Gene editing to improve joint function

Abstract

Provided herein are compositions and methods for treating musculoskeletal fibrosis and / or scarring by ablating intracellular signaling through specific cell surface receptors through gene editing. In some aspects, the compositions and methods are directed to TGFB1 ligands. In other aspects, the compositions and methods are directed to TGFB1 receptors (TGFBR1 / TGFBR2). In some aspects, the compositions and methods are for treating or preventing post-traumatic fibrosis and / or scarring. In some aspects, the compositions and methods are for treating or preventing post-operative fibrosis and / or scarring. In some aspects, the compositions and methods are for treating or preventing local pain, inflammation, degeneration, or morphological changes associated with fibrosis and / or scarring.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 303,472, filed January 26, 2022, U.S. Provisional Patent Application No. 63 / 303,388, filed January 26, 2022, and U.S. Provisional Patent Application No. 63 / 480,642, filed January 19, 2023, the contents of which are incorporated by reference in their entireties herein for all purposes. [Background technology]

[0002] Musculoskeletal tissues are particularly prone to fibrosis and / or scarring following trauma. In many cases, this trauma can be a surgical procedure. Fibrosis is the result of extracellular matrix (ECM) components accumulating at specific sites, contributing to the formation of scar tissue. See Sheets, K., et al (2022). Journal of Cellular Biochemistry. This musculoskeletal scar tissue can present several problems, such as limited range of motion or pain, and can further promote disease and complicate medical or surgical management of musculoskeletal tissues and joints.

[0003] Transforming growth factor-beta (TGFB) signals through canonical receptor-ligand interactions, which are involved in the transmission of extracellular signals across the plasma membrane of receptor-expressing cells. In many cases, circulating ligands bind to specific receptors anchored to the cell membrane, which leads to the transduction of signaling pathways from the cell surface to the interior through the activity of the receptor's cytoplasmic domain. TGFB signaling events can then affect a variety of cellular activities, which in turn lead to changes at the tissue and organism levels.

[0004] One such change is fibrosis of musculoskeletal tissue. Indeed, increased expression of TGFB1 is observed in fascial fibroblasts at the sites of such musculoskeletal trauma, among other factors. See Ihn, H. (2019). Allergology International, 68(4), 437-439. This expression pattern correlated with higher expression of collagen I and fibronectin, ECM components whose aggregation may contribute to fibrosis. TGFB, a known driver of pro-inflammatory signaling, provides a link between fibrosis and / or scarring and chronic inflammation.

[0005] Current treatments for such musculoskeletal conditions, such as conservative care, physical rehabilitation, or surgery, focus on treating symptoms rather than addressing the causes of fibrosis and / or scarring, which can lead to further trauma. Thus, there is a great need for new methods and compositions that address the causes of the condition. Summary of the Invention

[0006] Provided herein are compositions and methods for silencing the signaling function of TGFB1 or any of its cellular receptors in an animal in need thereof to treat a disease, illness, or condition caused by aberrant or excessive signaling through the receptor.

[0007] In some embodiments, receptor signaling is silenced by CRISPR editing of the gene encoding TGFBR1 receptor.In some embodiments, receptor signaling is silenced by CRISPR editing of the gene encoding TGFBR2 receptor.In some embodiments, receptor signaling is silenced by CRISPR editing of the gene encoding TGFB1 ligand.In some embodiments, CRISPR editing causes the removal of transmembrane domain (i.e., the generation of soluble receptor).In some embodiments, CRISPR editing causes the removal of cytoplasmic domain.

[0008] The ubiquity of the receptor-ligand paradigm in cell biology means that many diseases, illnesses, and conditions are caused, in whole or in part, by aberrant or excessive signaling through various cellular receptors, and a variety of approaches are available to address this. Small and large molecules can be used to disrupt receptor-ligand interactions, but problems of off-target effects and potential immunogenicity remain. More recently, genetic approaches have been explored to either transiently reduce (i.e., knock-down), for example with siRNA, or permanently eliminate (i.e., gene knock-out) the expression of a given ligand or receptor. However, more sophisticated genetic tools are needed to address the myriad diseases, illnesses, and conditions that involve such aberrant signaling.

[0009] In particular, the compositions and methods disclosed herein also encompass various routes of administration of CRISPR components, with a particular focus on post-traumatic and post-operative interventions to treat and prevent fibrotic tissues, such as musculoskeletal, cardiac, pulmonary, and renal tissues. [Brief description of the drawings]

[0010] Embodiments of the present disclosure will be further described with reference to the accompanying drawings. The drawings shown are not necessarily to scale, with emphasis instead generally on illustrating the principles of embodiments of the present disclosure.

[0011] [Figure 1A] SEQ ID NOs: 1-198 (A-D) collectively show the crRNA sequences generated by the bioinformatic methods described herein for targeting human TGFB1 to generate a gene knockout, and (E-H) additional information regarding the genomic coordinates of the bound DNA, the targeted DNA strand, the targeted exon, and several quality control parameters. [Figure 1B]SEQ ID NOs: 1-198 (A-D) collectively show the crRNA sequences generated by the bioinformatic methods described herein for targeting human TGFB1 to generate a gene knockout, and (E-H) additional information regarding the genomic coordinates of the bound DNA, the targeted DNA strand, the targeted exon, and several quality control parameters. [Figure 1C] SEQ ID NOs: 1-198 (A-D) collectively show the crRNA sequences generated by the bioinformatic methods described herein for targeting human TGFB1 to generate a gene knockout, and (E-H) additional information regarding the genomic coordinates of the bound DNA, the targeted DNA strand, the targeted exon, and several quality control parameters. [Figure 1D] SEQ ID NOs: 1-198 (A-D) collectively show the crRNA sequences generated by the bioinformatic methods described herein for targeting human TGFB1 to generate a gene knockout, and (E-H) additional information regarding the genomic coordinates of the bound DNA, the targeted DNA strand, the targeted exon, and several quality control parameters. [Figure 1E] SEQ ID NOs: 1-198 (A-D) collectively show the crRNA sequences generated by the bioinformatic methods described herein for targeting human TGFB1 to generate a gene knockout, and (E-H) additional information regarding the genomic coordinates of the bound DNA, the targeted DNA strand, the targeted exon, and several quality control parameters. [Figure 1F] SEQ ID NOs: 1-198 (A-D) collectively show the crRNA sequences generated by the bioinformatic methods described herein for targeting human TGFB1 to generate a gene knockout, and (E-H) additional information regarding the genomic coordinates of the bound DNA, the targeted DNA strand, the targeted exon, and several quality control parameters. [Figure 1G]SEQ ID NOs: 1-198 (A-D) collectively show the crRNA sequences generated by the bioinformatic methods described herein for targeting human TGFB1 to generate a gene knockout, and (E-H) additional information regarding the genomic coordinates of the bound DNA, the targeted DNA strand, the targeted exon, and several quality control parameters. [Figure 1H] SEQ ID NOs: 1-198 (A-D) collectively show the crRNA sequences generated by the bioinformatic methods described herein for targeting human TGFB1 to generate a gene knockout, and (E-H) additional information regarding the genomic coordinates of the bound DNA, the targeted DNA strand, the targeted exon, and several quality control parameters. [Figure 2A] SEQ ID NOs:199-320 collectively show (A-C) crRNA sequences generated by the bioinformatic methods described herein targeting human TGFBR1 to generate gene knockouts, soluble decoy receptors, or membrane-bound decoy receptors, and (D-F) additional information regarding the genomic coordinates of the bound DNA, the targeted DNA strand, the targeted exon, and several quality control parameters. [Figure 2B] SEQ ID NOs:199-320 collectively show (A-C) crRNA sequences generated by the bioinformatic methods described herein targeting human TGFBR1 to generate gene knockouts, soluble decoy receptors, or membrane-bound decoy receptors, and (D-F) additional information regarding the genomic coordinates of the bound DNA, the targeted DNA strand, the targeted exon, and several quality control parameters. [Figure 2C] SEQ ID NOs:199-320 collectively show (A-C) crRNA sequences generated by the bioinformatic methods described herein targeting human TGFBR1 to generate gene knockouts, soluble decoy receptors, or membrane-bound decoy receptors, and (D-F) additional information regarding the genomic coordinates of the bound DNA, the targeted DNA strand, the targeted exon, and several quality control parameters. [Figure 2D]SEQ ID NOs:199-320 collectively show (A-C) crRNA sequences generated by the bioinformatic methods described herein targeting human TGFBR1 to generate gene knockouts, soluble decoy receptors, or membrane-bound decoy receptors, and (D-F) additional information regarding the genomic coordinates of the bound DNA, the targeted DNA strand, the targeted exon, and several quality control parameters. [Figure 2E] SEQ ID NOs:199-320 collectively show (A-C) crRNA sequences generated by the bioinformatic methods described herein targeting human TGFBR1 to generate gene knockouts, soluble decoy receptors, or membrane-bound decoy receptors, and (D-F) additional information regarding the genomic coordinates of the bound DNA, the targeted DNA strand, the targeted exon, and several quality control parameters. [Figure 2F] SEQ ID NOs:199-320 collectively show (A-C) crRNA sequences generated by the bioinformatic methods described herein targeting human TGFBR1 to generate gene knockouts, soluble decoy receptors, or membrane-bound decoy receptors, and (D-F) additional information regarding the genomic coordinates of the bound DNA, the targeted DNA strand, the targeted exon, and several quality control parameters. [Figure 3A] SEQ ID NOs:321-519, (A-D) collectively show crRNA sequences generated by the bioinformatic methods described herein targeting human TGFBR2 to generate gene knockouts, soluble decoy receptors, or membrane-bound decoy receptors, and (E-H) additional information regarding the genomic coordinates of the bound DNA, the targeted DNA strand, the targeted exon, and several quality control parameters. [Figure 3B]SEQ ID NOs:321-519, (A-D) collectively show crRNA sequences generated by the bioinformatic methods described herein targeting human TGFBR2 to generate gene knockouts, soluble decoy receptors, or membrane-bound decoy receptors, and (E-H) additional information regarding the genomic coordinates of the bound DNA, the targeted DNA strand, the targeted exon, and several quality control parameters. [Figure 3C] SEQ ID NOs:321-519, (A-D) collectively show crRNA sequences generated by the bioinformatic methods described herein targeting human TGFBR2 to generate gene knockouts, soluble decoy receptors, or membrane-bound decoy receptors, and (E-H) additional information regarding the genomic coordinates of the bound DNA, the targeted DNA strand, the targeted exon, and several quality control parameters. [Figure 3D] SEQ ID NOs:321-519, (A-D) collectively show crRNA sequences generated by the bioinformatic methods described herein targeting human TGFBR2 to generate gene knockouts, soluble decoy receptors, or membrane-bound decoy receptors, and (E-H) additional information regarding the genomic coordinates of the bound DNA, the targeted DNA strand, the targeted exon, and several quality control parameters. [Figure 3E] SEQ ID NOs:321-519, (A-D) collectively show crRNA sequences generated by the bioinformatic methods described herein targeting human TGFBR2 to generate gene knockouts, soluble decoy receptors, or membrane-bound decoy receptors, and (E-H) additional information regarding the genomic coordinates of the bound DNA, the targeted DNA strand, the targeted exon, and several quality control parameters. [Figure 3F]SEQ ID NOs:321-519, (A-D) collectively show crRNA sequences generated by the bioinformatic methods described herein targeting human TGFBR2 to generate gene knockouts, soluble decoy receptors, or membrane-bound decoy receptors, and (E-H) additional information regarding the genomic coordinates of the bound DNA, the targeted DNA strand, the targeted exon, and several quality control parameters. [Figure 3G] SEQ ID NOs:321-519, (A-D) collectively show crRNA sequences generated by the bioinformatic methods described herein targeting human TGFBR2 to generate gene knockouts, soluble decoy receptors, or membrane-bound decoy receptors, and (E-H) additional information regarding the genomic coordinates of the bound DNA, the targeted DNA strand, the targeted exon, and several quality control parameters. [Figure 3H] SEQ ID NOs:321-519, (A-D) collectively show crRNA sequences generated by the bioinformatic methods described herein targeting human TGFBR2 to generate gene knockouts, soluble decoy receptors, or membrane-bound decoy receptors, and (E-H) additional information regarding the genomic coordinates of the bound DNA, the targeted DNA strand, the targeted exon, and several quality control parameters. [Figure 4A] (A) Schematic showing the targeting domain of TGFBR1 and the orientation of various sgRNAs predicted to generate knockouts, and (B) a set of parameters considered for designing sgRNAs against TGFBR1, as well as an exemplary crRNA sequence. [Figure 4B] (A) Schematic showing the targeting domain of TGFBR1 and the orientation of various sgRNAs predicted to generate knockouts, and (B) a set of parameters considered for designing sgRNAs against TGFBR1, as well as an exemplary crRNA sequence. [Figure 5A](A) Schematic showing the targeting domain of TGFBR1 and the orientation of various sgRNAs predicted to generate knockout, membrane-bound decoy receptor, and soluble decoy receptor (ECD: extracellular domain, TMD: transmembrane domain, ICD: intracellular domain, GSM: GS-rich motif), and (B) a set of parameters considered for the design of sgRNA against TGFBR1, as well as an exemplary crRNA sequence. [Figure 5B] (A) Schematic showing the targeting domain of TGFBR1 and the orientation of various sgRNAs predicted to generate knockout, membrane-bound decoy receptor, and soluble decoy receptor (ECD: extracellular domain, TMD: transmembrane domain, ICD: intracellular domain, GSM: GS-rich motif), and (B) a set of parameters considered for the design of sgRNA against TGFBR1, as well as an exemplary crRNA sequence. [Figure 6A] (A) Schematic showing the targeting domain of TGFBR2 and the orientation of various sgRNAs predicted to generate knockout, membrane-bound decoy receptor, and soluble decoy receptor (ECD: extracellular domain, TMD: transmembrane domain, ICD: intracellular domain, GSM: GS-rich motif), and (B) the set of parameters considered for the design of sgRNA against TGFBR2, as well as an exemplary crRNA sequence. [Figure 6B] (A) Schematic showing the targeting domain of TGFBR2 and the orientation of various sgRNAs predicted to generate knockout, membrane-bound decoy receptor, and soluble decoy receptor (ECD: extracellular domain, TMD: transmembrane domain, ICD: intracellular domain, GSM: GS-rich motif), and (B) the set of parameters considered for the design of sgRNA against TGFBR2, as well as an exemplary crRNA sequence. [Figure 7A] A summary of the identified guides and the efficiency for editing the human TGFBR1 gene in THP-1 cells using (A) wild-type SpCas9 or (B) ARCas9. [Figure 7B]A summary of the identified guides and the efficiency for editing the human TGFBR1 gene in THP-1 cells using (A) wild-type SpCas9 or (B) ARCas9. [Figure 8A] A summary of the identified guides and the efficiency for editing the human TGFBR1 gene in THP-1 cells using (A) wild-type SpCas9 or (B) ARCas9. [Figure 8B] A summary of the identified guides and the efficiency for editing the human TGFBR1 gene in THP-1 cells using (A) wild-type SpCas9 or (B) ARCas9. [Figure 9A] A summary of the identified guides and the efficiency for editing the human TGFBR2 gene in THP-1 cells using (A) wild-type SpCas9 or (B) ARCas9. [Figure 9B] A summary of the identified guides and the efficiency for editing the human TGFBR2 gene in THP-1 cells using (A) wild-type SpCas9 or (B) ARCas9. [Figure 10A] Shown are the relative levels of (A,C) TGFB1 and (B,D) TIMP1 gene expression in THP-1 cells that are either unedited (WT) or edited to knock out TGFB1 (OHTG), TGFBR1 (OHTIR), or TGFBR2 (OHTIIR) 6 hours after challenge with LPS (panels A and B) or TGF beta (panels C and D). [Figure 10B] Shown are the relative levels of (A,C) TGFB1 and (B,D) TIMP1 gene expression in THP-1 cells that are either unedited (WT) or edited to knock out TGFB1 (OHTG), TGFBR1 (OHTIR), or TGFBR2 (OHTIIR) 6 hours after challenge with LPS (panels A and B) or TGF beta (panels C and D). [Figure 10C]Shown are the relative levels of (A,C) TGFB1 and (B,D) TIMP1 gene expression in THP-1 cells that are either unedited (WT) or edited to knock out TGFB1 (OHTG), TGFBR1 (OHTIR), or TGFBR2 (OHTIIR) 6 hours after challenge with LPS (panels A and B) or TGF beta (panels C and D). [Figure 10D] Shown are the relative levels of (A,C) TGFB1 and (B,D) TIMP1 gene expression in THP-1 cells that are either unedited (WT) or edited to knock out TGFB1 (OHTG), TGFBR1 (OHTIR), or TGFBR2 (OHTIIR) 6 hours after challenge with LPS (panels A and B) or TGF beta (panels C and D). [Figure 11A] Collectively shown are human-directed crRNA sequences targeting (A) hTGFB1 (SEQ ID NOs: 520-527), (B) hTGFBR1 (SEQ ID NOs: 528-552), and (C) hTGFBR2 (SEQ ID NOs: 553-604) for use with sgRNA to validate in vitro editing with different delivery modes. [Figure 11B] Collectively shown are human-directed crRNA sequences targeting (A) hTGFB1 (SEQ ID NOs: 520-527), (B) hTGFBR1 (SEQ ID NOs: 528-552), and (C) hTGFBR2 (SEQ ID NOs: 553-604) for use with sgRNA to validate in vitro editing with different delivery modes. [Figure 11C] Collectively shown are human-directed crRNA sequences targeting (A) hTGFB1 (SEQ ID NOs: 520-527), (B) hTGFBR1 (SEQ ID NOs: 528-552), and (C) hTGFBR2 (SEQ ID NOs: 553-604) for use with sgRNA to validate in vitro editing with different delivery modes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] I. Introduction Provided herein are compositions and methods for silencing the signaling function of one or more cellular receptors in an animal in need thereof to treat a disease, illness, or condition caused by aberrant or excessive signaling through the receptor.

