid sequences are stably integrated into the chromosome of the cell. Another embodiment has the rep (replication) sequence and the capsid sequence transiently expressed in the host cell. For example, a nucleic acid molecule useful for such transfection includes, in the 5' to 3' direction, a promoter, an optional spacer interposed between the promoter and the start site of the rep (replication) gene sequence, an AAV rep (replication) gene sequence, and an AAV capsid gene sequence.

[0194] While the molecules providing rep (replication) and capsid can be present in the host cell transiently (i.e., via transfection), in some embodiments, one or both of the rep (replication) and capsid proteins and the promoters controlling their expression are stably expressed in the host cell, e.g., as an episome or by integration into the host cell chromosome. Methods used to construct embodiments of the present disclosure are conventional genetic or recombinant engineering techniques, such as those described in the references above.

[0195] Various methods for producing AAV virions are known in the art and can be used to produce AAV virions containing the AAV vectors described herein. Generally, these involve inserting or transducing an AAV vector of the present disclosure into a host cell capable of packaging the AAV vector into an AAV virion. Exemplary methods are described and referenced below, but any method known to those skilled in the art can be used to produce the AAV virions of the present disclosure.

[0196] AAV vectors containing heterologous nucleic acids (e.g., donor templates) and used to generate AAV virions can be constructed using methods well known in the art. See, for example, Koerber et al. (2009) Mol. Ther., 17:2088; Koerber et al. (2008) Mol. Ther., 16:1703-1709, and U.S. Patent 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 portions of the AAV genome can also be deleted, as long as sufficient portions of the ITRs remain to enable replication and packaging functions. Such constructs can be designed using techniques well known in the art. See, for example, U.S. Pat. Nos. 5,173,414 and 5,139,941, WO 92 / 01070 (published January 23, 1992) and WO 93 / 03769 (published March 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 Microbiol. Immunol. 158:97-129, Kotin, RM (1994) Human Gene Therapy 5:793-801, Shelling and See Smith (1994) Gene Therapy 1:165-169, and Zhou et al. (1994) J. Exp. Med. 179:1867-1875.

[0197] To produce AAV virions, the AAV vector is introduced into a suitable host cell using known techniques, such as by transfection. Several 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 a high-speed microprojectile (Klein et al. (1987) Nature 327:70-73).

[0198] Depending on the expression system used, any of several transcriptional and translational control elements, including promoters, transcriptional enhancers, transcriptional terminators, etc., can be used in the expression vector. Useful promoters can be derived from viruses or any organism, e.g., prokaryotic or eukaryotic. Suitable promoters include, but are not limited to, the SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter, adenovirus major late promoter (Ad MLP), herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoter (such as the 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).

[0199] In some embodiments, polynucleotides encoding Cas nucleases may be used in the present disclosure. Such polynucleotides (e.g., mRNA) may be obtained commercially, for example, from TriLink BioTechnologies, GE Dharmacon, ThermoFisher, etc.

[0200] 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, for example, in Hendel et al., Nat Biotechnol, 2015, 33(9):985-989 and Dever et al., Nature, 2016, 539:384-389, the disclosures of which are incorporated herein by reference in their entirety and for all purposes.

[0201] In some embodiments, a Cas nuclease (e.g., a Cas9 polypeptide) is complexed with an sgRNA to form a Cas ribonucleoprotein (e.g., a Cas9 ribonucleoprotein). The molar ratio of Cas nuclease to sgRNA can be any range that facilitates sequential homologous recombination of the targeting AAV vector and the target locus. In some embodiments, the molar ratio of Cas9 polypeptide to sgRNA is about 1:5, 1:4, 1:3, 1:2.5, 1:2, or 1:1. In other embodiments, the molar ratio of Cas9 polypeptide to sgRNA is about 1:2 to about 1:3. In certain embodiments, the molar ratio of Cas9 polypeptide to sgRNA is about 1:2.5.

[0202] 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 a subject) as Cas polypeptides or variants or fragments thereof, mRNA encoding Cas polypeptides or variants or fragments thereof, recombinant expression vectors comprising a nucleotide sequence encoding Cas polypeptides or variants or fragments thereof, or Cas ribonucleoproteins. One skilled in the art will recognize that any method of delivering exogenous polynucleotides, polypeptides, or ribonucleoproteins can be used. Non-limiting examples of such methods include electroporation, nucleofection, transfection, lipofection, transduction, microinjection, electroinjection, electrofusion, nanoparticle bombardment, transformation, conjugation, etc.

[0203] 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, e.g., Xu, C. F., et al. (2021). Advanced Drug Delivery Reviews, 168, 3-29; Buschmann et al. (2021). Vaccines 9:65; Kenjo, E., et al. (2021). Nature Communications, 12(1), 7101.

[0204] In some embodiments, the CRISPR components are delivered by nanoparticles. Without wishing to be bound by any particular theory, in certain embodiments, the nucleic acid, when present in the nanoparticles, is resistant to degradation by nucleases in aqueous solution. Lipid nanoparticles containing nucleic acids and methods for their preparation are described 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 / 0 46809, WO2019 / 089828, WO2020 / 061284, WO2020 / 061317, WO2020 / 081938, WO2020 / 097511, WO2020 / 097520, WO2020 / 0975 40, WO2020 / 097548, WO2020 / 214946, WO2020 / 219941, WO2020 / 232276, WO2020 / 227615, WO2020 / 061295, WO2021 / 007278, W O2021 / 016430, WO2021 / 021988, EP 2972360, US2020 / 0155691, US2020 / 0237671, U.S. Patent Nos. 8,058,069, 8,492,359, 8,822,668, 9,364,435, 9,404,127, 9,504,651, 9,593,077, 9,738,593, 9,868,691, and 9,868,692 Nos. 9,950,068, 10,138,213, 10,166,298, 10,221,127, 10,238,754, 10,266,485, 10,383,952, 10,730,924, 10,766,852, 11,141,378, and 11,246,933, which are incorporated herein by reference in their entirety for all purposes.

[0205] Lipid Nanoparticle Composition In some embodiments, the largest dimension of the nanoparticle composition is 1 micrometer or less (e.g., 1 micrometer, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, or less), as measured, for example, by dynamic light scattering (DLS), transmission electron microscopy, scanning electron microscopy, or another method. Nanoparticle compositions include, for example, lipid nanoparticles (LNPs), liposomes, lipid vesicles, and lipoplexes. In some embodiments, the nanoparticle composition is a vesicle comprising one or more lipid bilayers. In certain embodiments, the nanoparticle composition comprises two or more concentric bilayers separated by an aqueous compartment. The lipid bilayers may be functionalized and / or crosslinked to each other. The lipid bilayer may comprise one or more ligands, proteins, or channels.

[0206] In various embodiments, the lipid nanoparticles described herein have a 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 m, 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.

[0207] In certain embodiments, the lipid nanoparticles described herein comprise one or more components, including a lipid component and (optionally) a structural component. The lipid component comprises a lipid selected from ionic and / or cationic lipids (i.e., lipids that may have a positive or partial 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 comprises a single ionic lipid. In other embodiments, the lipid component comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 ionic lipids. In some embodiments, the lipid component comprises a single neutral lipid. In other embodiments, the lipid component comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 neutral lipids. In some embodiments, the lipid component comprises a single polymer-conjugated lipid. In other embodiments, the lipid component comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 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 foregoing components.

[0208] Ionic / cationic lipids In some embodiments, the lipid component comprises an ionic lipid. In some embodiments, the ionic lipid is anionic. In other embodiments, the ionic lipid is cationic. In some embodiments, the lipid component is selected from the group consisting of, but not limited to, 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylamine (KL26), 1,2-dilinoleyloxy-N,N-dimethylamine (KL27), 1,2-dilinoleyloxy-N,N-dimethylamine (KL28), 1,2-dilinoleyloxy-N,N-dimethylamine (KL29), 1,2-dilinoleyloxy-N,N-dimethylamine (KL30), 1,2-dilinoleyloxy-N,N-dimethylamine (KL31), 1,2-dilinoleyloxy-N,N-dimethylamine (KL32), 1,2-dilinoleyloxy-N,N-dimethylamine (KL33), 1,2-dilinoleyloxy-N,N-dimethylamine (KL34), 1,2-dilinoleyloxy-N,N-dimethylamine (KL35), 1,2-dilinoleyloxy-N,N-dimethylamine (KL36), 1,2-dilinoleyloxy-N,N-dimethylamine (KL37), 1,2-dilinoleyloxy-N,N-dimethylamine (KL38), 1,2-dilinoleyloxy-N,N-dimethylamine (KL39), 1,2-dilin DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane 2-({8-[(3.beta.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), (2R)- ...

[0023] The cationic lipid comprises a cationic lipid selected from the group consisting of (2S)-2-({8-[(3.beta)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z-,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA(2R)), (2S)-2-({8-[(3.beta)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z-,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA(2S)), lipids containing a cyclic amine group, and mixtures thereof.

[0209] Non-exhaustive and non-limiting examples of cationic lipids include: [ka] [ka] [ka] [ka] [ka]

[0210] Neutral lipids / phospholipids In some embodiments, the lipid component may be, but is not limited to, 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-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 Neutral lipids include 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin (SM), and mixtures thereof.

[0211] Polymer-conjugated lipids In some embodiments, the lipid component further comprises a 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 may be a PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DMA, or PEG-DSPE lipid.

[0212] Non-exhaustive and non-limiting examples of PEG-lipids include: [ka] [ka]

[0213] Structured Lipids / Sterols In some embodiments, the 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 a sterol, including, but not limited to, a sterol selected from the group consisting of cholesterol, fecosterol, stigmasterol, stigmastanol, sitosterol, β-sitosterol, lupeol, betulin, ursolic acid, oleanolic acid, campesterol, fucosterol, brassicasterol, ergosterol, 9,11-dehydroergosterol, tomatidine, tomatine, α-tocopherol, and mixtures thereof. In other embodiments, the structural lipid comprises cholesterol and a corticosteroid (e.g., prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof.

[0214] Non-exhaustive and non-limiting examples of structured lipids include: [ka]

[0215] formulation Nanoparticle compositions can include a lipid component and one or more additional components, such as a therapeutic and / or prophylactic agent. Nanoparticle compositions can be designed for one or more specific applications or targets. The components of the nanoparticle composition can be selected based on the specific application or target and / or based on the efficacy, toxicity, cost, ease of use, availability, or other characteristics of one or more components. Similarly, a particular formulation of a nanoparticle composition can be selected for a particular application or target, for example, according to the efficacy and toxicity of a particular combination of components.

[0216] The lipid component of the nanoparticle composition can include, for example, cationic lipids, phospholipids (unsaturated lipids such as DOPE or DSPC), PEG lipids, and structured lipids. Elements of the lipid component may be provided in specific fractions.

[0217] In some embodiments, the lipid component of the nanoparticle composition comprises an ionic lipid, a phospholipid, a PEG lipid, and a structured lipid. In certain embodiments, the lipid component of the nanoparticle composition comprises about 30 mol% to about 60 mol% of an ionic lipid, about 0 mol% to about 30 mol% of a phospholipid, about 0 mol% to about 10 mol% of a PEG lipid, and about 17.5 mol% to about 50 mol% of a structured lipid, provided that the total mol% does not exceed 100%. In some embodiments, the lipid component of the nanoparticle composition comprises about 35 mol% to about 55 mol% of an ionic lipid compound, about 5 mol% to about 25 mol% of a phospholipid, about 0 mol% to about 10 mol% of a PEG lipid, and about 30 mol% to about 40 mol% of a structured lipid. In certain embodiments, the lipid component comprises about 50 mol% of the compound, about 10 mol% of a phospholipid, about 38.5 mol% of a structured lipid, and about 1.5 mol% of a PEG lipid. In another embodiment, the lipid component comprises about 40 mol% of the compound, about 20 mol% of a phospholipid, about 38.5 mol% of a structural lipid, and about 1.5 mol% of a PEG lipid. In some embodiments, the phospholipid may be DOPE or DSPC. In other embodiments, the PEG lipid may be PEG-DMG and / or the structural lipid may be cholesterol.

[0218] In some embodiments, the ionic lipid comprises about 20 to about 60 mol% of the lipid component. In other embodiments, the ionic lipid comprises about 35 to about 55 mol% of the lipid component. In various embodiments, the ionic lipid comprises about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, or 60 mol% of the lipid component.

[0219] In some embodiments, the neutral lipids comprise about 0 to about 30 mol% of the lipid component. In other embodiments, the neutral lipids comprise about 5 to about 25 mol% of the lipid component. In various embodiments, the neutral lipids comprise 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.

[0220] In some embodiments, the polymer-conjugated lipid comprises about 0 to about 15 mol% of the lipid component. In other embodiments, the polymer-conjugated lipid comprises about 0.5 to about 10 mol% of the lipid component. In various embodiments, the polymer-conjugated lipid comprises 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, 9.5, 10, or 15 mol% of the lipid component.

[0221] In some embodiments, the structural component comprises about 17.5 mol% to about 50 mol% lipid components. In other embodiments, the structural component comprises about 30 mol% to about 40 mol% lipid components. In various embodiments, the structural component comprises 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 50 mol% lipid components.

[0222] Alternatively, the structural component can be expressed as a ratio to the lipid component. In some embodiments, the structural component is in a ratio of about 1:1 with the lipid component (sterol:lipid). In other embodiments, the structural component is in a ratio of about 1:5 with the lipid component (sterol:lipid). In various embodiments, the structural component is in a ratio of 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 with the lipid component (sterol:lipid).

[0223] Nanoparticle compositions can be designed for one or more specific applications or targets. For example, nanoparticle compositions can be designed to deliver therapeutic and / or prophylactic agents, such as RNA, to specific cells, tissues, organs, or systems, or groups thereof, within a mammalian body. The physiochemical properties of the nanoparticle composition can be modified to increase selectivity for specific bodily targets. For example, particle size can be adjusted based on the fenestration size of different organs. The therapeutic and / or prophylactic agents included in the nanoparticle composition can also be selected based on the desired delivery target. For example, therapeutic and / or prophylactic agents can be selected for a specific indication, condition, disease, or disorder and / or for delivery (e.g., localized or specific delivery) to specific cells, tissues, organs, or systems, or groups thereof. In certain embodiments, nanoparticle compositions can contain mRNA encoding a polypeptide of interest that can be translated intracellularly to produce the polypeptide of interest. Such compositions can be designed for specific delivery to a specific organ. In some embodiments, compositions can be designed for specific delivery to mammalian joints.

[0224] The amount of therapeutic and / or prophylactic agent in a nanoparticle composition can depend on the size, composition, desired target and / or use, or other characteristics of the nanoparticle composition, as well as the properties of the therapeutic and / or prophylactic agent. For example, the amount of RNA useful in a nanoparticle composition can depend on the size, sequence, and other characteristics of the RNA. The relative amounts of therapeutic and / or prophylactic agent and other components (e.g., lipids) in the nanoparticle composition can also vary. In some embodiments, the weight / weight ratio of lipid component to therapeutic and / or prophylactic agent in the nanoparticle composition can be from about 5:1 to about 60:1, e.g., 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 weight / weight ratio of lipid component to therapeutic and / or prophylactic agent can be from about 10:1 to about 40:1. In certain embodiments, the weight / weight ratio is about 20:1. The amount of therapeutic and / or prophylactic agent in the nanoparticle composition can be measured, for example, using absorption spectroscopy (e.g., UV-visible spectroscopy).

[0225] In some embodiments, the therapeutic and / or prophylactic 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-stranded RNA (sgRNA), short interfering RNA (siRNA), antisense oligonucleotides (ASO), 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.

[0226] In some embodiments, the nanoparticle composition comprises one or more RNAs, and the one or more RNAs, lipids, and amounts thereof can be selected to provide a particular N:P ratio. The N:P ratio of a composition refers to the molar ratio of nitrogen atoms in the one or more lipids to the number of phosphate groups in the RNA. Generally, lower N:P ratios are preferred. The one or more RNAs, lipids, and amounts thereof can be selected to provide an N:P ratio of about 2:1 to about 30:1, e.g., 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.

[0227] In some embodiments, the nucleic acid component is composed of a modified nucleic acid. For example, the RNA may be modified RNA. That is, the RNA may contain one or more non-naturally occurring nucleic acid bases, nucleosides, nucleotides, or linkers. "Modified" species may also be referred to herein as "altered" species. Species may be chemically, structurally, or functionally modified or altered. For example, modified nucleic acid base species may contain one or more non-naturally occurring substitutions.

[0228] In certain embodiments, the present disclosure includes a method for treating a joint disease or disorder. In other embodiments, the present disclosure includes a method for treating osteoarthritis. In some embodiments, the present disclosure includes a method for treating joint inflammation, the method comprising administering a therapeutically effective amount of a CRISPR-Cas composition encapsulated within or associated with a lipid nanoparticle (LNP), the composition comprising one or more non-naturally occurring polynucleotides encoding a Cas protein and at least one sgRNA. In some embodiments, the LNP is administered intra-articularly.