[0013] In some embodiments, receptor signaling is reduced or eliminated by using the compositions and methods described herein to edit the TGFB1, TGFBR1, and / or TGFBR2 genes. In some embodiments, the editing knocks out the TGFB1, TGFBR1, and / or TGFBR2 genes, which is editing of the genes such that the genes no longer encode functional proteins. In some embodiments, the editing results in the removal of the transmembrane domain of the protein encoded by the TGRBR1 or TGFBR2 gene, e.g., the generation of a soluble receptor decoy. In some embodiments, the editing results in the removal of the cytoplasmic domain of the protein encoded by the TGRBR1 or TGFBR2 gene, e.g., the generation of a membrane-bound receptor decoy. In some embodiments, the receptor is TGFBR1. In some embodiments, the receptor is TGFBR2. Receptor signaling is reduced or eliminated by using the compositions and methods described herein to edit the TGFB1 ligand.

[0014] II. 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 invention belongs. All patents and publications referenced herein are incorporated by reference in their entirety.

[0015] The term "transforming growth factor beta 1" or "TGFB1" refers to the gene (NCBI Gene ID: 7040 [human], NCBI Gene ID: 403998 [dog], NCBI Gene ID: 100033900 [horse], NCBI Gene ID: 768263 [cat]), or the encoded gene product (e.g., UniProt: P01137; AAH00125..1 [human], XP_038512824.1 [dog], XP_014716505.1 [horse], XP_006941294.1 [cat]), as well as sequence variants, proteins carrying conservative amino acid substitutions, and glycoforms thereof. Canonically, the cytokine proteins encoded by the genes listed above can bind to the TGFBR complex and mediate intracellular signaling pathways that control multiple physiological and pathological processes, including inflammatory processes, through the release of SMAD2 (which can then translocate to the nucleus), or activation of other cytoplasmic signaling mediators. In some cases, and simply for disambiguation, prefixes are added when referring to specific species of proteins or genes (h, c, e, and f refer to the human, canine, equine, and feline forms, respectively).

[0016] In certain embodiments, any region of the TGFB1 gene (e.g., the 5' untranslated region [UTR], exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, any intervening intron region, intron / exon junction, 3' UTR, or polyadenylation signal) is targeted by an RNA-guided nuclease to modify the gene. In some embodiments, the TGFB1 gene targeted by the RNA-guided nuclease is of mammalian origin. In some embodiments, the TGFB1 gene targeted by the RNA-guided nuclease is of human origin (hTGFB1). In some embodiments, the TGFB1 gene targeted by the RNA-guided nuclease is of canine origin (cTGFB1). In some embodiments, the TGFB1 gene targeted by the RNA-guided nuclease is of equine origin (eTGFB1). In some embodiments, the TGFB1 gene targeted by the RNA-guided nuclease is of feline origin (fTGFB1).

[0017] The term "transforming growth factor beta receptor 1" or "TGFBR1" refers to the gene (NCBI Gene ID: 7046 [human], NCBI Gene ID: 481628 [dog], NCBI Gene ID: 100034117 [horse], NCBI Gene ID: 101094057 [cat]), or the encoded gene product (e.g., UniProt: P36897; NP_004603.1 [human], XP_038538191.1 [dog], XP_023485510.1 [horse], XP_023098269.1 [cat]), as well as sequence variants, proteins carrying conservative amino acid substitutions, and glycoforms thereof. Canonically, the proteins encoded by the genes listed above are transmembrane serine / threonine kinases that, together with TGFBR2, form the natural receptors for the TGF-beta cytokines TGFB1, TGFB2, and TGFB3. Upon binding to its ligand, TGFBR1 is phosphorylated by TGFBR2 and activates intracellular signaling that controls multiple physiological and pathological processes through the release of SMAD2 (which can then translocate to the nucleus) or the activation of other cytoplasmic signaling mediators. In some cases, and simply for disambiguation, prefixes are added when referring to specific species of proteins or genes (h, c, e, and f refer to the human, canine, equine, and feline forms, respectively).

[0018] In certain embodiments, any region of the TGFBR1 gene (e.g., the 5' untranslated region [UTR], exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, any intervening intron region, intron / exon junction, 3'UTR, or polyadenylation signal) is targeted by an RNA-guided nuclease to modify the gene. In some embodiments, the TGFBR1 gene targeted by the RNA-guided nuclease is of mammalian origin. In some embodiments, the TGFBR1 gene targeted by the RNA-guided nuclease is of human origin (hTGFBR1). In some embodiments, the TGFBR1 gene targeted by the RNA-guided nuclease is of canine origin (cTGFBR1). In some embodiments, the TGFBR1 gene targeted by the RNA-guided nuclease is of equine origin (eTGFBR1). In some embodiments, the TGFBR1 gene targeted by the RNA-guided nuclease is of feline origin (fTGFBR1).

[0019] The term "transforming growth factor beta receptor 2" or "TGFBR2" refers to the gene (NCBI Gene ID: 7048 [human], NCBI Gene ID: 477039 [dog], NCBI Gene ID: 100033860 [horse], NCBI Gene ID: 101091725 [cat]), or the encoded gene product (e.g., UniProt: P37173; NP_003233.4 [human], XP_038288013.1 [dog], XP_023475502.1 [horse], XP_023116415.1 [cat]), as well as sequence variants, proteins carrying conservative amino acid substitutions, and glycoforms thereof. Canonically, the proteins encoded by the genes listed above are transmembrane serine / threonine kinases that, together with TGFBR2, form the natural receptors for the TGF-beta cytokines TGFB1, TGFB2, and TGFB3. Upon binding to its ligand, TGFBR1 is phosphorylated by TGFBR2 and activates intracellular signaling that controls multiple physiological and pathological processes through the release of SMAD2 (which can then translocate to the nucleus) or the activation of other cytoplasmic signaling mediators. In some cases, and simply for disambiguation, prefixes are added when referring to specific species of proteins or genes (h, c, e, and f refer to human, canine, equine, and feline forms, respectively). In some cases, and simply for disambiguation, prefixes are added when referring to specific species of proteins or genes (h, c, e, and f refer to human, canine, equine, and feline forms, respectively).

[0020] In certain embodiments, any region of the TGFBR2 gene (e.g., the 5' untranslated region [UTR], exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, any intervening intron region, intron / exon junction, 3'UTR, or polyadenylation signal) is targeted by an RNA-guided nuclease to modify the gene. In some embodiments, the TGFBR2 gene targeted by the RNA-guided nuclease is of mammalian origin. In some embodiments, the TGFBR2 gene targeted by the RNA-guided nuclease is of human origin (hTGFBR2). In some embodiments, the TGFBR2 gene targeted by the RNA-guided nuclease is of canine origin (cTGFBR2). In some embodiments, the TGFBR2 gene targeted by the RNA-guided nuclease is of equine origin (eTGFBR2). In some embodiments, the TGFBR2 gene targeted by the RNA-guided nuclease is of feline origin (fTGFBR2).

[0021] The term "treatment" refers 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 a partial or complete cure for the disease and / or adverse effects caused by the disease. For example, a composition, method, or system of the present disclosure may be administered as a prophylactic treatment to a subject having a predisposition to a given condition (e.g., arthritis). As used herein, "treatment" extends to 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 as having 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 relieving one or more disease symptoms.

[0022] "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" encompasses the delivery of a composition that may 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 treat all mammals, including, but not limited to, human, canine, feline, equine, and bovine subjects.

[0023] The term "therapeutically effective" refers to an amount of a composition or combination of compositions described herein that is sufficient to provide the intended use, including, but not limited to, disease treatment. The therapeutically effective amount may vary depending on the intended use (in vitro or in vivo), or the subject and disease state to be 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 induce a particular response in target cells (e.g., reducing platelet adhesion and / or cell migration). The 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 to which the composition is transported.

[0024] The terms "fibrosis" and "fibrous scarring" are used interchangeably and refer to the formation of abnormal amounts of fibrous tissue in an organ or tissue, typically resulting from inflammation, irritation, or healing of the tissue. Fibrosis encompasses the clinical phenomenon commonly referred to as scarring, e.g., tissue scarring or organ scarring, and adhesions that form, e.g., between organs or different tissues. Fibrosis may occur in joint tissue (e.g., knee joint, shoulder joint, e.g., adhesive capsulitis, elbow joint, etc.), kidney tissue (e.g., in chronic kidney disease, etc.), skin tissue (e.g., associated with wound healing, e.g., post-traumatic or post-operative, keloid disorders, nephrogenic systemic fibrosis, scleroderma / systemic sclerosis, etc.), lung tissue (e.g., in fibrothorax, pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, radiation-induced lung injury, progressive diffuse fibrosis, scleroderma / systemic sclerosis, etc.), liver tissue (e.g., in cirrhosis, bridging fibrosis, etc.), kidney tissue (e.g., in chronic kidney disease, etc.), heart tissue (e.g., in typical It may occur in any tissue, including, but not limited to, interstitial fibrosis typically associated with congestive heart failure, hypertension, and normal aging, replacement fibrosis typically associated with a previous myocardial infarction), brain tissue (e.g., glial scar), intestinal tissue (e.g., in Crohn's disease), the hand and / or fingers (e.g., in Dupuytren's contracture), lymphatic tissue (e.g., in mediastinal fibrosis), bone marrow tissue (e.g., in myelofibrosis), penile tissue (e.g., in Peyronie's disease), retroperitoneal soft tissue (e.g., retroperitoneal fibrosis), musculoskeletal tissue, and the like.

[0025] In some embodiments, fibrosis is caused by trauma to tissue. In some embodiments, fibrosis is associated with surgery, e.g., postoperative fibrosis. Non-limiting examples of procedures that can induce fibrosis include ligament reconstruction, anterior cruciate ligament (ACL) reconstruction, autograft ACL reconstruction, allograft ACL reconstruction, fracture repair, total knee arthroplasty (TKA), and microdiscectomy. In addition, fibrosis can be caused by any of the following conditions that can be induced or aggravated by surgery: knee arthrofibrosis, intra-articular fibrous nodules, or epidural fibrosis.

[0026] As used herein, the term "musculoskeletal trauma" refers to any injury affecting bones, muscles, ligaments, nerves, or tendons. In some embodiments, the trauma is the result of surgery.

[0027] The terms "polynucleotide", "nucleotide", and "nucleic acid" are used interchangeably herein and refer to all forms of nucleic acid, 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, for example, but not limited to, RNAi, miRNA, lncRNA, RNA antagonists, aptamers, and any other non-coding RNA known to those of skill in the art. Polynucleotides include natural, 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. A polynucleotide 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 includes both a double-stranded form and two complementary single-stranded forms that are known or predicted to constitute a double-stranded form. A polynucleotide may be single-, double-, or triple-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.

[0028] 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 includes a polynucleotide containing at least one open reading frame that is capable of encoding a particular protein or polypeptide after being transcribed and translated.

[0029] The term "extracellular domain", when referring to a transmembrane cellular receptor, is defined as the portion of a protein that is exposed to the extracellular environment and is capable of engaging and / or binding a ligand.

[0030] The terms "cytoplasmic domain" and "intracellular domain" may be used interchangeably and, when referring to transmembrane receptors, define the portions of a protein that are exposed to the cytoplasm. Often, these portions of a protein contain signaling domains to recruit and associate with various intracellular factors. After engagement with a ligand via the extracellular domain, the interaction effect may change, resulting in new association, dissociation, or recruitment of various cytoplasmic factors that help transmit the signal.

[0031] The term "transmembrane domain", when referring to a transmembrane receptor, is defined as the portion of a protein that is embedded within the plasma membrane (i.e., not exposed to either the extracellular environment or the cytosol). Transmembrane domains generally have a more hydrophobic character than either the extracellular or cytoplasmic portions, and often adopt a superhelical structure. Although its primary role is as an anchor, ligand-induced conformational changes to certain receptors have been shown to affect the transmembrane domain, making it essential for subsequent intracellular signal transduction.

[0032] The term "receptor" refers to a protein that can bind with high affinity to another cognate protein (i.e., its ligand). This receptor-ligand interaction can be 1:1 or can result in multimerization, where multiple proteins aggregate to bind to one or more ligands. Receptors are generally present on the cell surface so that they can most efficiently encounter ligands and initiate intracellular signaling.

[0033] The term "intracellular signaling" refers to cellular changes resulting from events occurring at the cell surface. Typically, a soluble ligand binds to its receptor at the cell surface, which can induce changes in the receptor, so that related intracellular factors are also affected. These factors can then affect others within the cell, and this cascade often continues until a particular factor can modify gene expression in the nucleus in response to a stimulus at the surface.

[0034] The term "RNA-guided nuclease" refers to an enzyme that can, for example, destroy the backbone of a DNA molecule. The activity of the RNA-guided nuclease is guided by a nucleic acid molecule (i.e., gRNA). When properly oriented to form a functional ribonucleoprotein complex, the enzyme is localized to a specific location within the target nucleic acid (e.g., a gene or locus) through sequence complementarity with a portion of the gRNA. Non-exhaustive examples of RNA-guided nucleases include Cas9, Cpf1, and Cas12.

[0035] The term "Cas9" refers to an RNA-guided nuclease, double-stranded DNA-binding nuclease protein or nickase protein, or variants thereof, and may be used to refer to either naturally occurring or recombinant Cas9 nuclease variants (e.g., ES-Cas9, HF-Cas9, PE-Cas9, and AR-Cas9). Wild-type Cas9 nuclease has two functional domains, e.g., RuvC and HNH, that simultaneously cleave different strands of double-stranded DNA, resulting in a double-stranded break. The Cas9 enzymes described herein may contain an HNH or HNH-like nuclease domain and / or a RuvC or RuvC-like nuclease domain without affecting their ability to induce a double-stranded break in genomic DNA (e.g., at a target locus) when both functional domains are active. The Cas9 enzyme may comprise one or more catalytic domains of a Cas9 protein 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 from different bacterial species.