[0229] In certain embodiments, the present disclosure includes methods for treating fibrosis or scarring. In some embodiments, the fibrosis and / or scarring is post-operative and / or post-surgical fibrosis and / or scarring. In some embodiments, the fibrosis and / or scarring is post-ligament reconstruction. In some embodiments, the fibrosis and / or scarring is post-anterior cruciate ligament (ACL) reconstruction. In some embodiments, the fibrosis and / or scarring is post-autograft anterior cruciate ligament (ACL) reconstruction. In some embodiments, the fibrosis and / or scarring is post-allograft anterior cruciate ligament (ACL) reconstruction. In some embodiments, the fibrosis and / or scarring is due to arthrofibrosis of the knee. In some embodiments, the fibrosis and / or scarring is due to intra-articular fibrous nodules. In some embodiments, the fibrosis and / or scarring is post-total knee arthroplasty (TKA). In some embodiments, the fibrosis and / or scarring is due to arthrofibrosis of the knee after TKA. In some embodiments, the fibrosis and / or scarring is post-discectomy. In some embodiments, the fibrosis and / or scarring is due to epidural fibrosis post-discectomy. 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 within or associated with a lipid nanoparticle (LNP), the composition comprising one or more non-naturally occurring polynucleotides encoding a Cas protein and at least one sgRNA. In some embodiments, the LNP is administered locally. In other embodiments, the LNP is administered intra-articularly. In some embodiments, the pharmaceutical composition is administered during and / or after surgery.

[0230] In certain embodiments, the present disclosure includes a method for treating lower back pain. In other embodiments, the present disclosure includes a method for treating a pathogenic disorder. In some embodiments, the present disclosure includes a method for treating local nociception, inflammation, or morphological changes associated with a back or spinal condition or disorder in a subject in need thereof, the method comprising administering a therapeutically effective amount of a CRISPR-Cas composition encapsulated within or associated with a lipid nanoparticle (LNP), the composition comprising one or more non-naturally occurring polynucleotides encoding a Cas protein and at least one sgRNA. In some embodiments, the LNP is administered intrathecally. In other embodiments, the LNP is administered epidurally.

[0231] As used herein, a "lipid component" is a component of a nanoparticle composition that includes one or more lipids. For example, the lipid component can include one or more cationic / ionic, PEGylated, structural, or other lipids, such as phospholipids.

[0232] 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 can involve administering a nanoparticle composition comprising the therapeutic and / or prophylactic agent to the subject (e.g., via intravenous, intramuscular, intradermal, subcutaneous, intraarticular, or intradiscal routes). Administering a nanoparticle composition to a mammal or mammalian cells can involve contacting one or more cells with the nanoparticle composition.

[0233] As used herein, "native" means occurring in nature without artificial assistance.

[0234] As used herein, "PEG lipid" or "PEGylated lipid" refers to a lipid that includes a polyethylene glycol moiety. These lipids may also be referred to as PEG-modified lipids.

[0235] As used herein, a "phospholipid" is a lipid that includes a phosphate moiety and one or more carbon chains, such as an unsaturated fatty acid chain. A phospholipid may include one or more multiple (e.g., double or triple) bonds (e.g., one or more unsaturations). Certain phospholipids can promote fusion with membranes. For example, cationic phospholipids can interact with one or more negatively charged phospholipids in a membrane (e.g., a cell membrane or an intracellular membrane). The fusion of a phospholipid with a membrane can allow one or more components of a lipid-containing composition to pass through the membrane, for example, allowing one or more components to be delivered to a cell.

[0236] physical properties The properties of a nanoparticle composition may depend on its components. For example, a nanoparticle composition containing cholesterol as a structural lipid may have different properties from a nanoparticle composition containing a different structural lipid. Similarly, the properties of a nanoparticle composition may depend on the absolute or relative amounts of its components. For example, a nanoparticle composition containing a higher molar fraction of phospholipids may have different properties from a nanoparticle composition containing a lower molar fraction of phospholipids. The characteristics may also vary depending on the method and conditions of preparation of the nanoparticle composition.

[0237] Nanoparticle compositions can be characterized by various methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of nanoparticle compositions. Dynamic light scattering or potentiometry (e.g., potentiometric titration) can be used to measure zeta potential. Dynamic light scattering can also be used to determine particle size. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can also be used to measure several properties of nanoparticle compositions, such as particle size, polydispersity index, and zeta potential.

[0238] The average size of the nanoparticle composition can be from 10 nm to 1 micrometer, as measured, for example, by dynamic light scattering (DLS). For example, the average size can be from about 40 nm to about 150 nm, such as about 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. In some embodiments, the average size of the nanoparticle composition can be about 50 nm to about 100 nm, about 50 nm to about 90 nm, about 50 nm to about 80 nm, about 50 nm to about 70 nm, about 50 nm to about 60 nm, about 60 nm to about 100 nm, about 60 nm to about 90 nm, about 60 nm to about 80 nm, about 60 nm to about 70 nm, about 70 nm to about 100 nm, about 70 nm to about 90 nm, about 70 nm to about 80 nm, about 80 nm to about 100 nm, about 80 nm to about 90 nm, or about 90 nm to about 100 nm. In certain embodiments, the average size of the nanoparticle composition can be about 70 nm to about 100 nm. In certain embodiments, the average size can be about 80 nm. In other embodiments, the average size can be about 100 nm.

[0239] The nanoparticle composition can be relatively homogeneous. The polydispersity index can be used to indicate the homogeneity of the nanoparticle composition, e.g., the particle size distribution of the nanoparticle composition. A small polydispersity index (e.g., less than 0.3) generally indicates a narrow particle size distribution. The nanoparticle composition can have a polydispersity index of about 0 to about 0.25, e.g., 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.

[0240] 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 a relatively low charge, positive or negative, are generally desirable because more highly charged species can interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of the nanoparticle composition can be about -10 mV to about +20 mV, about -10 mV to about +15 mV, about -10 mV to about +10 mV, about -10 mV to about +5 mV, about -10 mV to about 0 mV, about -10 mV to about -5 mV, about -5 mV to about +20 mV, about -5 mV to about +15 mV, about -5 mV to about +10 mV, about -5 mV to about +5 mV, about -5 mV to about 0 mV, about 0 mV to about +20 mV, about 0 mV to about +15 mV, about 0 mV to about +10 mV, about 0 mV to about +5 mV, about +5 mV to about +20 mV, about +5 mV to about +15 mV, or about +5 mV to about +10 mV.

[0241] The efficiency of encapsulation of a therapeutic and / or prophylactic agent describes the amount of therapeutic and / or prophylactic agent encapsulated in or otherwise associated with a nanoparticle composition after preparation, relative to the initial amount provided. A high encapsulation efficiency is desirable (e.g., approaching 100%). The encapsulation efficiency can be measured, for example, by comparing the amount of therapeutic and / or prophylactic agent in a solution containing the nanoparticle composition before and after disintegrating the nanoparticle composition and one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free therapeutic and / or prophylactic agent (e.g., RNA) in solution. For the nanoparticle compositions described herein, the encapsulation efficiency of a 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%.

[0242] The nanoparticle composition may optionally include one or more coatings. For example, the nanoparticle composition may be formulated into a capsule, film, or tablet with a coating. The capsule, film, or tablet containing the composition described herein may have any useful size, tensile strength, hardness, or density.

[0243] Non-limiting examples of genes that can be silenced or inhibited by permanently gene editing synovial cells via CRISPR technology include IL-1α, IL-1β, IL-4, IL-9, IL-10, IL-13, and TNF-α.

[0244] Non-limiting examples of genes that can be enhanced by permanently gene editing synovial cells via CRISPR include IL-1α, IL-1β, IL-4, IL-9, IL-10, IL-13, and TNF-α.

[0245] Examples of systems, methods and compositions for modifying the expression of target gene sequences by CRISPR method and that can be used according to embodiments of the present disclosure are described in United States Patent 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 implementing CRISPR method, such as the plasmids for expressing CRISPR-Cas9 and CRISPR-Cpf1, are commercially available from companies such as GenScript.