[0036] As used herein, "PAM" refers to a protospacer adjacent motif, which is necessary for an RNA-guided nuclease to bind to a target nucleic acid. In many cases, the PAM is immediately adjacent to the complementary sequence in the target. Native Cas9, e.g., molecules recognize a specific PAM sequence (see, e.g., Table 1). In some embodiments, the Cas9 molecule has the same PAM specificity as the native Cas9 molecule. In other embodiments, the Cas9 molecule has a PAM specificity that is not associated with the native Cas9 molecule. In other embodiments, the PAM specificity of the Cas9 molecule is not associated with the native Cas9 molecule with which it has the closest sequence homology. For example, a native Cas9 molecule can be modified such that PAM sequence recognition is modified 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, improve Cas9 specificity to a high level of identity, reduce off-target sites, and / or 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 engineered to ablate the PAM recognition.

[0037] The terms "guide RNA", "gRNA", or "sgRNA" may be used interchangeably and refer to an RNA molecule, preferably a synthetic RNA molecule, composed of a targeting (crRNA) sequence and a scaffold. These molecules, when loaded onto a functional RNA-guided nuclease, can direct sequence-specific cleavage of a target nucleic acid.

[0038] The sgRNA can be administered or formulated, for example, as a synthetic RNA or as a nucleic acid comprising a sequence encoding the gRNA, which is then expressed in the target cell. As will be apparent to one of skill in the art, various tools can be used in the design and / or optimization of the sgRNA, for example, to increase the specificity and / or precision of genome editing at a particular site.

[0039] Generally, candidate sgRNAs can be designed and identified by first locating a suitable PAM in the genome sequence. Additional calculations can then be used to predict on-target and off-target efficiency. Available web-based tools to aid in the initial setup and modeling of candidate sgRNAs include, but are not limited to, CRISPR seek, CRISPR Design Tool, Cas-OFFinder, E-CRISP, ChopChop, CasOT, CRISPR direct, CRISPOR, BREAKING-CAS, CrispRGold, and CCTop. See, for example, Safari, et al. Current Pharma. Biotechol. (2017) 18(13), which is incorporated herein by reference in its entirety for all purposes. Such tools are also described, for example, in PCT 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. Candidate sgRNAs can be further evaluated by experimental screening or other methodologies.

[0040] The term "CRISPR RNA" or "crRNA" refers to the portion of an sgRNA molecule that has complementarity to a target nucleic acid.

[0041] The phrase "pharmacologically acceptable" refers to those compounds, materials, compositions, and / or dosage forms that are within the scope of sound medical judgment 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.

[0042] 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 where any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic composition of the present disclosure is contemplated. Additional active pharmaceutical ingredients, such as other drugs, may also be incorporated into the compositions and methods described.

[0043] 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 pharma-ceutically active substances is well known in the art. Examples of such vehicles and carriers include solutions, solvents, dispersion media, retardants, emulsions, etc. Any other conventional carriers suitable for use with multibinding compounds also fall within the scope of this disclosure.

[0044] As used herein, the terms "a," "an," or "the" are generally intended to cover both the singular and the plural.

[0045] The terms "about" and "approximately" mean within a statistically significant range of values. Such ranges can be within an order of magnitude, preferably within 50%, more preferably within 20%, more preferably still within 10%, and even more preferably within 5% of a given value or range. 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, precise, but may be approximate and / or larger or smaller, as desired, to reflect tolerances, conversion factors, rounding, measurement errors, and the like, and other factors known to those of ordinary skill in the art. In general, a dimension, size, formulation, parameter, shape, or other quantity or characteristic is "about" or "approximately" whether or not it is explicitly stated as such. It should be noted that embodiments of very different sizes, shapes, and dimensions may adopt the arrangement described.

[0046] As used herein, the term "substantially" can refer to the 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.

[0047] When used in the appended claims, in their original and amended forms, the transitional phrases "comprising," "consisting essentially of," and "consisting of" define the claims as to which additional unrecited claim elements or steps, if any, are excluded from the claims. The term "comprising" is intended to be inclusive or open-ended and does not exclude any additional unrecited elements, methods, steps, or materials. The term "consisting of" excludes any 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 claims 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 be more specifically defined in alternative embodiments by any of the transitional phrases "comprising," "consisting essentially of," and "consisting of."

[0048] III. Method A. CRISPR Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled 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. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0049] In one aspect, the disclosure encompasses compositions related to clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated RNA-guided nucleases, and related methods, components, and compositions (hereinafter CRISPR / Cas systems). Such systems minimally require at least one isolated or non-naturally occurring RNA-guided nuclease (e.g., Cas9 protein) and at least one isolated or non-naturally occurring guide RNA (e.g., sgRNA) to effect augmentation of a nucleic acid sequence (e.g., genomic DNA).

[0050] In some embodiments, the CRISPR / Cas system effects modification of a gene or allele by effecting a modification of the sequence at a target location (e.g., an insertion or deletion (collectively, indels) resulting in a loss of function (i.e., a knockout) of the affected gene or allele; a nucleotide substitution resulting in, for example, a truncation, nonsense mutation, or other loss of function of the encoded product of, for example, one or more TGFB1, TGFBR1, or TGFBR2 genes (i.e., mRNA or protein); a deletion of one or more nucleotides resulting in, for example, a truncation, nonsense mutation, or other loss of function of the encoded product of, for example, one or more TGFB1, TGFBR1, or TGFBR2 genes 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, e.g., by causing a single nucleotide, double nucleotide, or other frameshift insertion, or an insertion resulting in a premature stop codon, resulting in a truncation, nonsense mutation, or other loss of function of the encoded gene product, e.g., the encoded gene product of one or more TGFB1, TGFBR1, or TGFBR2 genes (i.e., mRNA or protein), resulting in the modification of a target gene or locus in a eukaryotic cell. In some embodiments, the CRISPR / Cas system of the present disclosure provides for the modification of a gene and / or the encoded product of the gene, such that the modified product has a resulting loss of function and becomes a dominant negative or decoy (e.g., a transmembrane or soluble receptor unable to initiate intracellular signaling).

[0051] 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, an RNA-guided nuclease is located at a target location within a targeted gene or locus by sequence complementarity with a target genomic sequence (e.g., a complementary component of a CRISPR RNA (crRNA) or synthetic single guide RNA (sgRNA)) and cleaves the genomic DNA upon recognition of a specific nuclease-specific motif, called a protospacer adjacent motif (PAM). See generally, Collias, D., & Beisel, CL (2021). Nature Communications, 12(1), 1-12.

[0052] Nuclease activity (i.e., cleavage) induces double-strand breaks (DSBs) in the case of genomic DNA. The endogenous cellular mechanisms of DSB repair, i.e., non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and homologous recombination, result in erroneous repair at a given target position with some calculable frequency as a result of interference from the 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. With some frequency, these indels and / or substitutions can result in frameshifts, nonsense mutations (i.e., premature stop codons), or truncations that affect the availability of gene products such as mRNA and / or proteins. In certain embodiments, the CRISPR / Cas system can induce a homology-directed repair (HDR) mechanism that leads to the insertion of a non-random sequence at a target location through the use of a template (e.g., 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.

[0053] In general, the minimum requirements for a CRISPR / Cas system depend on the nuclease (i.e., Cas protein) provided therewith. To this extent, these bacterial nucleases are 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.

[0054] Class 1 CRISPR / Cas systems: The exact components, compositions, and methods for effecting alterations in a target nucleic acid sequence using a class 1 CRISPR / Cas system will vary, but should minimally include a nuclease (e.g., selected from at least type I and type III), at least one guide RNA, e.g., 1) selected from sgRNA, or 2) a combination of crRNA and tracrRNA. These CRISPR / Cas systems have been classified together as class 1 CRISPR / Cas systems due to their similarity in requirements and mode of action in eukaryotic cells. To this end, compositions, components, and methods between class 1 components may be considered functionally interchangeable, and the following details provided merely for illustrative purposes do not represent an exhaustive list of class members.

[0055] Cas3 (see Table 1) is a prototype type I DNA nuclease that functions as an effector protein as part of a larger complex (Cascade complex, including Cse1, 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 Cas3 nuclease via the Cascade complex, which then recruits Cas3 to cleave DNA upon binding and locating the 3'PAM. The Cascade complex is also responsible for processing crRNA so that it can be used to guide crRNA to the target location. Because of this functionality, Cascade has the ability 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.

[0056] Since the natural Cas3 substrate is ssDNA, its function in genome editing is thought to be as a nicase; however, when targeted in tandem, the resulting edit is the result of blunt-end cleavage on the opposing strand to approximate a blunt-cutting endonuclease such as Cas9. See Pickar-Oliver, A., & Gersbach, CA (2019). Nature Reviews Molecular Cell Biology, 20(8), 490-507.

[0057] Similar to type I nucleases, type III systems rely on a complex of proteins to effect nucleic acid cleavage. In particular, Cas10 has nuclease activity to cleave ssDNA in prokaryotes. See Tamulaitis, G. Trends in Microbiology, 25(1), 49-61. Interestingly, this CRISPR / Cas system of archaeal origin exhibits dual specificity, targeting both ssDNA and ssRNA. Except for this change, the system functions very similarly to type I in that the crRNA targets the 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, since both genomic DNA and, in some cases, mRNA with the same sequence can be targeted to silence specific targeted genes.

[0058] Class 2 CRISPR / Cas systems: The exact components, compositions, and methods for effecting changes in 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 sgRNA, or 2) a combination of crRNA and tracrRNA. These CRISPR / Cas systems have been classified together as class 2 CRISPR / Cas systems due to their similarity in requirements and mode of action in eukaryotic cells. To this end, compositions, components, and methods between class 2 components may be considered functionally interchangeable, and the following details provided merely for illustrative purposes do not represent an exhaustive list of class members.

[0059] Type II nucleases are the best characterized CRISPR / Cas systems, especially the canonical genome editing nuclease Cas9 (see Table 1). Multiple Cas9 proteins from various bacterial species have been isolated. The main difference between these nucleases is the required recognition site, PAM, in the targeting dsDNA. After association with the gRNA molecule, the crRNA (or the targeting domain of the sgRNA) orients the nuclease to the appropriate position, while the protein recognition of the PAM induces a cleavage event near the site, resulting in a blunt DSB.

[0060] In addition to the naturally occurring Cas9 protein, several engineered variants have been reported as well. These range from Cas9s with enhanced specificity (i.e., lower off-target activity), such as espCas9. Others are catalytically modified via point mutations in the RuvC (e.g., D10A) and HNH (e.g., H840A) domains, so that they only induce single-strand breaks (i.e., Cas9 nickase). See Frock, R. et al. (2015). Nature Biotechnology, 33(2), 179-186. They have also been shown to be less error-prone in editing. Such mitigation of off-target effects is paramount when selecting the desired insertion (i.e., knock-in mutations in which the desired nucleotide sequence is introduced into the target nucleic acid molecule) rather than a deletion. Indeed, fewer off-target effects may aid in favorable DNA repair mechanisms (most often HDR for knock-in mutations). See generally, Naeem, M., et al. (2020). Cells, 9(7), 1608.

[0061] Additional exemplary engineered variants (e.g., mutants, chimeras) of canonical Cas proteins include the following (each of which is incorporated by reference herein in their entirety for all purposes): WO2015 / 035162A2, WO2019 / 126716A1, WO2019 / 126774A1, WO2014 / 093694A1, WO2014 / 150624A1, US2019 / 0225955A1, U.S. Pat. No. 11427818, U.S. Pat. No. 11242542, U.S. Pat. No. 11098297, U.S. Pat. No. 10876100, U.S. Pat. No. 10767193, U.S. Pat. No. 10494621, and U.S. Pat. No. 10100291.

[0062] 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.

[0063] 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, for example, the blunt cut of Cas9 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 that simultaneously nicks 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 to mismatches. See Yang H, et al. Cell. 2016;167(7):1814-1828.e12. [Table 1]

[0064] 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.

[0065] In one aspect, the CRISPR / Cas system of the present disclosure comprises at least one RNA-guided nuclease (e.g., 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.

[0066] Some aspects of the disclosure provide strategies, methods, compositions, and therapeutic modalities for modifying a targeted sequence within a genetic locus (e.g., modifying the sequence of a wild-type and / or mutant sequence within a cell or mammal) by insertion or deletion of one or more nucleotides mediated by an RNA-guided nuclease and one or more guide RNAs (gRNAs), resulting in loss of function of the targeted gene product. In some embodiments, the loss of function results in "knocking out" a gene of interest by eliminating gene expression (i.e., producing a "knockout"). In some embodiments, the loss of function results in a non-functional gene product (i.e., a gene product that does not have all the functionality of the wild-type gene product). In some embodiments, the loss of function results in the expression of a gene product with different properties (e.g., different binding affinity or different cellular localization).

[0067] In certain embodiments, the targeted gene is selected from TGFB1, TGFBR1, TGFBR2, and combinations thereof. 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 RNA-guided nuclease to modify the gene. In some embodiments, non-coding regions of the targeted gene (e.g., enhancer region, promoter region, intron, 5' UTR, 3' UTR, polyadenylation signal) are targeted to modify the gene.

[0068] 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 an RNA-guided nuclease. In certain embodiments, the gRNA is an sgRNA that includes a crRNA sequence that includes a nucleotide sequence that is complementary to a sequence in a target nucleic acid. In some embodiments, the sgRNA further includes an RNA scaffold portion (tracrRNA) that interacts with the RNA-guided nuclease, such that the crRNA is positioned to scan the target nucleic acid for complementarity. In some embodiments, the system further comprises an oligonucleotide-HDR template that optionally has homology on either side of the target position. See Bloh, K., & Rivera-Torres, N, at 3836.

[0069] In one embodiment, the RNA-guided nuclease and sgRNA are configured to orient the associated nuclease such that a cleavage event (e.g., a double-stranded or single-stranded cleavage) occurs in sufficient proximity to a complementary sequence in the targeted nucleic acid, thereby facilitating modification of the nucleic acid sequence. In some embodiments, the crRNA is 20 nucleotides in length. In some embodiments, the crRNA is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0070] In some embodiments, the crRNA directs the RNA-guided nuclease such 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 complementary sequence in the targeting nucleic acid. The cleavage of double-stranded or single-stranded DNA can be located upstream or downstream of the complementary sequence in the targeting nucleic acid. In some embodiments, the cleavage event occurs within the targeted gene. In some embodiments, the cleavage event occurs upstream of the targeted gene.

[0071] In certain embodiments, the second gRNA molecule, including the second crRNA, orients the second RNA-guided nuclease such that a cleavage event occurs in sufficient proximity to a complementary sequence in the targeted nucleic acid, thereby facilitating modification of the nucleic acid sequence. In some embodiments, the first gRNA and the second gRNA facilitate a cleavage event in a single targeted gene. In some embodiments, the first gRNA and the second gRNA facilitate cleavage events in different targeted genes. In some embodiments, the second crRNA is 20 nucleotides in length. In some embodiments, the second crRNA is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0072] In some embodiments, the second crRNA directs the RNA-guided nuclease such 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 complementary sequence in the targeting nucleic acid. The cleavage of double-stranded or single-stranded DNA can be located upstream or downstream of the complementary sequence in the targeting nucleic acid. In some embodiments, the cleavage event occurs within the targeted gene. In some embodiments, the cleavage event occurs upstream of the targeted gene.