[0246] In one embodiment, the genetic modification of at least a portion of the synovial cells of a joint described herein may be performed using the CRISPR-Cpf1 system described in U.S. Patent No. 9,790,490, the disclosure of which is incorporated herein by reference. In one embodiment, the genetic modification of at least a portion of the synovial cells of a joint described herein may be performed using a CRISPR-Cas system, including the single vector system described in U.S. Patent No. 9,907,863, the disclosure of which is incorporated herein by reference.

[0247] A pharmaceutical composition for treating or preventing a joint disease or condition, comprising a gene editing system, wherein the gene editing system targets at least one genetic locus associated with joint function, and the method further comprises gene editing at least a portion of joint synovial cells by the TALE method. According to certain embodiments, the TALE method is used to target at least one genetic locus associated with joint function, whereby at least a portion of joint synovial cells are gene-edited. Alternatively, the TALE method is used during targeting at least one genetic locus associated with joint function, whereby at least a portion of joint synovial cells are gene-edited to enhance expression of at least one genetic locus associated with joint function genes in at least a portion of joint synovial cells.

[0248] TALE stands for "Transcription Activator-Like Effector" protein, including TALENs ("Transcription Activator-Like Effector Nucleases"). Methods using the TALE system for gene editing may also be referred to herein as the TALE method. TALEs are naturally occurring proteins derived from plant pathogenic bacteria of the genus Xanthomonas. They contain a DNA-binding domain composed of a series of 33-35 amino acid repeat domains, each of which recognizes a single base pair. TALE specificity is determined by two hypervariable amino acids known as repeat variable dinucleotides (RVDs). Modular TALE repeats are linked together to recognize consecutive DNA sequences. Specific RVDs within the DNA-binding domain recognize bases within the target locus, providing the structural features that assemble a predictable DNA-binding domain. The DNA-binding domain of a TALE is fused to the catalytic domain of a type IIS FokI endonuclease to create a targetable TALE nuclease. To induce site-specific mutagenesis, two individual TALEN arms, separated by a 14-20 base pair spacer region, bring the FokI monomers into close proximity, allowing them to dimerize and generate the target double-strand break.

[0249] Several large-scale systematic studies utilizing various assembly methods have shown that TALE repeats can be combined to recognize virtually any user-defined sequence. Custom-designed TALE arrays are also commercially available through 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. 2011 / 0201118A1, 2013 / 0117869A1, 2013 / 0315884A1, 2015 / 0203871A1, and 2016 / 0120906A1, the disclosures of which are incorporated herein by reference.

[0250] Non-limiting examples of genes that can be silenced or inhibited by permanently gene editing synovial cells via the TALE method include IL-1α, IL-1β, IL-4, IL-9, IL-10, IL-13, and TNF-α.

[0251] Non-limiting examples of genes that can be enhanced by permanently gene editing synovial cells via the TALE method include IL-1α, IL-1β, IL-4, IL-9, IL-10, IL-13, and TNF-α.

[0252] Examples of systems, methods, and compositions for modifying expression of target gene sequences by the TALE method and that may be used in accordance with embodiments of the present disclosure are described in U.S. Patent No. 8,586,526, which is incorporated herein by reference.

[0253] Zinc finger method A pharmaceutical composition for treating or preventing a joint disease or condition, comprising a gene editing system, wherein the gene editing system targets at least one genetic locus associated with joint function, and the method further comprises gene editing at least a portion of joint synovial cells using zinc finger or zinc finger nuclease technology. According to certain embodiments, the method comprises using zinc finger technology to target at least one genetic locus associated with joint function, whereby at least a portion of joint synovial cells are gene-edited. Alternatively, the method comprises using zinc finger technology while targeting at least one genetic locus associated with joint function, whereby at least a portion of joint synovial cells are gene-edited to enhance expression of at least one genetic locus associated with joint function genes in at least a portion of the joint synovial cells.

[0254] Each zinc finger contains approximately 30 amino acids in a conserved ββα configuration. Several amino acids on the surface of the α-helix typically contact 3 bp of the major groove of DNA with varying levels of selectivity. Zinc fingers have two protein domains. The first domain is a DNA-binding domain that contains zinc fingers and is found in eukaryotic transcription factors. The second domain is a nuclease domain that contains the FokI restriction enzyme and is responsible for catalytic cleavage of DNA.

[0255] The DNA-binding domain of an individual ZFN typically contains three to six individual zinc finger repeats, each capable of recognizing 9 to 18 base pairs. If the zinc finger domains are specific for their intended target sites, a pair of three-finger ZFNs recognizing a total of 18 base pairs could theoretically target a single locus in a mammalian genome. One method for generating new zinc finger arrays is to combine smaller zinc finger "modules" of known specificity. The most common modular assembly process involves combining three distinct zinc fingers, each capable of recognizing a 3-base pair DNA sequence, to generate a three-finger array capable of recognizing a 9-base pair target site. Alternatively, selection-based approaches such as oligomerization pool engineering (OPEN) can be used to select new zinc finger arrays from randomized libraries that consider context-dependent interactions between adjacent fingers. Engineered zinc fingers are commercially available, and Sangamo Biosciences (Richmond, CA, USA) has partnered with Sigma-Aldrich (St. Louis, MO, USA) to develop a unique platform (CompoZr®) for zinc finger construction.

[0256] Non-limiting examples of genes that can be silenced or inhibited by permanently gene editing synovial cells via zinc finger technology 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, ASC (apoptosis-associated speck-like protein containing CARD), caspase-1, and combinations thereof.

[0257] Non-limiting examples of genes that may be enhanced by permanently gene editing synovial cells via zinc finger technology 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.

[0258] Examples of systems, methods, and compositions for modifying expression of target gene sequences by zinc finger technology and that may be used in accordance with embodiments of the present disclosure are described in U.S. Patent Nos. 6,534,261, 6,607,882, 6,746,838, 6,794,136, 6,824,978, 6,866,997, 6,933,113, Nos. 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.

[0259] In some embodiments, the cells may be gene-edited ex vivo, where the gene editing targets one or more anti-inflammatory cytokine loci. In some embodiments, the cells are synovial cells. In some embodiments, the cells are mesenchymal stem cells. In some embodiments, the cells are macrophages. In some embodiments, the present disclosure provides a pharmaceutical composition for treating or preventing a joint disease or condition, comprising a population of gene-edited cells, where the gene-edited cells are edited by a gene-editing system that targets at least one locus associated with joint function. In one embodiment, the population of gene-edited cells is injected into a synovial joint.

[0260] Other examples of systems, methods, and compositions for modifying expression of target gene sequences by zinc finger technology and that may be used in accordance with embodiments of the present disclosure are described in Beane, et al., Mol. Therapy, 2015, 23 1380-1390, the disclosure of which is incorporated herein by reference.

[0261] Illustrative Embodiments In some embodiments, the present disclosure provides a pharmaceutical composition for treating or preventing a disease or condition of a joint, 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 comprising 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 adjacent motif (PAM) sequence of the Cas9 protein.

[0262] In some embodiments, at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence that is complementary to a target sequence in exon 2 of the IL-1α gene. In some embodiments, the crRNA sequence forms five or fewer mismatches with the target sequence in exon 2 of the human IL-1α gene. In some embodiments, the crRNA sequence forms four or fewer mismatches with the target sequence in exon 2 of the human IL-1α gene. In some embodiments, the crRNA sequence forms three or fewer mismatches with the target sequence in exon 2 of the human IL-1α gene. In some embodiments, the crRNA sequence forms two or fewer mismatches with the target sequence in exon 2 of the human IL-1α gene. In some embodiments, the crRNA sequence forms one or fewer mismatches with the target sequence in exon 2 of the human IL-1α gene. In some embodiments, the crRNA sequence does not form any mismatches with the target sequence in exon 2 of the human IL-1α gene.

[0263] In some embodiments, at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence that is complementary to a target sequence in exon 3 of the IL-1α gene. In some embodiments, the crRNA sequence forms five or fewer mismatches with the target sequence in exon 3 of the human IL-1α gene. In some embodiments, the crRNA sequence forms four or fewer mismatches with the target sequence in exon 3 of the human IL-1α gene. In some embodiments, the crRNA sequence forms three or fewer mismatches with the target sequence in exon 3 of the human IL-1α gene. In some embodiments, the crRNA sequence forms two or fewer mismatches with the target sequence in exon 3 of the human IL-1α gene. In some embodiments, the crRNA sequence forms one or fewer mismatches with the target sequence in exon 3 of the human IL-1α gene. In some embodiments, the crRNA sequence does not form any mismatches with the target sequence in exon 3 of the human IL-1α gene.

[0264] In some embodiments, at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence that is complementary to a target sequence in exon 4 of the IL-1α gene. In some embodiments, the crRNA sequence forms five or fewer mismatches with the target sequence in exon 4 of the human IL-1α gene. In some embodiments, the crRNA sequence forms four or fewer mismatches with the target sequence in exon 4 of the human IL-1α gene. In some embodiments, the crRNA sequence forms three or fewer mismatches with the target sequence in exon 4 of the human IL-1α gene. In some embodiments, the crRNA sequence forms two or fewer mismatches with the target sequence in exon 4 of the human IL-1α gene. In some embodiments, the crRNA sequence forms one or fewer mismatches with the target sequence in exon 4 of the human IL-1α gene. In some embodiments, the crRNA sequence does not form any mismatches with the target sequence in exon 4 of the human IL-1α gene.

[0265] In some embodiments, at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence that is complementary to a target sequence in exon 5 of the IL-1α gene. In some embodiments, the crRNA sequence forms five or fewer mismatches with the target sequence in exon 5 of the human IL-1α gene. In some embodiments, the crRNA sequence forms four or fewer mismatches with the target sequence in exon 5 of the human IL-1α gene. In some embodiments, the crRNA sequence forms three or fewer mismatches with the target sequence in exon 5 of the human IL-1α gene. In some embodiments, the crRNA sequence forms two or fewer mismatches with the target sequence in exon 5 of the human IL-1α gene. In some embodiments, the crRNA sequence forms one or fewer mismatches with the target sequence in exon 5 of the human IL-1α gene. In some embodiments, the crRNA sequence does not form any mismatches with the target sequence in exon 5 of the human IL-1α gene.

[0266] In some embodiments, at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence that is complementary to a target sequence in exon 6 of the IL-1α gene. In some embodiments, the crRNA sequence forms five or fewer mismatches with the target sequence in exon 6 of the human IL-1α gene. In some embodiments, the crRNA sequence forms four or fewer mismatches with the target sequence in exon 6 of the human IL-1α gene. In some embodiments, the crRNA sequence forms three or fewer mismatches with the target sequence in exon 6 of the human IL-1α gene. In some embodiments, the crRNA sequence forms two or fewer mismatches with the target sequence in exon 6 of the human IL-1α gene. In some embodiments, the crRNA sequence forms one or fewer mismatches with the target sequence in exon 6 of the human IL-1α gene. In some embodiments, the crRNA sequence does not form any mismatches with the target sequence in exon 6 of the human IL-1α gene.

[0267] In some embodiments, at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence that is complementary to a target sequence in exon 7 of the IL-1α gene. In some embodiments, the crRNA sequence forms five or fewer mismatches with the target sequence in exon 7 of the human IL-1α gene. In some embodiments, the crRNA sequence forms four or fewer mismatches with the target sequence in exon 7 of the human IL-1α gene. In some embodiments, the crRNA sequence forms three or fewer mismatches with the target sequence in exon 7 of the human IL-1α gene. In some embodiments, the crRNA sequence forms two or fewer mismatches with the target sequence in exon 7 of the human IL-1α gene. In some embodiments, the crRNA sequence forms one or fewer mismatches with the target sequence in exon 7 of the human IL-1α gene. In some embodiments, the crRNA sequence does not form any mismatches with the target sequence in exon 7 of the human IL-1α gene.

[0268] In some embodiments, at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence that is complementary to a target sequence in exon 2 or exon 3 of the IL-1α gene. In some embodiments, at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence that is complementary to a target sequence in exon 2 or exon 4 of the IL-1α gene. In some embodiments, at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence that is complementary to a target sequence in exon 2 or exon 5 of the IL-1α gene. In some embodiments, at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence that is 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 that is complementary to a target sequence in exon 2 or exon 7 of the IL-1α gene.

[0269] In some embodiments, at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence that is complementary to a target sequence in exon 3 or exon 4 of the IL-1α gene. In some embodiments, at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence that is complementary to a target sequence in exon 3 or exon 5 of the IL-1α gene. In some embodiments, at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence that is complementary to a target sequence in exon 3 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 that is complementary to a target sequence in exon 3 or exon 7 of the IL-1α gene.

[0270] In some embodiments, at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence that is complementary to a target sequence in exon 4 or exon 5 of the IL-1α gene. In some embodiments, at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence that is complementary to a target sequence in exon 4 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 that is complementary to a target sequence in exon 4 or exon 7 of the IL-1α gene.

[0271] In some embodiments, at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence that is complementary to a target sequence in exon 5 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 that is complementary to a target sequence in exon 5 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 that is complementary to a target sequence in exon 6 or exon 7 of the IL-1α gene.

[0272] In some embodiments, at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence that is complementary to a target sequence in exon 2, exon 3, or exon 4 of the IL-1α gene. In some embodiments, at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence that is complementary to a target sequence in exon 2, exon 3, or exon 5 of the IL-1α gene. In some embodiments, at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence that is complementary to a target sequence in exon 2, exon 3, 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 that is complementary to a target sequence in exon 2, exon 3, 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 that is complementary to a target sequence in exon 2, exon 4, or exon 5 of the IL-1α gene. In some embodiments, at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence that is complementary to a target sequence in exon 2, exon 4, 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 that is complementary to a target sequence in exon 2, 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 that is complementary to a target sequence in exon 2, exon 5, 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 that is complementary to a target sequence in exon 2, exon 5, 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 that is complementary to a target sequence in exon 2, exon 6, or exon 7 of the IL-1α gene.

[0273] In some embodiments, at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence that is complementary to a target sequence in exon 3, exon 4, or exon 5 of the IL-1α gene. In some embodiments, at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence that is complementary to a target sequence in exon 3, exon 4, 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 that is 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 that is complementary to a target sequence in exon 3, exon 5, 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 that is complementary to a target sequence in exon 3, exon 5, 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 that is complementary to a target sequence in exon 3, exon 6, or exon 7 of the IL-1α gene.

[0274] In some embodiments, at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence that is complementary to a target sequence in exon 4, exon 5, 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 that is complementary to a target sequence in exon 4, exon 5, 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 that is complementary to a target sequence in exon 4, exon 6, 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 that is complementary to a target sequence in exon 5, exon 6, or exon 7 of the IL-1α gene.

[0275] In some embodiments, at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence that is complementary to a target sequence in exon 2, exon 3, exon 4, or exon 5 of the IL-1α gene. In some embodiments, at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence that is complementary to a target sequence in exon 2, exon 3, exon 4, 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 that is complementary to a target sequence in exon 2, 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 that is complementary to a target sequence in exon 2, exon 4, exon 5, 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 that is complementary to a target sequence in exon 2, exon 4, exon 5, 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 that is complementary to a target sequence in exon 2, exon 4, exon 6, 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 that is complementary to a target sequence in exon 2, exon 5, exon 6, or exon 7 of the IL-1α gene.

[0276] In some embodiments, at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence that is complementary to a target sequence in exon 3, exon 4, exon 5, 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 that is complementary to a target sequence in exon 3, exon 4, exon 5, 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 that is complementary to a target sequence in exon 3, exon 4, exon 6, 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 that is complementary to a target sequence in exon 3, exon 5, exon 6, 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 that is complementary to a target sequence in exon 4, exon 5, exon 6, or exon 7 of the IL-1α gene.

[0277] In some embodiments, at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence that is 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, at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence that is 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, at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence that is 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, at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence that is 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, at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence that is 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, at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence that is 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, at least one guide RNA targets the human IL-1α gene and comprises a crRNA sequence that is complementary to a target sequence in exon 2, exon 3, exon 4, exon 5, exon 6, or exon 7 of the IL-1α gene.

[0278] 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: 298-387. In some embodiments, the crRNA sequence has at least 80% identity to a sequence selected from the group consisting of SEQ ID NOs: 298-387. In some embodiments, the crRNA sequence has at least 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 298-387. In some embodiments, the crRNA sequence has at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 298-387. In some embodiments, the crRNA sequence has at least 95% identity to a sequence selected from the group consisting of SEQ ID NOs: 298-387. In some embodiments, the crRNA sequence is selected from the group consisting of SEQ ID NOs: 298-387.

[0279] 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.

[0280] 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: 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.

[0281] 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 a sequence selected from the group consisting of SEQ ID NOs: 188-201, 388-496, and 711-740. In some embodiments, the crRNA sequence has at least 80% identity 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 has at least 85% identity 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 has at least 90% identity 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 has at least 95% identity 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.