[0073] In some embodiments, the targeting domains of the first gRNA and the second gRNA are configured such that the cleavage events are 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 other cleavage event, independently for each gRNA molecule. In some embodiments, the first gRNA and the second gRNA modify the targeting nucleic acid sequence simultaneously. In some embodiments, the first gRNA and the second gRNA modify the targeting nucleic acid sequence sequentially.

[0074] In some embodiments, the single-stranded cleavage is accompanied by a second single-stranded cleavage positioned by the crRNA of the first gRNA and the second gRNA, respectively. For example, the crRNA may orient the associated RNA-guided nuclease such that the cleavage events (e.g., the two single-stranded cleavage events) are 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 each other. In some embodiments, the first crRNA and the second crRNA are configured to orient their associated RNA-guided nucleases such that, for example, two single-stranded breaks occur on opposite strands of genomic DNA at the same position 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.

[0075] In some embodiments, the nucleic acid encoding the one or more crRNAs is selected from any of the sequences disclosed in Table 2. In some embodiments, the nucleic acid encoding the one or more crRNAs is selected from any of the sequences disclosed in Table 3. In some embodiments, the nucleic acid encoding the one or more crRNAs is selected from any of the sequences disclosed in Table 4. In some embodiments, the nucleic acid encodes a second sgRNA molecule. In some embodiments, the nucleic acid encodes a third sgRNA molecule. In some embodiments, the nucleic acid encodes a fourth sgRNA molecule. In certain embodiments, the one or more sgRNAs are encoded by a nucleic acid selected from any of SEQ ID NOs: 1-519.

[0076] In certain embodiments, the nucleic acid may include (a) a sequence encoding a first sgRNA comprising a crRNA that is complementary to a sequence in a targeting gene, (b) a sequence encoding a second sgRNA comprising a crRNA that is complementary to a sequence in a second targeting gene, and (c) a sequence encoding an RNA-guided nuclease (e.g., Cas9). Optionally, (d) and (e) are sequences encoding a third sgRNA and a fourth sgRNA, respectively. In some embodiments, the second targeting gene is the same as the first targeting gene. In other embodiments, the second targeting gene is different from the first targeting gene. In some embodiments, (a), (b), and (c) are encoded within the same nucleic acid molecule (e.g., the same 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 sgRNAs are used, any combination of (a), (b), (c), (d), and (e) can be encoded within a single or separate nucleic acid molecule.

[0077] 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).

[0078] In some embodiments, contacting a target cell comprises delivering said RNA-guided nuclease of (c) as a protein together with at least one said nucleic acid molecule selected from (a), (b), (d), and (e). In some embodiments, contacting a target cell comprises delivering said encoded RNA-guided nuclease of (c) as DNA together with at least one said nucleic acid molecule selected from (a), (b), (d), and (e). In some embodiments, contacting a target cell comprises delivering said encoded RNA-guided nuclease of (c) as mRNA together with at least one said nucleic acid molecule selected from (a), (b), (d), and (e).

[0079] In certain embodiments, CRISPR components are delivered to target cells via nanoparticles. Exemplary nanoparticles that may 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.

[0080] B.TALEN In one aspect, the present disclosure contemplates the use of methods, components, and compositions relating to nucleic acid sequences (e.g., transcription activator-like effector nucleases (TALENs) to effect targeted gene enhancement.

[0081] TALE is an abbreviation for "Transcription Activator-Like Effector" proteins, including TALENs ("Transcription Activator-Like Effector Nucleases"). Methods using the TALE system for gene editing may also be referred to herein as TALE methods. TALEs are naturally occurring proteins from plant pathogenic bacteria of the genus Xanthomonas that contain a DNA-binding domain composed of a series of 33-35 amino acid repeat domains that each recognize a single base pair. TALE specificity is determined by two hypervariable amino acids known as repeat variable diresidues (RVDs). Modular TALE repeats are linked together to recognize consecutive DNA sequences. Specific RVDs within the DNA-binding domain provide the structural features that recognize bases within the target locus and assemble predictable DNA-binding domains. 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 FokI monomers into close proximity to dimerize and generate the targeted double-stranded break.

[0082] 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.

[0083] Non-limiting examples of genes that can be silenced or inhibited by permanent gene editing via the TALE method include TGFB1, TGFBR1, TGFBR2, and combinations thereof. Non-limiting examples of genes that can be enhanced by permanent gene editing via the TALE method such that their resulting products function as decoys or dominant negatives include TGFB1, TGFBR1, TGFBR2, and combinations thereof. Non-limiting examples of genes that can be enhanced by permanent gene editing via the TALE method include TGFB1, TGFBR1, TGFBR2, and combinations thereof. In one aspect, the present disclosure provides compositions for upregulation of protein receptors (including wild type or gene edited), including those that bind anti-inflammatory cytokines via the TALE method.

[0084] 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. Pat. No. 8,586,526, which is incorporated herein by reference.

[0085] C. Zinc finger nucleases (ZFNs) In one aspect, the present disclosure contemplates the use of zinc finger nuclease (ZFN)-related methods, components, and compositions to effect the enhancement of nucleic acid sequences (e.g., targeting genes).

[0086] 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 3bp of the major groove of DNA with varying levels of selectivity. Zinc fingers have two protein domains. The first domain is the DNA binding domain, which includes eukaryotic transcription factors and contains the zinc finger. The second domain is the nuclease domain, which includes the FokI restriction enzyme and is responsible for the catalytic cleavage of DNA.

[0087] The DNA binding domain of an individual ZFN typically contains 3-6 individual zinc finger repeats, each capable of recognizing 9-18 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 way to generate new zinc finger arrays is to combine smaller zinc finger "modules" of known specificity. The most common modular assembly process involves combining three separate zinc fingers, each capable of recognizing a three base pair DNA sequence, to generate a three-finger array capable of recognizing a nine 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 take into account context-dependent interactions between adjacent fingers. Engineered zinc fingers are commercially available, and Sangamo Biosciences (Richmond, Calif., USA) has partnered with Sigma-Aldrich (St. Louis, Mo., USA) to develop a unique platform for zinc finger construction (CompoZr®).

[0088] Non-limiting examples of genes that can be silenced or inhibited by permanent gene editing via zinc finger methodology include TGFB1, TGFBR1, TGFBR2, and combinations thereof. Non-limiting examples of genes that can be enhanced by permanent gene editing via zinc finger methodology so that their resulting products function as decoys or dominant negatives include TGFB1, TGFBR1, TGFBR2, and combinations thereof. Non-limiting examples of genes that can be enhanced by permanent gene editing via zinc finger methodology include TGFB1, TGFBR1, TGFBR2, and combinations thereof. In one aspect, the present disclosure provides compositions for upregulation of protein receptors (including wild type or gene editing), including those that bind anti-inflammatory cytokines via zinc finger methodology.

[0089] 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. Pat. 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.

[0090] 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.

[0091] IV. Fibrosis / Scarring A. Introduction Musculoskeletal tissues are particularly prone to fibrosis and / or scarring following trauma. In many cases, this trauma can be a surgical procedure. Indeed, the formation of musculoskeletal scar tissue presents several problems, including limiting the range of motion, causing pain, and complicating subsequent surgical access to the site of injury. Without wishing to be bound by a particular theory, increased expression of TGFB1 is observed in fascial fibroblasts at the site of such musculoskeletal trauma, among other factors. See Ihn, H. (2019). Allergology International, 68(4), 437-439. This expression pattern correlated with higher expression of type I collagen and fibronectin, contributors to the fibrotic process.

[0092] In one aspect, the compositions and methods described herein are directed to the treatment of excessive fibrosis and / or scarring. In some embodiments, the fibrosis and / or scarring is a result of surgery. In some embodiments, the surgery is a ligament reconstruction. In some embodiments, the surgery is anterior cruciate ligament (ACL) reconstruction. In some embodiments, the surgery is an autograft ACL reconstruction. In some embodiments, the surgery is an allograft ACL reconstruction. In some embodiments, the surgery is a fracture repair. In some embodiments, the surgery is a total knee arthroplasty (TKA). In some embodiments, the surgery is a microdiscectomy.

[0093] In some embodiments, the fibrosis and / or scarring is the result of a condition that may be induced or exacerbated by surgery. In some embodiments, the condition that contributes to musculoskeletal fibrosis and / or scarring is knee joint fibrosis. In some embodiments, the condition that contributes to musculoskeletal fibrosis and / or scarring is intra-articular fibrous nodules. In some embodiments, the condition that contributes to musculoskeletal fibrosis and / or scarring is epidural fibrosis.

[0094] B. Fibrosis In one aspect, the present disclosure encompasses a treatment for fibrosis. In some embodiments, the CRISPR gene editing system for treating fibrosis comprises one or more sgRNAs targeting TGFB1. In some embodiments, the CRISPR gene editing system for treating fibrosis comprises one or more sgRNAs targeting hTGFB1. In some embodiments, the CRISPR gene editing system for treating fibrosis comprises one or more sgRNAs targeting cTGFB1. In some embodiments, the CRISPR gene editing system for treating fibrosis comprises one or more sgRNAs targeting eTGFB1. In some embodiments, the CRISPR gene editing system for treating fibrosis comprises one or more sgRNAs targeting fTGFB1.

[0095] In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 1 of hTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 2 of hTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 3 of hTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 4 of hTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 5 of hTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 6 of hTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 7 of hTGFB1.

[0096] In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 1 of cTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 2 of cTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 3 of cTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 4 of cTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 5 of cTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 6 of cTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 7 of cTGFB1.

[0097] In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 1 of eTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 2 of eTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 3 of eTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 4 of eTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 5 of eTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 6 of eTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 7 of eTGFB1.

[0098] In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 1 of fTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 2 of fTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 3 of fTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 4 of fTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 5 of fTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 6 of fTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 7 of fTGFB1.

[0099] In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting TGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting cTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting fTGFBR1.

[0100] In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 1 of hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 2 of hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 3 of hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 4 of hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 5 of hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 6 of hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 7 of hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 8 of hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 9 of hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 10 of hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 11 of hTGFBR1.

[0101] In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 1 of cTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 2 of cTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 3 of cTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 4 of cTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 5 of cTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 6 of cTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 7 of cTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 8 of cTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 9 of cTGFBR1.

[0102] In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 1 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 2 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 3 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 4 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 5 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 6 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 7 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 8 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 9 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 10 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 11 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 12 of eTGFBR1.

[0103] In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 1 of fTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 2 of fTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 3 of fTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 4 of fTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 5 of fTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 6 of fTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 7 of fTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 8 of fTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 9 of fTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 10 of fTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 11 of fTGFBR1.

[0104] In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting TGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting hTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting cTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting eTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting fTGFBR2.

[0105] In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 1 of hTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 2 of hTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 3 of hTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 4 of hTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 5 of hTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 6 of hTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 7 of hTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 8 of hTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 9 of hTGFBR2.

[0106] In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 1 of cTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 2 of cTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 3 of cTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 4 of cTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 5 of cTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 6 of cTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 7 of cTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 8 of cTGFBR2.

[0107] In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 1 of eTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 2 of eTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 3 of eTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 4 of eTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 5 of eTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 6 of eTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 7 of eTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 8 of eTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 9 of eTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 10 of eTGFBR2.

[0108] In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 1 of fTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 2 of fTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 3 of fTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 4 of fTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 5 of fTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 6 of fTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 7 of fTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis comprises one or more sgRNAs targeting exon 8 of fTGFBR2.

[0109] C. Musculoskeletal scarring In one aspect, the present disclosure encompasses a treatment for musculoskeletal scarring. In some embodiments, a CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting TGFB1. In some embodiments, a CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting hTGFB1. In some embodiments, a CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting cTGFB1. In some embodiments, a CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting eTGFB1. In some embodiments, a CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting fTGFB1.

[0110] In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 1 of hTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 2 of hTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 3 of hTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 4 of hTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 5 of hTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 6 of hTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 7 of hTGFB1.

[0111] In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 1 of cTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 2 of cTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 3 of cTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 4 of cTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 5 of cTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 6 of cTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 7 of cTGFB1.

[0112] In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 1 of eTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 2 of eTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 3 of eTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 4 of eTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 5 of eTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 6 of eTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 7 of eTGFB1.

[0113] In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 1 of fTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 2 of fTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 3 of fTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 4 of fTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 5 of fTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 6 of fTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 7 of fTGFB1.

[0114] In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting TGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting cTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting fTGFBR1.

[0115] In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 1 of hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 2 of hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 3 of hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 4 of hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 5 of hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 6 of hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 7 of hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 8 of hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 9 of hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 10 of hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 11 of hTGFBR1.

[0116] In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 1 of cTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 2 of cTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 3 of cTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 4 of cTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 5 of cTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 6 of cTGFBR1. In some embodiments, a CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 7 of cTGFBR1. In some embodiments, a CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 8 of cTGFBR1. In some embodiments, a CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 9 of cTGFBR1.

[0117] In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 1 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 2 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 3 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 4 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 5 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 6 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 7 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 8 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 9 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 10 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 11 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 12 of eTGFBR1.

[0118] In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 1 of fTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 2 of fTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 3 of fTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 4 of fTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 5 of fTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 6 of fTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 7 of fTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 8 of fTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 9 of fTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 10 of fTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 11 of fTGFBR1.

[0119] In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting TGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting hTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting cTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting eTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting fTGFBR2.

[0120] In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 1 of hTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 2 of hTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 3 of hTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 4 of hTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 5 of hTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 6 of hTGFBR2. In some embodiments, a CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 7 of hTGFBR2. In some embodiments, a CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 8 of hTGFBR2. In some embodiments, a CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 9 of hTGFBR2.

[0121] In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 1 of cTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 2 of cTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 3 of cTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 4 of cTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 5 of cTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 6 of cTGFBR2. In some embodiments, a CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 7 of cTGFBR2. In some embodiments, a CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 8 of cTGFBR2.

[0122] In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 1 of eTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 2 of eTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 3 of eTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 4 of eTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 5 of eTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 6 of eTGFBR2. In some embodiments, a CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 7 of eTGFBR2. In some embodiments, a CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 8 of eTGFBR2. In some embodiments, a CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 9 of eTGFBR2. In some embodiments, a CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 10 of eTGFBR2.

[0123] In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 1 of fTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 2 of fTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 3 of fTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 4 of fTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 5 of fTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 6 of fTGFBR2. In some embodiments, a CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 7 of fTGFBR2. In some embodiments, a CRISPR gene editing system for the treatment of musculoskeletal scarring comprises one or more sgRNAs targeting exon 8 of fTGFBR2.

[0124] V. Delivery A. Viral Vectors In one aspect, the disclosure encompasses a method of delivery of a CRISPR gene editing system targeting a gene selected from TGFB1, TGFBR1, TGFBR2, and combinations thereof using one or more recombinant viral particles. In some embodiments, one of the more viral vectors comprises a recombinant virus selected from retrovirus, adenovirus, adeno-associated virus, lentivirus, and herpes simplex virus-1. In some embodiments, one of the more viral vectors comprises 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). 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 encoding a Cas protein, and a second viral vector comprising a second nucleic acid of the one or more nucleic acids encoding at least one guide RNA. In some embodiments, the one or more viral vectors comprise a viral vector comprising a single nucleic acid, the single nucleic acid encoding a Cas9 protein and at least one guide RNA.

[0125] 1. Adeno-associated virus (AAV) A useful viral vector system for delivery of nucleic acids is the adeno-associated virus (AAV), a naturally defective virus that requires another virus, such as an adenovirus or a herpesvirus, as a helper virus for efficient replication and a productive life cycle. (For 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 are able to integrate. Space for exogenous DNA is limited to approximately 4.5 kb. Tratschin et al. AAV vectors such as those described in (Hermonat et al., Mol. Cell. Biol. 5:3251-3260 (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.