[0282] In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 2 of the IL-1β gene. In some embodiments, the crRNA sequence forms five or fewer mismatches with the target sequence in exon 2 of the human IL-1β gene. In some embodiments, the crRNA sequence forms four or fewer mismatches with the target sequence in exon 2 of the human IL-1β gene. In some embodiments, the crRNA sequence forms three or fewer mismatches with the target sequence in exon 2 of the human IL-1β gene. In some embodiments, the crRNA sequence forms two or fewer mismatches with the target sequence in exon 2 of the human IL-1β gene. In some embodiments, the crRNA sequence forms one or fewer mismatches with the target sequence in exon 2 of the human IL-1β gene. In some embodiments, the crRNA sequence does not form any mismatches with the target sequence in exon 2 of the human IL-1β gene.

[0283] In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 3 of the IL-1β gene. In some embodiments, the crRNA sequence forms five or fewer mismatches with the target sequence in exon 3 of the human IL-1β gene. In some embodiments, the crRNA sequence forms four or fewer mismatches with the target sequence in exon 3 of the human IL-1β gene. In some embodiments, the crRNA sequence forms three or fewer mismatches with the target sequence in exon 3 of the human IL-1β gene. In some embodiments, the crRNA sequence forms two or fewer mismatches with the target sequence in exon 3 of the human IL-1β gene. In some embodiments, the crRNA sequence forms one or fewer mismatches with the target sequence in exon 3 of the human IL-1β gene. In some embodiments, the crRNA sequence does not form any mismatches with the target sequence in exon 3 of the human IL-1β gene.

[0284] In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 4 of the IL-1β gene. In some embodiments, the crRNA sequence forms five or fewer mismatches with the target sequence in exon 4 of the human IL-1β gene. In some embodiments, the crRNA sequence forms four or fewer mismatches with the target sequence in exon 4 of the human IL-1β gene. In some embodiments, the crRNA sequence forms three or fewer mismatches with the target sequence in exon 4 of the human IL-1β gene. In some embodiments, the crRNA sequence forms two or fewer mismatches with the target sequence in exon 4 of the human IL-1β gene. In some embodiments, the crRNA sequence forms one or fewer mismatches with the target sequence in exon 4 of the human IL-1β gene. In some embodiments, the crRNA sequence does not form any mismatches with the target sequence in exon 4 of the human IL-1β gene.

[0285] In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 5 of the IL-1β gene. In some embodiments, the crRNA sequence forms five or fewer mismatches with the target sequence in exon 5 of the human IL-1β gene. In some embodiments, the crRNA sequence forms four or fewer mismatches with the target sequence in exon 5 of the human IL-1β gene. In some embodiments, the crRNA sequence forms three or fewer mismatches with the target sequence in exon 5 of the human IL-1β gene. In some embodiments, the crRNA sequence forms two or fewer mismatches with the target sequence in exon 5 of the human IL-1β gene. In some embodiments, the crRNA sequence forms one or fewer mismatches with the target sequence in exon 5 of the human IL-1β gene. In some embodiments, the crRNA sequence does not form any mismatches with the target sequence in exon 5 of the human IL-1β gene.

[0286] In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 6 of the IL-1β gene. In some embodiments, the crRNA sequence forms five or fewer mismatches with the target sequence in exon 6 of the human IL-1β gene. In some embodiments, the crRNA sequence forms four or fewer mismatches with the target sequence in exon 6 of the human IL-1β gene. In some embodiments, the crRNA sequence forms three or fewer mismatches with the target sequence in exon 6 of the human IL-1β gene. In some embodiments, the crRNA sequence forms two or fewer mismatches with the target sequence in exon 6 of the human IL-1β gene. In some embodiments, the crRNA sequence forms one or fewer mismatches with the target sequence in exon 6 of the human IL-1β gene. In some embodiments, the crRNA sequence does not form any mismatches with the target sequence in exon 6 of the human IL-1β gene.

[0287] In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 7 of the IL-1β gene. In some embodiments, the crRNA sequence forms five or fewer mismatches with the target sequence in exon 7 of the human IL-1β gene. In some embodiments, the crRNA sequence forms four or fewer mismatches with the target sequence in exon 7 of the human IL-1β gene. In some embodiments, the crRNA sequence forms three or fewer mismatches with the target sequence in exon 7 of the human IL-1β gene. In some embodiments, the crRNA sequence forms two or fewer mismatches with the target sequence in exon 7 of the human IL-1β gene. In some embodiments, the crRNA sequence forms one or fewer mismatches with the target sequence in exon 7 of the human IL-1β gene. In some embodiments, the crRNA sequence does not form any mismatches with the target sequence in exon 7 of the human IL-1β gene.

[0288] In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 2 or exon 3 of the IL-1β gene. In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 2 or exon 4 of the IL-1β gene. In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 2 or exon 5 of the IL-1β gene. In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 2 or exon 6 of the IL-1β gene. In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 2 or exon 7 of the IL-1β gene.

[0289] In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 3 or exon 4 of the IL-1β gene. In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 3 or exon 5 of the IL-1β gene. In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 3 or exon 6 of the IL-1β gene. In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 3 or exon 7 of the IL-1β gene.

[0290] In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 4 or exon 5 of the IL-1β gene. In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 4 or exon 6 of the IL-1β gene. In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 4 or exon 7 of the IL-1β gene.

[0291] In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 5 or exon 6 of the IL-1β gene. In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 5 or exon 7 of the IL-1β gene. In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 6 or exon 7 of the IL-1β gene.

[0292] In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 2, exon 3, or exon 4 of the IL-1β gene. In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 2, exon 3, or exon 5 of the IL-1β gene. In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 2, exon 3, or exon 6 of the IL-1β gene. In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 2, exon 3, or exon 7 of the IL-1β gene. In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 2, exon 4, or exon 5 of the IL-1β gene. In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 2, exon 4, or exon 6 of the IL-1β gene. In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 2, exon 4, or exon 7 of the IL-1β gene. In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 2, exon 5, or exon 6 of the IL-1β gene. In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 2, exon 5, or exon 7 of the IL-1β gene. In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 2, exon 6, or exon 7 of the IL-1β gene.

[0293] In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 3, exon 4, or exon 5 of the IL-1β gene. In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 3, exon 4, or exon 6 of the IL-1β gene. In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 3, exon 4, or exon 7 of the IL-1β gene. In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 3, exon 5, or exon 6 of the IL-1β gene. In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 3, exon 5, or exon 7 of the IL-1β gene. In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 3, exon 6, or exon 7 of the IL-1β gene.

[0294] In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 4, exon 5, or exon 6 of the IL-1β gene. In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 4, exon 5, or exon 7 of the IL-1β gene. In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 4, exon 6, or exon 7 of the IL-1β gene. In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 5, exon 6, or exon 7 of the IL-1β gene.

[0295] In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 2, exon 3, exon 4, or exon 5 of the IL-1β gene. In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 2, exon 3, exon 4, or exon 6 of the IL-1β gene. In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 2, exon 3, exon 4, or exon 7 of the IL-1β gene. In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 2, exon 4, exon 5, or exon 6 of the IL-1β gene. In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 2, exon 4, exon 5, or exon 7 of the IL-1β gene. In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 2, exon 4, exon 6, or exon 7 of the IL-1β gene. In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 2, exon 5, exon 6, or exon 7 of the IL-1β gene.

[0296] In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 3, exon 4, exon 5, or exon 6 of the IL-1β gene. In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 3, exon 4, exon 5, or exon 7 of the IL-1β gene. In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 3, exon 4, exon 6, or exon 7 of the IL-1β gene. In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 3, exon 5, exon 6, or exon 7 of the IL-1β gene. In some embodiments, at least one guide RNA targets the human IL-1β 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-1β gene.

[0297] In some embodiments, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is 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, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is 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, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is 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, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is 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, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is 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, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is 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, at least one guide RNA targets the human IL-1β gene and comprises a crRNA sequence that is complementary to a target sequence in exon 2, exon 3, exon 4, exon 5, exon 6, or exon 7 of the IL-1β gene.

[0298] 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: 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.

[0299] 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.

[0300] 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: 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.

[0301] 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: 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.

[0302] 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: 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.

[0303] In some embodiments, at least one guide RNA targets the 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, 522-590, 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, 522-590, 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, 522-590, 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, 522-590, and 741-770. In some embodiments, the crRNA sequence has at least 95% identity to a sequence selected from the group consisting of SEQ ID NOs: 202-216, 522-590, and 741-770. In some embodiments, the crRNA sequence is selected from the group consisting of SEQ ID NOs: 202-216, 522-590, and 741-770.