[0126] According to certain embodiments, the CRISPR gene editing system for targeting a gene selected from TGFB1, TGFBR1, TGFBR2, and combinations thereof further comprises a recombinant AAV vector. In some embodiments, the CRISPR gene editing system is encoded by a nucleic acid, and the nucleic acid is a recombinant AAV genome. In some embodiments, the AAV vector is selected from an AAV1 vector, an AAV2 vector, an AAV3 vector, an AAV4 vector, an AAV5 vector, an AAV6 vector, an AAV7 vector, an AAV8 vector, an AAV9 vector, and an AAV10 vector.

[0127] 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).

[0128] In one aspect, the use of the CRISPR gene editing system further comprising one or more AAV vectors for targeting TGFB1, TGFBR1, and / or TGFBR2 is therapeutic. In some embodiments, the use of the system treats fibrosis and / or scarring. In some embodiments, the use of the system treats one or more musculoskeletal diseases, conditions, and diseases, including, but not limited to, Loeys-Dietz syndrome, osteoarthritis, Marfan syndrome, aortic aneurysms (e.g., familial thoracic 3 aortic aneurysms), craniofacial abnormalities, and combinations thereof. In other embodiments, the use of the system treats neoplastic diseases, conditions, and diseases, including pancreatic cancer, multiple self-healing squamous cell carcinoma (Ferguson-Smith disease), gastrointestinal stromal tumors (GIST), hereditary nonpolyposis colorectal cancer (Lynch syndrome), metastatic colorectal cancer, bone neoplasms, anaplastic carcinomas, spindle cell carcinomas, lung neoplasms, brain neoplasms, and combinations thereof.

[0129] 2. Lentivirus 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).

[0130] Lentiviral transduction systems are known in the art and 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. Pat. No. 6,627,442, the disclosures of each of which are incorporated herein by reference.

[0131] In one aspect, the use of the CRISPR gene editing system further comprising one or more lentiviral vectors for targeting TGFB1, TGFBR1, and / or TGFBR2 is therapeutic. In some embodiments, the use of the system treats fibrosis and / or scarring. In some embodiments, the use of the system treats one or more musculoskeletal diseases, conditions, and diseases, including, but not limited to, Loeys-Dietz syndrome, osteoarthritis, Marfan syndrome, aortic aneurysms (e.g., familial thoracic 3 aortic aneurysms), craniofacial abnormalities, and combinations thereof. In other embodiments, the use of the system treats neoplastic diseases, conditions, and diseases, including pancreatic cancer, multiple self-healing squamous cell carcinoma (Ferguson-Smith disease), gastrointestinal stromal tumors (GIST), hereditary nonpolyposis colorectal cancer (Lynch syndrome), metastatic colorectal cancer, bone neoplasms, anaplastic carcinomas, spindle cell carcinomas, lung neoplasms, brain neoplasms, and combinations thereof.

[0132] B. Lipid Nanoparticles (LNPs) In some embodiments, the CRISPR gene editing system is delivered by nanoparticles. Without wishing to be bound by any particular theory, in certain embodiments, nucleic acids are resistant in aqueous solution to degradation by nucleases when present in nanoparticles. In other embodiments, proteins are protected from protease degradation. In some embodiments, proteins and nucleic acids encapsulated by nanoparticles can permeate cell plasma membranes.

[0133] Lipid nanoparticles containing nucleic acids and methods for preparing the same 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 / 0468 09, WO2019 / 089828, WO2020 / 061284, WO2020 / 061317, WO2020 / 081938, WO2020 / 097511, WO2020 / 097520, WO2020 / 097540, W O2020 / 097548, WO2020 / 214946, WO2020 / 219941, WO2020 / 232276, WO2020 / 227615, WO2020 / 061295, WO2021 / 007278, WO2021 / 016430, WO2021 / 021988, European Patent No. 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, 9,868,692, and 9,95 Nos. 0,068, 10,138,213, 10,166,298, 10,221,127, 10,238,754, 10,266,485, 10,383,952, 10,730,924, 10,766,852, 11,079,379, 11,141,378, and 11,246,933, which are incorporated herein by reference in their entireties for all purposes.

[0134] Lipid Nanoparticle Compositions In some embodiments, the maximum 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), for example, as measured 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 bilayers may comprise one or more ligands, proteins, or channels. 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, 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.

[0135] 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 an ionic lipid and / or a cationic lipid (i.e., a lipid that may have a positive or partial positive charge at physiological pH), a neutral lipid (e.g., a phospholipid or a sphingolipid), and a polymer-conjugated lipid (e.g., a PEGylated lipid). 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.

[0136] 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 a cationic lipid. In some embodiments, the lipid component may be, but is 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-dimethylaza (KL26), 1,2-dilinoleyloxy-N,N-dimethylaza (KL27), 1,2-dilinoleyloxy-N,N-dimethylaza (KL28), 1,2-dilinoleyloxy-N,N-dimethylaza (KL29), 1,2-dilinoleyloxy-N,N-dimethylaza (KL30), 1,2-dilinoleyloxy-N,N-dimethylaza (KL31), 1,2-dilinoleyloxy-N,N-dimethylaza (KL32), 1,2-dilinoleyloxy-N,N-dimethylaza (KL33), 1,2-dilinoleyloxy-N,N-dimethylaza (KL34), 1,2-dilinoleyloxy-N,N-dimethylaza (KL35), 1,2-dilinoleyloxy-N,N-dimethylaza (KL36), 1,2-dilinoleyloxy-N,N-dimethylaza (KL37), 1,2-dilinoleyloxy-N,N-dimethylaza (KL38), 1,2-dilinoleyloxy-N,N-dimethylaza ( 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 (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), In one embodiment, 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.

[0137] Non-exhaustive and non-limiting examples of cationic lipids include: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0138] 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 and mixtures thereof.

[0139] 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 PEG-c-DOMG, PEG-DMG, PEG 2000 -c-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DMA, or PEG-DSPE lipids.

[0140] Non-exhaustive and non-limiting examples of PEG-lipids include: [ka]

[0141] PEG-C-DMA [ka] [ka] [ka] [ka] [ka]

[0142] 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 structured lipid comprises cholesterol and a corticosteroid (e.g., prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof.

[0143] Non-exhaustive and non-limiting examples of structured lipids include: [ka] [ka] [ka]

[0144] formulation The nanoparticle composition may include a lipid component and one or more additional components, such as a therapeutic and / or prophylactic agent. The nanoparticle composition may be designed for one or more specific applications or targets. The components of the nanoparticle composition may 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 the nanoparticle composition may be selected for a particular application or target, for example, according to the efficacy and toxicity of a particular combination of components.

[0145] 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.

[0146] In some embodiments, the lipid components of the nanoparticle composition include ionic lipids, phospholipids, PEG lipids, and structured lipids. In certain embodiments, the lipid components of the nanoparticle composition include about 30 mol% to about 60 mol% ionic lipids, about 0 mol% to about 30 mol% phospholipids, about 0 mol% to about 10 mol% PEG lipids, and about 17.5 mol% to about 50 mol% structured lipids, provided that the total mol% does not exceed 100%. In some embodiments, the lipid components of the nanoparticle composition include about 35 mol% to about 55 mol% ionic lipid compounds, about 5 mol% to about 25 mol% phospholipids, about 0 mol% to about 10 mol% PEG lipids, and about 30 mol% to about 40 mol% structured lipids. In certain embodiments, the lipid components include about 50 mol% of the compounds, about 10 mol% phospholipids, about 38.5 mol% structured lipids, and about 1.5 mol% PEG lipids. In another embodiment, the lipid component comprises about 40 mol% of the compound, about 20 mol% of phospholipid, about 38.5 mol% of structured lipid, and about 1.5 mol% of PEG lipid. In some embodiments, the phospholipid can be DOPE or DSPC. In other embodiments, the PEG lipid can be PEG-DMG and / or the structured lipid can be cholesterol.

[0147] 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.

[0148] In some embodiments, the neutral lipid comprises about 0 to about 30 mol% of the lipid component. In other embodiments, the neutral lipid comprises about 5 to about 25 mol% of the lipid component. In various embodiments, the neutral lipid comprises 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.

[0149] 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, 9.5, 10, or 15 mol% of the lipid component.

[0150] In some embodiments, the structural component comprises about 17.5 mol% to about 50 mol% of the lipid component. In other embodiments, the structural component comprises about 30 to about 40 mol% of the lipid component. 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% of the lipid component.

[0151] Alternatively, the structural component may 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).

[0152] Nanoparticle compositions may be designed for one or more specific applications or targets. For example, nanoparticle compositions may be designed to deliver therapeutic and / or prophylactic agents, such as RNA, to specific cells, tissues, organs, or systems, or groups thereof, within the mammalian body. The physiochemical properties of the nanoparticle compositions may be altered to increase selectivity for specific body targets. For example, particle size may be adjusted based on fenestration size of different organs. The therapeutic and / or prophylactic agents included in the nanoparticle compositions may also be selected based on the desired delivery target. For example, therapeutic and / or prophylactic agents may 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 may include mRNA encoding a polypeptide of interest that can be translated in a cell to produce the polypeptide of interest. Such compositions may be designed to be specifically delivered to a specific organ. In some embodiments, the compositions may be designed to be specifically delivered to a mammalian joint.

[0153] The amount of therapeutic and / or prophylactic agent in the nanoparticle composition may depend on the size, composition, desired target and / or use, or other characteristics of the nanoparticle composition, and the characteristics of the therapeutic and / or prophylactic agent. For example, the amount of RNA useful in a nanoparticle composition may 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 may also vary. In some embodiments, the weight / weight ratio of lipid components to therapeutic and / or prophylactic agent in the nanoparticle composition may be 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).

[0154] 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.

[0155] 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 the nitrogen atoms in the one or more lipids to the number of phosphate groups in the RNA. In general, 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.

[0156] In some embodiments, the nucleic acid component is composed of modified nucleic acids. For example, the RNA may be modified RNA. That is, the RNA may contain one or more nucleobases, nucleosides, nucleotides, or linkers that are non-natural. "Modified" species may also be referred to as "altered" species herein. Species may be chemically, structurally, or functionally modified or altered. For example, modified nucleobase species may contain one or more substitutions that are not natural.

[0157] In certain embodiments, the disclosure includes a method for treating a back or spinal condition or disorder. In other embodiments, the disclosure includes a method for treating a pathogenic disorder. In some embodiments, the disclosure includes a method for treating a subject in need of treatment for localized pain, inflammation, or morphological changes associated with a back or spinal condition or disorder, the method includes administering a therapeutically effective amount of a CRISPR-Cas composition encapsulated in or associated with a lipid nanoparticle (LNP), the composition comprising one or more non-naturally occurring polynucleotides encoding a Cas9 protein and at least one sgRNA. In some embodiments, the LNP is administered intrathecally. In other embodiments, the LNP is administered epidurally. In some embodiments, the LNP is administered peridiscally. In some embodiments, the LNP is administered perivertebrally.

[0158] physical properties The properties of a nanoparticle composition may depend on its components. For example, a nanoparticle composition that contains cholesterol as a structural lipid may have different properties from a nanoparticle composition that contains 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 that contains a higher molar fraction of phospholipids may have different properties from a nanoparticle composition that contains a lower molar fraction of phospholipids. The properties may also vary depending on the method and conditions of preparation of the nanoparticle composition.

[0159] Nanoparticle compositions can be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of the nanoparticle composition. Dynamic light scattering or potentiometry (e.g., potentiometric titration) can be used to measure the zeta potential. Dynamic light scattering can 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.

[0160] The average size of the nanoparticle composition can be from 10 nm to 1 micrometer, for example, as measured by dynamic light scattering (DLS). For example, the average size can be from about 40 nm to about 150 nm, for example, 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.

[0161] The nanoparticle composition may be relatively homogeneous. The polydispersity index may be used to indicate the homogeneity of the nanoparticle composition, e.g., the particle size distribution of the nanoparticle composition. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. The nanoparticle composition may 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 may be about 0.10 to about 0.20.

[0162] 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.

[0163] The efficiency of encapsulation of a therapeutic and / or prophylactic agent describes the amount of therapeutic and / or prophylactic agent that is encapsulated or otherwise associated with a nanoparticle composition after preparation relative to the initial amount provided. It is desirable for the encapsulation efficiency to be high (e.g., close to 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 the solution. For the nanoparticle compositions described herein, the encapsulation efficiency of a therapeutic and / or prophylactic agent can be at least 50%, for example, 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 may be at least 80%. In certain embodiments, the encapsulation efficiency may be at least 90%.

[0164] Nanoparticle compositions may optionally include one or more coatings.For example, nanoparticle compositions may be formulated in capsules, films, or tablets with coatings.Capsules, films, or tablets containing compositions described herein may have any useful size, tensile strength, hardness, or density.

[0165] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled 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. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0166] In some embodiments, the CRISPR gene editing system comprises one or more RNA-containing compositions. In some embodiments, the CRISPR gene editing system further comprises one or more nanoparticles. In some embodiments, the one or more RNA-containing compositions comprise a guide RNA. In some embodiments, the one or more RNA-containing compositions comprise an mRNA. In some embodiments, the one or more RNA-containing compositions comprise an RNP (e.g., Cas9 and a guide RNA). In some embodiments, the one or more nanoparticles are lipid nanoparticles (LNPs).

[0167] In some embodiments, the CRISPR gene editing system comprises one or more LNPs that collectively encapsulate (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease, and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA. In some embodiments, the one or more LNPs comprise a first plurality of LNPs encapsulating an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease, and a second plurality of LNPs encapsulating at least one guide RNA or a nucleic acid encoding at least one guide RNA.

[0168] In some embodiments, one or more LNPs are 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 (KL26), 1,2-diaminopropane (KL27), 1,2-diaminopropane (KL28), 1,2-diaminopropane (KL29), 1,2-diaminopropane (KL30), 1,2-diaminopropane (KL31), 1,2-diaminopropane (KL32), 1,2-diaminopropane (KL33), 1,2-diaminopropane (KL34), 1,2-diaminopropane (KL35), 1,2-diaminopropane (KL36), 1,2-diaminopropane (KL37), 1,2-diaminopropane (KL38), 1,2-diaminopropane (KL39 ... Pan (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 propane (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), 2-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.

[0169] In some embodiments, the one or more LNPs are 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- 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 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.

[0170] In some embodiments, one or more LNPs comprise a component selected from the group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, the PEG lipid may be a PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DMA, PEG-DSPE lipid, and mixtures thereof.

[0171] In some embodiments, one or more LNPs comprise a component 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.

[0172] In one aspect, the use of the CRISPR gene editing system further comprising one or more LNPs for targeting TGFB1, TGFBR1, and / or TGFBR2 is therapeutic. In some embodiments, the use of the system treats fibrosis and / or scarring. In some embodiments, the use of the system treats one or more musculoskeletal diseases, conditions, and diseases, including, but not limited to, Loeys-Dietz syndrome, osteoarthritis, Marfan syndrome, aortic aneurysms (e.g., familial thoracic 3 aortic aneurysms), craniofacial abnormalities, and combinations thereof. In other embodiments, the use of the system treats neoplastic diseases, conditions, and diseases, including pancreatic cancer, multiple self-healing squamous cell carcinoma (Ferguson-Smith disease), gastrointestinal stromal tumors (GIST), hereditary nonpolyposis colorectal cancer (Lynch syndrome), metastatic colorectal cancer, bone neoplasms, anaplastic carcinomas, spindle cell carcinomas, lung neoplasms, brain neoplasms, and combinations thereof.

[0173] C. Virus-like particles In one aspect, the present disclosure encompasses a means for delivering a CRISPR gene editing system to a mammalian cell via a virus-like particle (VLP). In some embodiments, the CRISPR gene editing system is delivered by the VLP. Without wishing to be bound by any particular theory, in certain embodiments, the nucleic acid is resistant in aqueous solution to degradation by nucleases when present in the particle. In other embodiments, the protein is protected from protease degradation while present in the particle. In some embodiments, the protein and nucleic acid encapsulated by the VLP can penetrate the cell plasma membrane.

[0174] In some embodiments, the CRISPR gene editing system comprises one or more RNA-containing compositions. In some embodiments, the CRISPR gene editing system further comprises one or more VLPs. In some embodiments, the one or more RNA-containing compositions comprise a guide RNA. In some embodiments, the one or more RNA-containing compositions comprise an mRNA. In some embodiments, the one or more RNA-containing compositions comprise an RNP (e.g., Cas9 and a guide RNA).

[0175] In some embodiments, the CRISPR gene editing system comprises one or more virus-like particles collectively encapsulating (i) an RNA-guided nuclease or a nucleic acid encoding the RNA-guided nuclease, and (ii) at least one guide RNA or a nucleic acid encoding the at least one guide RNA. In some embodiments, the one or more virus-like particles comprise a first plurality of virus-like particles encapsulating the RNA-guided nuclease or a nucleic acid encoding the RNA-guided nuclease, and a second plurality of virus-like particles encapsulating at least one guide RNA or a nucleic acid encoding the at least one guide RNA.

[0176] In one aspect, the use of the CRISPR gene editing system further comprising one or more VLPs for targeting TGFB1, TGFBR1, and / or TGFBR2 is therapeutic. In some embodiments, the use of the system treats fibrosis and / or scarring. In some embodiments, the use of the system treats one or more musculoskeletal diseases, conditions, and diseases, including, but not limited to, Loeys-Dietz syndrome, osteoarthritis, Marfan syndrome, aortic aneurysms (e.g., familial thoracic 3 aortic aneurysms), craniofacial abnormalities, and combinations thereof. In other embodiments, the use of the system treats neoplastic diseases, conditions, and diseases, including pancreatic cancer, multiple self-healing squamous cell carcinoma (Ferguson-Smith disease), gastrointestinal stromal tumors (GIST), hereditary nonpolyposis colorectal cancer (Lynch syndrome), metastatic colorectal cancer, bone neoplasms, anaplastic carcinomas, spindle cell carcinomas, lung neoplasms, brain neoplasms, and combinations thereof.

[0177] D. Other Methods of Delivery 1. Liposomes In some embodiments, the nucleic acid (e.g., Cas9 or gRNA) encoding the CRISPR gene editing system targeting a gene selected from TGFB1, TGFBR1, TGFBR2, and combinations thereof is entrapped in liposomes (e.g., lipofectin) that have a positive charge on their surface and can be tagged with an antibody against a cell surface antigen of the target cell. These delivery vehicles can also be used to deliver the Cas9 protein / gRNA complex.

[0178] In some embodiments, the CRISPR gene editing system comprises one or more RNA-containing compositions. In some embodiments, the CRISPR gene editing system further comprises one or more liposomes. In some embodiments, the one or more RNA-containing compositions comprise a guide RNA. In some embodiments, the one or more RNA-containing compositions comprise an mRNA. In some embodiments, the one or more RNA-containing compositions comprise an RNP (e.g., Cas9 and a guide RNA).

[0179] In some embodiments, the composition comprises one or more liposomes that collectively encapsulate (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease, and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA. In some embodiments, the one or more liposomes comprise a first plurality of liposomes encapsulating the RNA-guided nuclease or a nucleic acid encoding the RNA-guided nuclease, and a second plurality of liposomes encapsulating at least one guide RNA or a nucleic acid encoding at least one guide RNA.

[0180] In one aspect, the use of the CRISPR gene editing system further comprising one or more liposomes for targeting TGFB1, TGFBR1, and / or TGFBR2 is therapeutic. In some embodiments, the use of the system treats fibrosis and / or scarring. In some embodiments, the use of the system treats one or more musculoskeletal diseases, conditions, and diseases, including, but not limited to, Loeys-Dietz syndrome, osteoarthritis, Marfan syndrome, aortic aneurysms (e.g., familial thoracic 3 aortic aneurysms), craniofacial abnormalities, and combinations thereof. In other embodiments, the use of the system treats neoplastic diseases, conditions, and diseases, including pancreatic cancer, multiple self-healing squamous cell carcinomas (Ferguson-Smith disease), gastrointestinal stromal tumors (GIST), hereditary nonpolyposis colorectal cancer (Lynch syndrome), metastatic colorectal cancer, bone neoplasms, anaplastic carcinomas, spindle cell carcinomas, lung neoplasms, brain neoplasms, and combinations thereof.

[0181] 2. Lipid Nanocrystals (LNCs) In one aspect, the present disclosure encompasses a means for delivering a CRISPR gene editing system to a mammalian cell via lipid nanocrystals (LNCs). In some embodiments, the CRISPR gene editing system is delivered by LNCs. Without wishing to be bound by any particular theory, in certain embodiments, nucleic acids are resistant in aqueous solution to degradation by nucleases when present in nanocrystals. In other embodiments, proteins are protected from protease degradation while present in nanocrystals. In some embodiments, proteins and nucleic acids encapsulated by nanocrystals can penetrate cell plasma membranes.

[0182] In some embodiments, the CRISPR gene editing system comprises one or more RNA-containing compositions. In some embodiments, the CRISPR gene editing system further comprises one or more nanocrystals. In some embodiments, the one or more RNA-containing compositions comprise a guide RNA. In some embodiments, the one or more RNA-containing compositions comprise an mRNA. In some embodiments, the one or more RNA-containing compositions comprise an RNP (e.g., Cas9 and a guide RNA). In some embodiments, the one or more nanocrystals are lipid nanocrystals (LNCs).

[0183] In some embodiments, the CRISPR gene editing system comprises one or more LNCs that collectively encapsulate (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease, and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA. In some embodiments, the one or more LNCs comprise a first plurality of LNCs encapsulating an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease, and a second plurality of LNCs encapsulating at least one guide RNA or a nucleic acid encoding at least one guide RNA.

[0184] In one aspect, the use of the CRISPR gene editing system further comprising one or more LNCs for targeting TGFB1, TGFBR1, and / or TGFBR2 is therapeutic. In some embodiments, the use of the system treats fibrosis and / or scarring. In some embodiments, the use of the system treats one or more musculoskeletal diseases, conditions, and diseases, including, but not limited to, Loeys-Dietz syndrome, osteoarthritis, Marfan syndrome, aortic aneurysms (e.g., familial thoracic 3 aortic aneurysms), craniofacial abnormalities, and combinations thereof. In other embodiments, the use of the system treats neoplastic diseases, conditions, and diseases, including pancreatic cancer, multiple self-healing squamous cell carcinoma (Ferguson-Smith disease), gastrointestinal stromal tumors (GIST), hereditary nonpolyposis colorectal cancer (Lynch syndrome), metastatic colorectal cancer, bone neoplasms, anaplastic carcinomas, spindle cell carcinomas, lung neoplasms, brain neoplasms, and combinations thereof.

[0185] VI. Pharmaceutical Compositions In one aspect, the present disclosure encompasses a pharmaceutical composition comprising a CRISPR gene editing system for treatment in a mammal in need of treatment. In some embodiments, the CRISPR gene editing system targets a gene selected from TGFB1, TGFBR1, TGFBR2, and combinations thereof. In some embodiments, the mammal is selected from a human, a dog, a horse, and a cat.

[0186] A.TGFB1 In some embodiments, the pharmaceutical composition comprising the CRISPR gene editing system targets TGFB1. In some embodiments, the CRISPR gene editing system targeting TGFB1 is delivered to mammalian cells via a viral vector. In some embodiments, the CRISPR gene editing system targeting TGFB1 is delivered to mammalian cells via an AAV vector. In some embodiments, the CRISPR gene editing system targeting TGFB1 is delivered to mammalian cells via a lentiviral vector. In some embodiments, the CRISPR gene editing system targeting TGFB1 is delivered to mammalian cells via lipid nanoparticles. In some embodiments, the CRISPR gene editing system targeting TGFB1 is delivered to mammalian cells via virus-like particles. In some embodiments, the CRISPR gene editing system targeting TGFB1 is delivered to mammalian cells via liposomes. In some embodiments, the CRISPR gene editing system targeting TGFB1 is delivered to mammalian cells via lipid nanocrystals.

[0187] In various embodiments, a pharmaceutical composition comprising a CRISPR gene editing system targeting the TGFB1 gene is used in a method of treatment in a mammal in need of such treatment. In some embodiments, the method treats fibrosis and / or scarring. In some embodiments, the method treats one or more musculoskeletal diseases, conditions, and diseases, including, but not limited to, Loeys-Dietz syndrome, osteoarthritis, Marfan syndrome, aortic aneurysms (e.g., familial thoracic 3 aortic aneurysms), craniofacial abnormalities, and combinations thereof. In other embodiments, the method treats neoplastic diseases, conditions, and diseases, including pancreatic cancer, multiple self-healing squamous cell carcinomas (Ferguson-Smith disease), gastrointestinal stromal tumors (GIST), hereditary nonpolyposis colorectal cancer (Lynch syndrome), metastatic colorectal cancer, bone neoplasms, anaplastic carcinomas, spindle cell carcinomas, lung neoplasms, brain neoplasms, and combinations thereof.

[0188] 1. Treating musculoskeletal disorders by targeting the transforming growth factor beta gene In one aspect, methods and pharmaceutical compositions are provided for treating musculoskeletal disorders using CRISPR pharmaceuticals. In one aspect, methods and pharmaceutical compositions are provided for treating musculoskeletal disorders using CRISPR pharmaceutical compositions targeting transforming growth factor beta gene. In some embodiments, the CRISPR pharmaceutical composition comprises (i) an RNA-guided nuclease or a nucleic acid encoding the RNA-guided nuclease, and (ii) at least one guide RNA (gRNA) or a nucleic acid encoding at least one gRNA targeting the transforming growth factor beta gene, where the gRNA specifically binds to a target sequence adjacent to a protospacer adjacent motif (PAM) sequence of the RNA-guided nuclease. In some embodiments, the CRISPR pharmaceutical composition comprises (i) an mRNA encoding the RNA-guided nuclease, and (ii) at least one gRNA targeting the transforming growth factor beta gene. In some embodiments, the mRNA encoding the RNA-guided nuclease and at least one gRNA is packaged in a lipid nanoparticle (LNP). In some embodiments, the musculoskeletal disorder is Loeys-Dietz syndrome, osteoarthritis, Marfan syndrome, aortic aneurysm (familial thoracic 3), or craniofacial abnormality and is treated with a CRISPR pharmaceutical composition targeting the TGFB1 gene.

[0189] In some embodiments, the transforming growth factor beta receptor gene is the TGFB1 gene. In some embodiments, the TGFB1 gene is a human TGFB1 gene and the guide RNA comprises a crRNA sequence selected from SEQ ID NOs: 1-198.

[0190] 2. Treatment of fibrosis and / or scarring by targeting the transforming growth factor beta gene In one aspect, methods and pharmaceutical compositions are provided for treating fibrosis and / or scarring using CRISPR pharmaceuticals. In one aspect, methods and pharmaceutical compositions are provided for treating fibrosis and / or scarring using CRISPR pharmaceutical compositions targeting transforming growth factor beta gene. In some embodiments, the CRISPR pharmaceutical composition comprises (i) an RNA-guided nuclease or a nucleic acid encoding the RNA-guided nuclease, and (ii) at least one guide RNA (gRNA) or a nucleic acid encoding at least one gRNA that targets the transforming growth factor beta gene, where the gRNA specifically binds to a target sequence adjacent to the protospacer adjacent motif (PAM) sequence of the RNA-guided nuclease. In some embodiments, the CRISPR pharmaceutical composition comprises (i) an mRNA encoding the RNA-guided nuclease, and (ii) at least one gRNA that targets the transforming growth factor beta gene. In some embodiments, the fibrosis and / or scarring is due to knee joint fibrosis, intra-articular fibrous nodules, or epidural fibrosis, and is treated with a CRISPR pharmaceutical composition targeting the TGFB1 gene. In some embodiments, the CRISPR pharmaceutical composition described herein is a post-operative treatment to prevent or reduce fibrosis and / or scarring. For example, in some embodiments, the surgical procedure is selected from ligament reconstruction, anterior cruciate ligament (ACL) reconstruction, autograft ACL reconstruction, allograft ACL reconstruction, fracture repair, total knee arthroplasty (TKA), and microdiscectomy, and then the fibrosis and / or scarring is treated with a CRISPR pharmaceutical composition targeting the TGFB1 gene. In some embodiments, the fibrosis and / or scarring is the result of a condition that may be induced or exacerbated by surgery.

[0191] In some embodiments, the transforming growth factor beta receptor gene is the TGFB1 gene. In some embodiments, the TGFB1 gene is a human TGFB1 gene, and the guide RNA comprises a crRNA sequence selected from SEQ ID NOs: 1-198.

[0192] B. TGFBR1 In some embodiments, the pharmaceutical composition comprising the CRISPR gene editing system targets TGFBR1. In some embodiments, the CRISPR gene editing system targeting TGFBR1 is delivered to mammalian cells via a viral vector. In some embodiments, the CRISPR gene editing system targeting TGFBR1 is delivered to mammalian cells via an AAV vector. In some embodiments, the CRISPR gene editing system targeting TGFBR1 is delivered to mammalian cells via a lentiviral vector. In some embodiments, the CRISPR gene editing system targeting TGFBR1 is delivered to mammalian cells via lipid nanoparticles. In some embodiments, the CRISPR gene editing system targeting TGFBR1 is delivered to mammalian cells via virus-like particles. In some embodiments, the CRISPR gene editing system targeting TGFBR1 is delivered to mammalian cells via liposomes. In some embodiments, the CRISPR gene editing system targeting TGFBR1 is delivered to mammalian cells via lipid nanocrystals.

[0193] In various embodiments, a pharmaceutical composition comprising a CRISPR gene editing system targeting the TGFBR1 gene is used in a method of treatment in a mammal in need of such treatment. In some embodiments, the method treats fibrosis and / or scarring. In some embodiments, the method treats one or more musculoskeletal diseases, conditions, and diseases, including, but not limited to, Loeys-Dietz syndrome, osteoarthritis, Marfan syndrome, aortic aneurysms (e.g., familial thoracic 3 aortic aneurysms), craniofacial abnormalities, and combinations thereof. In other embodiments, the method treats neoplastic diseases, conditions, and diseases, including pancreatic cancer, multiple self-healing squamous cell carcinoma (Ferguson-Smith disease), gastrointestinal stromal tumors (GIST), hereditary nonpolyposis colorectal cancer (Lynch syndrome), metastatic colorectal cancer, bone neoplasms, anaplastic carcinomas, spindle cell carcinomas, lung neoplasms, brain neoplasms, and combinations thereof.

[0194] 1. Treating musculoskeletal disorders by targeting the transforming growth factor beta receptor 1 gene In one aspect, methods and pharmaceutical compositions are provided for treating musculoskeletal disorders using CRISPR pharmaceuticals. In one aspect, methods and pharmaceutical compositions are provided for treating musculoskeletal disorders using CRISPR pharmaceutical compositions targeting transforming growth factor beta receptor gene. In some embodiments, the CRISPR pharmaceutical composition comprises (i) an RNA-guided nuclease or a nucleic acid encoding the RNA-guided nuclease, and (ii) at least one guide RNA (gRNA) or a nucleic acid encoding at least one gRNA targeting transforming growth factor beta receptor 1 gene, where the gRNA specifically binds to a target sequence adjacent to a protospacer adjacent motif (PAM) sequence of the RNA-guided nuclease. In some embodiments, the CRISPR pharmaceutical composition comprises (i) an mRNA encoding the RNA-guided nuclease, and (ii) at least one gRNA targeting transforming growth factor beta receptor gene. In some embodiments, the mRNA encoding the RNA-guided nuclease and at least one gRNA is packaged in a lipid nanoparticle (LNP). In some embodiments, the musculoskeletal disorder is Loeys-Dietz syndrome, osteoarthritis, Marfan syndrome, aortic aneurysm (familial thoracic 3), or craniofacial abnormality and is treated with a CRISPR pharmaceutical composition targeting the TGFBR1 or TGFBR2 gene.