[0304] In some embodiments, at least one guide RNA targets the canine IL-1α gene and comprises a crRNA sequence having at least 75% identity to SEQ ID NO: 552. In some embodiments, the crRNA sequence has at least 80% identity to SEQ ID NO: 552. In some embodiments, the crRNA sequence has at least 85% identity to SEQ ID NO: 552. In some embodiments, the crRNA sequence has at least 90% identity to SEQ ID NO: 552. In some embodiments, the crRNA sequence has at least 95% identity to SEQ ID NO: 552. In some embodiments, the crRNA sequence is SEQ ID NO: 552.

[0305] In some embodiments, at least one guide RNA targets the canine IL-1α gene and comprises a crRNA sequence having at least 75% identity to SEQ ID NO: 554. In some embodiments, the crRNA sequence has at least 80% identity to SEQ ID NO: 554. In some embodiments, the crRNA sequence has at least 85% identity to SEQ ID NO: 554. In some embodiments, the crRNA sequence has at least 90% identity to SEQ ID NO: 554. In some embodiments, the crRNA sequence has at least 95% identity to SEQ ID NO: 554. In some embodiments, the crRNA sequence is SEQ ID NO: 554.

[0306] In some embodiments, at least one guide RNA targets the canine IL-1α gene and comprises a crRNA sequence having at least 75% identity to SEQ ID NO: 578. In some embodiments, the crRNA sequence has at least 80% identity to SEQ ID NO: 578. In some embodiments, the crRNA sequence has at least 85% identity to SEQ ID NO: 578. In some embodiments, the crRNA sequence has at least 90% identity to SEQ ID NO: 578. In some embodiments, the crRNA sequence has at least 95% identity to SEQ ID NO: 578. In some embodiments, the crRNA sequence is SEQ ID NO: 578.

[0307] In some embodiments, at least one guide RNA targets the canine IL-1α gene and comprises a crRNA sequence having at least 75% identity to SEQ ID NO: 579. In some embodiments, the crRNA sequence has at least 80% identity to SEQ ID NO: 579. In some embodiments, the crRNA sequence has at least 85% identity to SEQ ID NO: 579. In some embodiments, the crRNA sequence has at least 90% identity to SEQ ID NO: 579. In some embodiments, the crRNA sequence has at least 95% identity to SEQ ID NO: 579. In some embodiments, the crRNA sequence is SEQ ID NO: 579.

[0308] In some embodiments, at least one guide RNA targets the 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 has at least 95% 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 selected from the group consisting of SEQ ID NOs: 217-235, 497-551, and 771-800.

[0309] In some embodiments, at least one guide RNA targets the canine IL-1β gene and comprises a crRNA sequence having at least 75% identity to SEQ ID NO: 498. In some embodiments, the crRNA sequence has at least 80% identity to SEQ ID NO: 498. In some embodiments, the crRNA sequence has at least 85% identity to SEQ ID NO: 498. In some embodiments, the crRNA sequence has at least 90% identity to SEQ ID NO: 498. In some embodiments, the crRNA sequence has at least 95% identity to SEQ ID NO: 498. In some embodiments, the crRNA sequence is SEQ ID NO: 498.

[0310] In some embodiments, at least one guide RNA targets the canine IL-1β gene and comprises a crRNA sequence having at least 75% identity to SEQ ID NO: 506. In some embodiments, the crRNA sequence has at least 80% identity to SEQ ID NO: 506. In some embodiments, the crRNA sequence has at least 85% identity to SEQ ID NO: 506. In some embodiments, the crRNA sequence has at least 90% identity to SEQ ID NO: 506. In some embodiments, the crRNA sequence has at least 95% identity to SEQ ID NO: 506. In some embodiments, the crRNA sequence is SEQ ID NO: 506.

[0311] In some embodiments, the pharmaceutical composition comprises one or more viral vectors collectively comprising 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 a 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).

[0312] In some embodiments, the one or more viral vectors comprise a first viral vector comprising a first nucleic acid of the one or more nucleic acids that encodes a Cas9 protein and a second viral vector comprising a second nucleic acid of the one or more nucleic acids that encodes at least one guide RNA. In some embodiments, the one or more viral vectors comprise a viral vector comprising a single nucleic acid, where the single nucleic acid encodes the Cas9 protein and at least one guide RNA.

[0313] In some embodiments, the composition comprises one or more liposomes that collectively comprise one or more nucleic acids. In some embodiments, the one or more nucleic acids are present in a naked state.

[0314] In some embodiments, the Cas9 protein is a S. pyogenes Cas9 polypeptide. In some embodiments, the Cas9 protein is a S. aureus Cas9 polypeptide.

[0315] In some embodiments, the composition is formulated for parenteral administration, hi some embodiments, the composition is formulated for intra-articular injection into a joint of a subject.

[0316] In another aspect, the disclosure provides a method for treating or preventing a joint disease or condition in a subject in need thereof. The method comprises administering to a joint of the subject a pharmaceutical composition comprising a pharmaceutically effective amount of a composition comprising 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 the IL-1α or IL-1β gene, wherein the target sequence is adjacent to the protospacer adjacent motif (PAM) sequence of the Cas9 protein.

[0317] 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 has at least 95% 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 selected from the group consisting of SEQ ID NOs: 168-187, 298-387, and 681-710.

[0318] 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.

[0319] 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: 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.

[0320] 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 a sequence selected from the group consisting of SEQ ID NOs: 188-201, 388-496, and 711-740. In some embodiments, the crRNA sequence has at least 80% identity 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 has at least 85% identity 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 has at least 90% identity 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 has at least 95% identity 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.

[0321] 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: 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.

[0322] 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.

[0323] 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: 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.

[0324] 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: 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.

[0325] 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: 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.

[0326] In some embodiments, at least one guide RNA targets the 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 has at least 95% 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 selected from the group consisting of SEQ ID NOs: 202-216, 552-590, and 741-770.

[0327] In some embodiments, at least one guide RNA targets the canine IL-1α gene and comprises a crRNA sequence having at least 75% identity to SEQ ID NO: 552. In some embodiments, the crRNA sequence has at least 80% identity to SEQ ID NO: 552. In some embodiments, the crRNA sequence has at least 85% identity to SEQ ID NO: 552. In some embodiments, the crRNA sequence has at least 90% identity to SEQ ID NO: 552. In some embodiments, the crRNA sequence has at least 95% identity to SEQ ID NO: 552. In some embodiments, the crRNA sequence is SEQ ID NO: 552.

[0328] In some embodiments, at least one guide RNA targets the canine IL-1α gene and comprises a crRNA sequence having at least 75% identity to SEQ ID NO: 554. In some embodiments, the crRNA sequence has at least 80% identity to SEQ ID NO: 554. In some embodiments, the crRNA sequence has at least 85% identity to SEQ ID NO: 554. In some embodiments, the crRNA sequence has at least 90% identity to SEQ ID NO: 554. In some embodiments, the crRNA sequence has at least 95% identity to SEQ ID NO: 554. In some embodiments, the crRNA sequence is SEQ ID NO: 554.

[0329] In some embodiments, at least one guide RNA targets the canine IL-1α gene and comprises a crRNA sequence having at least 75% identity to SEQ ID NO: 578. In some embodiments, the crRNA sequence has at least 80% identity to SEQ ID NO: 578. In some embodiments, the crRNA sequence has at least 85% identity to SEQ ID NO: 578. In some embodiments, the crRNA sequence has at least 90% identity to SEQ ID NO: 578. In some embodiments, the crRNA sequence has at least 95% identity to SEQ ID NO: 578. In some embodiments, the crRNA sequence is SEQ ID NO: 578.

[0330] In some embodiments, at least one guide RNA targets the canine IL-1α gene and comprises a crRNA sequence having at least 75% identity to SEQ ID NO: 579. In some embodiments, the crRNA sequence has at least 80% identity to SEQ ID NO: 579. In some embodiments, the crRNA sequence has at least 85% identity to SEQ ID NO: 579. In some embodiments, the crRNA sequence has at least 90% identity to SEQ ID NO: 579. In some embodiments, the crRNA sequence has at least 95% identity to SEQ ID NO: 579. In some embodiments, the crRNA sequence is SEQ ID NO: 579.