[0195] In some embodiments, the transforming growth factor beta receptor gene is the TGFBR1 gene. In some embodiments, the TGFBR1 gene is a human TGFBR1 gene and the guide RNA comprises a crRNA sequence selected from SEQ ID NOs: 199-320. In some embodiments, editing of the TGFBR1 gene results in knockout of the TGFBR1 gene. In some such embodiments, the TGFBR1 gene is a human TGFBR1 gene and the guide RNA comprises a crRNA sequence selected from SEQ ID NOs: 199-228. In some embodiments, editing of the TGFBR1 gene results in a gene expressing a membrane-bound receptor decoy protein. In some such embodiments, the TGFBR1 gene is a human TGFBR1 gene and the guide RNA comprises a crRNA sequence selected from SEQ ID NOs: 229-308 and 317-318. In some embodiments, editing of the TGFBR1 gene results in a gene expressing a soluble receptor decoy protein. In some such embodiments, the TGFBR1 gene is a human TGFBR1 gene and the guide RNA comprises a crRNA sequence selected from SEQ ID NOs: 309-318.

[0196] 2. Treating fibrosis by targeting the transforming growth factor beta receptor 1 gene In one aspect, methods and pharmaceutical compositions are provided for treating fibrosis and / or scarring using CRISPR pharmaceuticals. In one aspect, methods and pharmaceutical compositions are provided for treating fibrosis and / or scarring using CRISPR pharmaceutical compositions targeting transforming growth factor beta receptor gene. In some embodiments, the CRISPR pharmaceutical composition comprises (i) an RNA-guided nuclease or a nucleic acid encoding the RNA-guided nuclease, and (ii) at least one guide RNA (gRNA) or a nucleic acid encoding at least one gRNA that targets the transforming growth factor beta receptor gene, where the gRNA specifically binds to a target sequence adjacent to the protospacer adjacent motif (PAM) sequence of the RNA-guided nuclease. In some embodiments, the CRISPR pharmaceutical composition comprises (i) an mRNA encoding the RNA-guided nuclease, and (ii) at least one gRNA that targets the transforming growth factor beta receptor gene. In some embodiments, the fibrosis and / or scarring is due to knee joint fibrosis, intra-articular fibrous nodules, or epidural fibrosis, and is treated with a CRISPR pharmaceutical composition targeting the TGFBR1 or TGFBR2 gene. In some embodiments, the CRISPR pharmaceutical composition described herein is a post-operative treatment to prevent or reduce fibrosis and / or scarring. For example, in some embodiments, the surgical procedure is selected from ligament reconstruction, anterior cruciate ligament (ACL) reconstruction, autograft ACL reconstruction, allograft ACL reconstruction, fracture repair, total knee arthroplasty (TKA), and microdiscectomy, and then the fibrosis and / or scarring is treated with a CRISPR pharmaceutical composition targeting the TGFBR1 or TGFBR2 gene. In some embodiments, the fibrosis and / or scarring is the result of a condition that may be induced or exacerbated by surgery.

[0197] In some embodiments, the transforming growth factor beta receptor gene is the TGFBR1 gene. In some embodiments, the TGFBR1 gene is a human TGFBR1 gene and the guide RNA comprises a crRNA sequence selected from SEQ ID NOs: 199-320. In some embodiments, editing of the TGFBR1 gene results in knockout of the TGFBR1 gene. In some such embodiments, the TGFBR1 gene is a human TGFBR1 gene and the guide RNA comprises a crRNA sequence selected from SEQ ID NOs: 199-228. In some embodiments, editing of the TGFBR1 gene results in a gene expressing a membrane-bound receptor decoy protein. In some such embodiments, the TGFBR1 gene is a human TGFBR1 gene and the guide RNA comprises a crRNA sequence selected from SEQ ID NOs: 229-308 and 317-318. In some embodiments, editing of the TGFBR1 gene results in a gene expressing a soluble receptor decoy protein. In some such embodiments, the TGFBR1 gene is a human TGFBR1 gene and the guide RNA comprises a crRNA sequence selected from SEQ ID NOs: 309-318.

[0198] C.TGFBR2 In some embodiments, the pharmaceutical composition comprising the CRISPR gene editing system targets TGFBR1. In some embodiments, the CRISPR gene editing system targeting TGFBR1 is delivered to mammalian cells via a viral vector. In some embodiments, the CRISPR gene editing system targeting TGFBR1 is delivered to mammalian cells via an AAV vector. In some embodiments, the CRISPR gene editing system targeting TGFBR1 is delivered to mammalian cells via a lentiviral vector. In some embodiments, the CRISPR gene editing system targeting TGFBR1 is delivered to mammalian cells via lipid nanoparticles. In some embodiments, the CRISPR gene editing system targeting TGFBR1 is delivered to mammalian cells via virus-like particles. In some embodiments, the CRISPR gene editing system targeting TGFBR1 is delivered to mammalian cells via liposomes. In some embodiments, the CRISPR gene editing system targeting TGFBR1 is delivered to mammalian cells via lipid nanocrystals.

[0199] In various embodiments, a pharmaceutical composition comprising a CRISPR gene editing system targeting the TGFBR1 gene is used in a method of treatment in a mammal in need of such treatment. In some embodiments, the method treats fibrosis and / or scarring. In some embodiments, the method treats one or more musculoskeletal diseases, conditions, and diseases, including, but not limited to, Loeys-Dietz syndrome, osteoarthritis, Marfan syndrome, aortic aneurysms (e.g., familial thoracic 3 aortic aneurysms), craniofacial abnormalities, and combinations thereof. In other embodiments, the method treats neoplastic diseases, conditions, and diseases, including pancreatic cancer, multiple self-healing squamous cell carcinoma (Ferguson-Smith disease), gastrointestinal stromal tumors (GIST), hereditary nonpolyposis colorectal cancer (Lynch syndrome), metastatic colorectal cancer, bone neoplasms, anaplastic carcinomas, spindle cell carcinomas, lung neoplasms, brain neoplasms, and combinations thereof.

[0200] 1. Treating musculoskeletal disorders by targeting the transforming growth factor beta receptor 2 gene In one aspect, methods and pharmaceutical compositions are provided for treating musculoskeletal disorders using CRISPR pharmaceuticals. In one aspect, methods and pharmaceutical compositions are provided for treating musculoskeletal disorders using CRISPR pharmaceutical compositions targeting transforming growth factor beta receptor gene. In some embodiments, the CRISPR pharmaceutical composition comprises (i) an RNA-guided nuclease or a nucleic acid encoding the RNA-guided nuclease, and (ii) at least one guide RNA (gRNA) or a nucleic acid encoding at least one gRNA targeting the transforming growth factor beta receptor gene, where the gRNA specifically binds to a target sequence adjacent to a protospacer adjacent motif (PAM) sequence of the RNA-guided nuclease. In some embodiments, the CRISPR pharmaceutical composition comprises (i) an mRNA encoding the RNA-guided nuclease, and (ii) at least one gRNA targeting the transforming growth factor beta receptor gene. In some embodiments, the mRNA encoding the RNA-guided nuclease and at least one gRNA is packaged in a lipid nanoparticle (LNP). In some embodiments, the musculoskeletal disorder is Loeys-Dietz syndrome, osteoarthritis, Marfan syndrome, aortic aneurysm (familial thoracic 3), or craniofacial abnormality and is treated with a CRISPR pharmaceutical composition targeting the TGFBR1 or TGFBR2 gene.

[0201] In some embodiments, the transforming growth factor beta receptor gene is the TGFBR2 gene. In some embodiments, the TGFBR2 gene is the human TGFBR2 gene and the guide RNA comprises a crRNA sequence selected from SEQ ID NOs: 321-519. In some embodiments, editing of the TGFBR2 gene results in knockout of the TGFBR2 gene. In some such embodiments, the TGFBR2 gene is the human TGFBR2 gene and the guide RNA comprises a crRNA sequence selected from SEQ ID NOs: 321-354. In some embodiments, editing of the TGFBR2 gene results in a gene expressing a membrane-bound receptor decoy protein. In some such embodiments, the TGFBR2 gene is the human TGFBR2 gene and the guide RNA comprises a crRNA sequence selected from SEQ ID NOs: 355-498 and SEQ ID NOs: 503-508. In some embodiments, editing of the TGFBR2 gene results in a gene expressing a soluble receptor decoy protein. In some such embodiments, the TGFBR2 gene is a human TGFBR2 gene and the guide RNA comprises a crRNA sequence selected from SEQ ID NOs: 499-508.

[0202] 2. Treatment of fibrosis and / or scarring by targeting the transforming growth factor beta receptor 2 gene In one aspect, methods and pharmaceutical compositions are provided for treating fibrosis and / or scarring using CRISPR pharmaceuticals. In one aspect, methods and pharmaceutical compositions are provided for treating fibrosis and / or scarring using CRISPR pharmaceutical compositions targeting transforming growth factor beta receptor gene. In some embodiments, the CRISPR pharmaceutical composition comprises (i) an RNA-guided nuclease or a nucleic acid encoding the RNA-guided nuclease, and (ii) at least one guide RNA (gRNA) or a nucleic acid encoding at least one gRNA that targets the transforming growth factor beta receptor gene, where the gRNA specifically binds to a target sequence adjacent to the protospacer adjacent motif (PAM) sequence of the RNA-guided nuclease. In some embodiments, the CRISPR pharmaceutical composition comprises (i) an mRNA encoding the RNA-guided nuclease, and (ii) at least one gRNA that targets the transforming growth factor beta receptor gene. In some embodiments, the fibrosis and / or scarring is due to knee joint fibrosis, intra-articular fibrous nodules, or epidural fibrosis, and is treated with a CRISPR pharmaceutical composition targeting the TGFBR1 or TGFBR2 gene. In some embodiments, the CRISPR pharmaceutical composition described herein is a post-operative treatment to prevent or reduce fibrosis and / or scarring. For example, in some embodiments, the surgical procedure is selected from ligament reconstruction, anterior cruciate ligament (ACL) reconstruction, autograft ACL reconstruction, allograft ACL reconstruction, fracture repair, total knee arthroplasty (TKA), and microdiscectomy, and then the fibrosis and / or scarring is treated with a CRISPR pharmaceutical composition targeting the TGFBR1 or TGFBR2 gene. In some embodiments, the fibrosis and / or scarring is the result of a condition that may be induced or exacerbated by surgery.

[0203] In some embodiments, the transforming growth factor beta receptor gene is the TGFBR2 gene. In some embodiments, the TGFBR2 gene is the human TGFBR2 gene and the guide RNA comprises a crRNA sequence selected from SEQ ID NOs: 321-519. In some embodiments, editing of the TGFBR2 gene results in knockout of the TGFBR2 gene. In some such embodiments, the TGFBR2 gene is the human TGFBR2 gene and the guide RNA comprises a crRNA sequence selected from SEQ ID NOs: 321-354. In some embodiments, editing of the TGFBR2 gene results in a gene expressing a membrane-bound receptor decoy protein. In some such embodiments, the TGFBR2 gene is the human TGFBR2 gene and the guide RNA comprises a crRNA sequence selected from SEQ ID NOs: 355-498 and SEQ ID NOs: 503-508. In some embodiments, editing of the TGFBR2 gene results in a gene expressing a soluble receptor decoy protein. In some such embodiments, the TGFBR2 gene is a human TGFBR2 gene and the guide RNA comprises a crRNA sequence selected from SEQ ID NOs: 499-508.

[0204] VII. Route of Administration The methods and compositions described herein encompass the use of pharmaceutical compositions that include a CRISPR gene editing system as an active ingredient.

[0205] Depending on the method / route of administration, pharmaceutical dosage forms come in several types. These include many kinds of liquid, solid, and semi-solid dosage forms. Common pharmaceutical dosage forms include pills, tablets, or capsules, beverages or syrups, and natural or herbal forms such as plants or foods, among many others. In particular, the route of administration (ROA) of drug delivery depends on the dosage form of the substance in question. A liquid pharmaceutical dosage form is a liquid form of a dose of a compound used as a drug or medicine that is intended to be administered or consumed.

[0206] As described below, compositions of the present disclosure can be delivered to a subject subcutaneously (e.g., intra-articular or intradiscal injection), dermally (e.g., transdermally via a patch), and / or via an implant. Exemplary pharmaceutical dosage forms include, for example, pills, osmotic delivery systems, elixirs, emulsions, hydrogels, suspensions, syrups, capsules, tablets, orally dissolving tablets (ODTs), gel capsules, thin films, adhesive topical patches, lollipops, lozenges, chewing gums, dry powder inhalers (DPIs), vaporizers, nebulizers, metered dose inhalers (MDIs), ointments, transdermal patches, and intradermal implants.

[0207] As used herein, "dermal delivery" or "dermal administration" may refer to a route of administration in which a pharmaceutical dosage form is administered into or through the dermis (i.e., the layer of skin between the epidermis (which constitutes the skin) and the subcutaneous tissue). "Subcutaneous delivery" may refer to a route of administration in which a pharmaceutical dosage form is to or below the subcutaneous tissue layer.

[0208] Methods for formulating suitable pharmaceutical compositions are known in the art, see, for example, Remington: The Science and Practice of Pharmacy, 21st ed., 2005, and the books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following components: sterile diluents such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents, antibacterial agents such as benzyl alcohol or methylparabens, antioxidants such as ascorbic acid or sodium bisulfate, chelating agents such as ethylenediaminetetraacetic acid, buffers such as acetates, citrates, or phosphates, agents for the regulation of toxicity such as sodium chloride or glucose. pH may be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0209] Pharmaceutical compositions suitable for injectable use may include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride, etc. in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0210] Sterile injectable solution can be prepared by incorporating active compound in the required amount in suitable solvent with one or combination of the above-listed components, and then sterilized by filtration if necessary.Generally, dispersion is prepared by incorporating active compound containing basic dispersion medium and other required components from above-listed components into sterile vehicle.For the preparation of sterile powder for sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying, which obtains powder of active ingredient by adding any additional desired components from the solution that has been previously sterilized and filtered.

[0211] The therapeutic compound that is or contains nucleic acid can be administered by any method suitable for administration of nucleic acid agents, such as DNA vaccines. These methods include gene guns, bioinjectors, and skin patches, as well as needle-free methods, such as the microparticle DNA vaccine technology disclosed in U.S. Pat. No. 6,194,389, and mammalian transdermal needle-free vaccination with powder form vaccines disclosed in U.S. Pat. No. 6,168,587. In addition, intranasal delivery is possible, particularly as described in Hamajima et al., Clin. Immunol. Immunopathol., 88(2), 205-10 (1998). Liposomes (e.g., as described in U.S. Pat. No. 6,472,375) and microencapsulation can also be used. Biodegradable targetable microparticle delivery systems can also be used (e.g., as described in U.S. Pat. No. 6,471,996).