[0331] In some embodiments, at least one guide RNA targets the 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 five or fewer mismatches with the target sequence in exon 2 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms four or fewer mismatches with the target sequence in exon 2 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms three or fewer mismatches with the target sequence in exon 2 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms two or fewer mismatches with the target sequence in exon 2 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms one or fewer mismatches with the target sequence in exon 2 of the canine IL-1α gene. In some embodiments, the crRNA sequence does not form any mismatches with the target sequence in exon 2 of the canine IL-1α gene.

[0332] In some embodiments, at least one guide RNA targets the 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 five or fewer mismatches with the target sequence in exon 3 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms four or fewer mismatches with the target sequence in exon 3 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms three or fewer mismatches with the target sequence in exon 3 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms two or fewer mismatches with the target sequence in exon 3 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms one or fewer mismatches with the target sequence in exon 3 of the canine IL-1α gene. In some embodiments, the crRNA sequence does not form any mismatches with the target sequence in exon 3 of the canine IL-1α gene.

[0333] In some embodiments, at least one guide RNA targets the 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 five or fewer mismatches with the target sequence in exon 4 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms four or fewer mismatches with the target sequence in exon 4 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms three or fewer mismatches with the target sequence in exon 4 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms two or fewer mismatches with the target sequence in exon 4 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms one or fewer mismatches with the target sequence in exon 4 of the canine IL-1α gene. In some embodiments, the crRNA sequence does not form any mismatches with the target sequence in exon 4 of the canine IL-1α gene.

[0334] In some embodiments, at least one guide RNA targets the canine IL-1α gene and comprises a crRNA sequence that is complementary to a target sequence in exon 5 of the IL-1α gene. In some embodiments, the crRNA sequence forms five or fewer mismatches with the target sequence in exon 5 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms four or fewer mismatches with the target sequence in exon 5 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms three or fewer mismatches with the target sequence in exon 5 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms two or fewer mismatches with the target sequence in exon 5 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms one or fewer mismatches with the target sequence in exon 5 of the canine IL-1α gene. In some embodiments, the crRNA sequence does not form any mismatches with the target sequence in exon 5 of the canine IL-1α gene.

[0335] In some embodiments, at least one guide RNA targets the 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 five or fewer mismatches with the target sequence in exon 6 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms four or fewer mismatches with the target sequence in exon 6 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms three or fewer mismatches with the target sequence in exon 6 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms two or fewer mismatches with the target sequence in exon 6 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms one or fewer mismatches with the target sequence in exon 6 of the canine IL-1α gene. In some embodiments, the crRNA sequence does not form any mismatches with the target sequence in exon 6 of the canine IL-1α gene.

[0336] In some embodiments, at least one guide RNA targets the canine IL-1α gene and comprises a crRNA sequence that is complementary to a target sequence in exon 7 of the IL-1α gene. In some embodiments, the crRNA sequence forms five or fewer mismatches with the target sequence in exon 7 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms four or fewer mismatches with the target sequence in exon 7 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms three or fewer mismatches with the target sequence in exon 7 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms two or fewer mismatches with the target sequence in exon 7 of the canine IL-1α gene. In some embodiments, the crRNA sequence forms one or fewer mismatches with the target sequence in exon 7 of the canine IL-1α gene. In some embodiments, the crRNA sequence does not form any mismatches with the target sequence in exon 7 of the canine IL-1α gene.

[0337] In some embodiments, at least one guide RNA targets the canine IL-1α gene and comprises a crRNA sequence that is complementary to a target sequence in exon 2 or exon 3 of the IL-1α gene. In some embodiments, at least one guide RNA targets the canine IL-1α gene and comprises a crRNA sequence that is complementary to a target sequence in exon 2 or exon 4 of the IL-1α gene. In some embodiments, at least one guide RNA targets the canine IL-1α gene and comprises a crRN...

Claims

**Claim 1** A composition for the treatment or prevention of 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 targeting the IL-1α or IL-1β gene, or a nucleic acid encoding at least one guide RNA, wherein the guide RNA specifically binds to a target sequence adjacent to the protospacer adjacent motif (PAM) sequence of the RNA-guided nuclease; and one or more lipid nanoparticles (LNP) encapsulating the above components collectively. **Claim 2** The composition according to claim 1, wherein the at least one guide RNA targets the human IL-1α gene and comprises a sequence selected from the group consisting of SEQ ID NOs: 168-187, 298-387, and 681-710. **Claim 3** The composition according to claim 1, wherein the at least one guide RNA targets the human IL-1β gene and comprises a sequence selected from the group consisting of SEQ ID NOs: 188-201, 388-496, and 711-740. **Claim 4** The composition according to claim 1, wherein the at least one guide RNA targets the canine IL-1α gene and comprises a sequence selected from the group consisting of SEQ ID NOs: 202-216, 552-590, and 741-770. **Claim 5** The composition according to claim 1, wherein the at least one guide RNA targets the canine IL-1β gene and comprises a sequence selected from the group consisting of SEQ ID NOs: 217-235, 497-551, and 771-800. **Claim 6** The composition according to claim 1, wherein the at least one guide RNA targets the equine IL-1α gene and comprises a sequence selected from the group consisting of SEQ ID NOs: 236-255 and 801-830. **Claim 7** The composition according to claim 1, wherein the at least one guide RNA targets the equine IL-1β gene and comprises a sequence selected from the group consisting of SEQ ID NOs: 256-262 and 831-860. **Claim 8** The composition according to claim 1, wherein the at least one guide RNA targets the feline IL-1α gene and comprises a sequence selected from the group consisting of SEQ ID NOs: 861-890. **Claim 9** The composition according to claim 1, wherein the at least one guide RNA targets the feline IL-1β gene and comprises a sequence selected from the group consisting of SEQ ID NOs: 891 to 920.

10. The composition according to any one of claims 1 to 9, wherein the RNA-guided nuclease is a CRISPR-associated protein 9 (Cas9) protein, and optionally, the Cas9 protein is an enhanced specificity Cas9 variant protein.

11. The composition according to claim 10, wherein the Cas9 protein is a S. pyogenes Cas9 protein or a S. aureus Cas9 protein.

12. The composition according to any one of claims 1 to 9, wherein the RNA-guided nuclease or the nucleic acid encoding the RNA-guided nuclease is the mRNA encoding the RNA-guided nuclease, and the at least one guide RNA or the nucleic acid encoding the at least one guide RNA is the at least one guide RNA.

13. The composition according to any one of claims 1 to 9, wherein the at least one guide RNA is a single guide RNA (sgRNA).

14. The composition is (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, and comprises one or more lipid nanoparticles (LNPs) that collectively encapsulate them. The composition according to any one of claims 1 to 9.

15. The one or more LNPs include components selected from the group consisting of 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butanoate (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.beta.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)--octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3.beta.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z-,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2R)), (2S)-2-({8-[(3.beta.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z-,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2S)), lipids containing a cyclic amine group, and mixtures thereof. Optionally, the LNP comprises a component 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-didodecanoyl-sn-glycero-phosphocholine (DUPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesteryl hemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lysophosphatidylcholine), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin (SM), and mixtures thereof. Optionally, 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, and Optionally, the LNP comprises a component selected from the group consisting of cholesterol, fecosterol, stigmasterol, stigmasteranol, sitosterol, β-sitosterol, lupenol, betulin, ursolic acid, oleanolic acid, campesterol, fucosterol, brassicasterol, ergosterol, 9,11-dehydroergosterol, tomatidine, tomatin, α-tocopherol, and mixtures thereof. The composition according to claim 14. **Claim 16** Use of a lipid nanoparticle (LNP) in the manufacture of a medicament for treating a joint disease or condition, wherein the LNP comprises: (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease; (ii) at least one guide RNA targeting the IL-1α or IL-1β gene or a nucleic acid encoding at least one guide RNA, wherein the guide RNA specifically binds to a target sequence adjacent to the protospacer adjacent motif (PAM) sequence of the RNA-guided nuclease, and at least one guide RNA or a nucleic acid encoding at least one guide RNA; The use as defined above, wherein the LNP encapsulates the components (i) and (ii) collectively. **Claim 17** The use according to claim 16, wherein the joint disease or condition is arthritis, and optionally, the arthritis is osteoarthritis. **Claim 18** The use according to claim 16, wherein the joint disease or disorder is post-traumatic arthritis, gout, or pseudogout. **Claim 19** The use according to any one of claims 16 to 18, wherein the treatment of the joint disease or condition comprises intra-articular injection of the medicament into the joint of the subject. **Claim 20** The use according to any one of claims 16 to 18, wherein the medicament is administered during surgery or after surgery.

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