[0212] The therapeutic compound may be prepared with a carrier that will protect the therapeutic compound against rapid elimination from the body, such as sustained release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers may be used, such as collagen, ethylene vinyl acetate, polyanhydrides (e.g., poly[1,3-bis(carboxyphenoxy)propane-co-sebacic acid] (PCPP-SA) matrix, fatty acid dimer-sebacic acid (FAD-SA) copolymer, poly(lactide-co-glycolide)), polyglycolic acid, collagen, polyol triesters, polyethylene glycol-coated liposomes, hyaluronic acid, and polylactic acid. Such formulations may be prepared using standard techniques or obtained commercially, for example, from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to selected cells with monoclonal antibodies against cellular antigens) may also be used as pharmaceutically acceptable carriers. These may be prepared by methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811. Semi-solid, gelling, soft gel, or other formulations (including sustained release) may be used, for example, when administration to a surgical site is desired. Methods for making such formulations are known in the art and may include the use of biodegradable, biocompatible polymers. See, for example, Sawyer et al., Yale J Biol Med. 2006 December, 79(3-4):141-152.

[0213] The pharmaceutical compositions described herein can be included in a container, kit, pack, or dispenser together with instructions for administration.

[0214] A. Systemic Administration In some embodiments, the pharmaceutical composition comprising the CRISPR gene editing system is administered systemically to a mammal in need thereof. In some embodiments, the composition is formulated for intravenous injection. In some embodiments, the composition is formulated for oral administration. In some embodiments, the composition is formulated for parenteral administration.

[0215] B. Topical Administration In some embodiments, the pharmaceutical composition comprising the CRISPR gene editing system is administered locally to a mammal in need thereof. In some embodiments, the local administration is an intra-articular injection. In some embodiments, the composition is formulated for intradiscal injection. In some embodiments, the composition is formulated for epidural injection. In some embodiments, the composition is formulated for peridiscal injection. In some embodiments, the composition is formulated for perivertebral injection. In some embodiments, the composition is formulated for perivertebral injection. In some embodiments, the composition is formulated for administration to a facet joint of the spine.

[0216] In some embodiments, the pharmaceutical composition comprising the CRISPR gene editing system is locally administered to a mammal in need thereof during surgery. In some embodiments, the pharmaceutical composition comprising the CRISPR gene editing system is locally administered to a mammal in need thereof 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or 30 days after surgery.

[0217] 1. Administration after musculoskeletal trauma In some embodiments, the compositions and methods disclosed herein are administered to a subject following musculoskeletal trauma. In some embodiments, the musculoskeletal trauma is the result of surgery (i.e., a surgical incision). In some embodiments, the musculoskeletal trauma is a wound.

[0218] In some embodiments, the composition containing the RNA-guided nuclease and the sgRNA is applied to a wound or incision. In some embodiments, the composition is a spray. In some embodiments, the composition is a patch. In further embodiments, the patch is a bioabsorbable patch. In some embodiments, the composition is a mesh. In some embodiments, the composition is a hydrogel.

[0219] Additional exemplary compositions in which the RNA-guided nuclease and sgRNA are applied to a wound or incision include the following (each of which is incorporated by reference herein in its entirety for all purposes): U.S. Patent Nos. 8,574,627, 9,204,953, 9,272,073, 9,592,324, 9,597,426, 9,993,298, 10,471,181, 10,765,423, 10,835,235, 11,338,062, 11,426,156, and U.S. Patent Application Nos. 2008 / 0039877A1, 2020 / 0345366A1, and 2020 / 0069478A1.

[0220] In some embodiments, the disclosure provides a pharmaceutical composition for treating or preventing a disorder having a symptom caused at least in part by intercellular signaling mediated through the transforming growth factor beta (TGFβ) signaling pathway, the composition comprising: (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease; and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA targeting the TGFB1 gene, the TGFBR1 gene, the TGFBR2 gene, or a combination thereof.

[0221] In some embodiments, the disorder is fibrosis.

[0222] In some embodiments, the fibrosis is post-operative fibrosis.

[0223] In some embodiments, the fibrosis is post-ligament reconstruction fibrosis.

[0224] In some embodiments, the fibrosis is post-anterior cruciate ligament (ACL) reconstruction fibrosis, post-autograft ACL reconstruction fibrosis, or post-allograft ACL reconstruction fibrosis.

[0225] In some embodiments, the fibrosis is knee arthrofibrosis.

[0226] In some embodiments, the fibrosis results from intra-articular fibrous nodules.

[0227] In some embodiments, the fibrosis is following total knee arthroplasty (TKA).

[0228] In some embodiments, the fibrosis is following total knee arthroplasty (TKA).

[0229] In some embodiments, the fibrosis is post-fracture repair fibrosis.

[0230] In some embodiments, the fibrosis is post-microdiscectomy fibrosis.

[0231] In some embodiments, the fibrosis is epidural fibrosis following microdiscectomy.

[0232] In some embodiments, the fibrosis is post-lumbar laminectomy fibrosis.

[0233] In some embodiments, the fibrosis is epidural fibrosis following lumbar laminectomy.

[0234] In some embodiments, the fibrosis is post-anterior acromioplasty fibrosis.

[0235] In some embodiments, the fibrosis is post-subacromial decompression fibrosis.

[0236] In some embodiments, the fibrosis is soft tissue fibrosis following anterior acromioplasty.

[0237] In some embodiments, the fibrosis is joint contracture following anterior acromioplasty.

[0238] In some embodiments, the fibrosis is post-arthroscopic fibrosis.

[0239] In some embodiments, the fibrosis is post-arthroscopic arthrofibrosis.

[0240] In some embodiments, the fibrosis is fibrosis of the trabecular meshwork.

[0241] In some embodiments, the fibrosis of the trabecular meshwork is post-glaucoma surgery fibrosis.

[0242] In some embodiments, the fibrosis is epidural fibrosis.

[0243] In some embodiments, the fibrosis is atrial fibrosis, cardiac fibrosis, myocardial fibrosis, and / or post-open heart surgery fibrosis.

[0244] In some embodiments, the fibrosis is post-liposuction fibrosis.

[0245] In some embodiments, the fibrosis is idiopathic pulmonary fibrosis (IPF).

[0246] In some embodiments, the fibrosis is fibrosis in the knee, shoulder, or elbow.

[0247] In some embodiments, the disorder comprises fibrosis in kidney tissue.

[0248] In some embodiments, the disorder is chronic kidney disease.

[0249] In some embodiments, the disorder comprises fibrosis in skin tissue.

[0250] In some embodiments, the disorder is scarring after wound healing, a keloid disorder, nephrogenic systemic fibrosis, or scleroderma / systemic sclerosis.

[0251] In some embodiments, the disorder comprises fibrosis in lung tissue.

[0252] In some embodiments, the disorder is fibrothorax, pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, radiation-induced lung injury, progressive diffuse fibrosis, or scleroderma / systemic sclerosis.

[0253] In some embodiments, the disorder comprises fibrosis in liver tissue.

[0254] In some embodiments, the disorder is cirrhosis or bridging fibrosis.

[0255] In some embodiments, the disorder comprises fibrosis in cardiac tissue.

[0256] In some embodiments, the fibrosis is interstitial fibrosis.

[0257] In some embodiments, the disorder is congestive heart failure or hypertension.

[0258] In some embodiments, the fibrosis is replacement fibrosis.

[0259] In some embodiments, the disorder is myocardial infarction.

[0260] In some embodiments, the disorder comprises fibrosis in brain tissue.

[0261] In some embodiments, the fibrosis is a glial scar.

[0262] In some embodiments, the disorder comprises fibrosis in intestinal tissue.

[0263] In some embodiments, the disorder is Crohn's disease.

[0264] In some embodiments, the disorder comprises fibrosis in the hand or finger.

[0265] In some embodiments, the disorder is Dupuytren's contracture.

[0266] In some embodiments, the disorder comprises fibrosis in lymphatic tissue.

[0267] In some embodiments, the fibrosis is mediastinal fibrosis.

[0268] In some embodiments, the disorder comprises fibrosis in bone marrow tissue.

[0269] In some embodiments, the fibrosis is myelofibrosis.

[0270] In some embodiments, the disorder comprises fibrosis in penile tissue.

[0271] In some embodiments, the disorder is Peyronie's disease.

[0272] In some embodiments, the disorder comprises fibrosis in the retroperitoneal soft tissue.

[0273] In some embodiments, the fibrosis is retroperitoneal fibrosis.

[0274] In some embodiments, the disorder comprises fibrosis in musculoskeletal tissue.

[0275] In some embodiments, the disorder is a musculoskeletal disorder.

[0276] In some embodiments, the musculoskeletal disorder is a genetic musculoskeletal disease.

[0277] In some embodiments, the musculoskeletal disorder is a muscular dystrophy.

[0278] In some embodiments, the musculoskeletal disorder is selected from Duchenne muscular dystrophy (DMD), myotonic dystrophy (DM), facioscapulohumeral muscular dystrophy (FSHD), and limb-girdle muscular dystrophy (LGMD).

[0279] In some embodiments, the musculoskeletal disorder is a type II collagen disorder.

[0280] In some embodiments, the musculoskeletal disorder is selected from achondroplasia type II, hypochondroplasia, Stickler syndrome, and Czech dysplasia.

[0281] In some embodiments, the musculoskeletal disorder is osteogenesis imperfecta (OI).

[0282] In some embodiments, the musculoskeletal disorder is an autoimmune musculoskeletal disease.

[0283] In some embodiments, the musculoskeletal disorder is an autoimmune musculoskeletal disease with a polygenic susceptibility trait.

[0284] In some embodiments, the musculoskeletal disorder is spondyloarthropathy (SA).

[0285] In some embodiments, the musculoskeletal disorder is ankylosing spondylitis.

[0286] In some embodiments, the musculoskeletal disorder is rheumatoid arthritis.

[0287] In some embodiments, the musculoskeletal disorder is autoimmune osteoarthritis.

[0288] In some embodiments, the musculoskeletal disorder is osteoarthritis (OA).

[0289] In some embodiments, the musculoskeletal disorder is Kamuracci-Engelmann disease.

[0290] In some embodiments, the musculoskeletal disorder is Sjogren's syndrome.

[0291] In some embodiments, the musculoskeletal disorder is a mechanical musculoskeletal injury.

[0292] In some embodiments, the musculoskeletal disorder is selected from chronic muscle injury, tendon rupture, post-traumatic osteoarthritis, and post-traumatic arthrofibrosis.

[0293] In some embodiments, the at least one guide RNA or a nucleic acid encoding at least one guide RNA targets a mammalian TGFB1 gene.

[0294] In some embodiments, the mammalian TGFB1 gene is a canine, equine, or feline TGFB1 gene.

[0295] In some embodiments, the mammalian TGFB1 gene is a human TGFB1 gene.

[0296] In some embodiments, at least one guide RNA comprises a crRNA sequence selected from the group consisting of sequences depicted in Figures 1A-1D (SEQ ID NOs: 1-198).

[0297] In some embodiments, at least one guide RNA comprises a crRNA sequence selected from the group consisting of sequences depicted in FIG. 12A (SEQ ID NOs: 520-527).

[0298] In some embodiments, at least one guide RNA comprises the crRNA sequence of OHTG03 (SEQ ID NO: 522) or OHTG04 (SEQ ID NO: 523).

[0299] In some embodiments, the at least one guide RNA or the nucleic acid encoding the at least one guide RNA targets a mammalian TGFBR1 gene.

[0300] In some embodiments, the mammalian TGFBR1 gene is a canine, equine, or feline TGFBR1 gene.

[0301] In some embodiments, the mammalian TGFBR1 gene is a human TGFBR1 gene.

[0302] In some embodiments, at least one guide RNA comprises a crRNA sequence selected from the group consisting of the sequences depicted in Figures 2A-2C (SEQ ID NOs: 199-320).

[0303] In some embodiments, at least one guide RNA comprises a crRNA sequence selected from the group consisting of sequences depicted in Figure 12B (SEQ ID NOs: 528-552).

[0304] In some embodiments, at least one guide RNA comprises a crRNA sequence selected from the group consisting of sequences depicted in Figure 5B.

[0305] In some embodiments, at least one guide RNA comprises a crRNA sequence of OHTIR04 (SEQ ID NO: 532) or OHTIR08 (SEQ ID NO: 539).

[0306] In some embodiments, the at least one guide RNA or the nucleic acid encoding at least one guide RNA targets a mammalian TGFBR2 gene.

[0307] In some embodiments, the mammalian TGFBR2 gene is a canine, equine, or feline TGFBR2 gene.

[0308] In some embodiments, the mammalian TGFBR2 gene is a human TGFBR2 gene.

[0309] In some embodiments, at least one guide RNA comprises a crRNA sequence selected from the group consisting of the sequences depicted in Figures 3A-3D (SEQ ID NOs: 321-519).

[0310] In some embodiments, at least one guide RNA comprises a crRNA sequence selected from the group consisting of sequences depicted in Figure 12C (SEQ ID NOs: 553-604).

[0311] In some embodiments, at least one guide RNA comprises a crRNA sequence selected from the group consisting of sequences depicted in Figure 6B.

[0312] In some embodiments, at least one guide RNA comprises the crRNA sequence of OHTIIR04 (SEQ ID NO:563).

[0313] In some embodiments, the RNA-guided nuclease or the nucleic acid encoding an RNA-guided nuclease is an RNA-guided nuclease.

[0314] In some embodiments, the RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease is a DNA encoding an RNA-guided nuclease.

[0315] In some embodiments, the RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease is an mRNA encoding an RNA-guided nuclease.

[0316] In some embodiments, the RNA-guided nuclease is a Cas protein.

[0317] In some embodiments, the Cas protein is a Cas9 protein.

[0318] In some embodiments, the Cas9 protein is a S. pyogenes Cas9 polypeptide.

[0319] In some embodiments, the Cas9 protein is selected from the group consisting of esCas9, hfCas9, peCas9, and ARCas9.

[0320] In some embodiments, the at least one guide RNA or the nucleic acid encoding at least one guide RNA is at least one guide RNA.

[0321] In some embodiments, the at least one guide RNA or the nucleic acid encoding at least one guide RNA is DNA encoding at least one guide RNA.

[0322] In some embodiments, the pharmaceutical composition comprises a nucleic acid encoding both an RNA-guided nuclease and at least one guide RNA.

[0323] In some embodiments, at least one guide RNA is a single guide RNA (sgRNA).

[0324] In some embodiments, the compositions comprise one or more viral vectors that collectively comprise: (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease; and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA that targets a gene encoding a transmembrane receptor.

[0325] In some embodiments, one of the more viral vectors comprises a recombinant virus selected from a retrovirus, an adenovirus, an adeno-associated virus, a lentivirus, and herpes simplex virus-1.

[0326] In some embodiments, one of the more viral vectors comprises a recombinant adeno-associated virus (AAV).

[0327] In some embodiments, the recombinant AAV is of serotype 5 (AAV5).

[0328] In some embodiments, the recombinant AAV is of serotype 6 (AAV6).

[0329] In some embodiments, the composition comprises one or more lipid nanoparticles (LNPs) that collectively comprise: (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease; and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA that targets a gene encoding a transmembrane receptor.

[0330] In some embodiments, the one or more LNPs comprise a first plurality of LNPs encapsulating an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease, and a second plurality of LNPs encapsulating at least one guide RNA or a nucleic acid encoding at least one guide RNA.

[0331] In some embodiments, the one or more LNPs comprise a plurality of LNPs encapsulating both (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease and (ii) at least one guide RNA targeting a gene encoding a transmembrane receptor or a nucleic acid encoding at least one guide RNA.

[0332] In some embodiments, one or more LNPs are 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 (KL26), 1,2-diaminopropane (KL27), 1,2-diaminopropane (KL28), 1,2-diaminopropane (KL29), 1,2-diaminopropane (KL30), 1,2-diaminopropane (KL31), 1,2-diaminopropane (KL32), 1,2-diaminopropane (KL33), 1,2-diaminopropane (KL34), 1,2-diaminopropane (KL35), 1,2-diaminopropane (KL36), 1,2-diaminopropane (KL37), 1,2-diaminopropane (KL38), 1,2-diaminopropane (KL39 ... Pan (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 propane (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), 2-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.

[0333] In some embodiments, the LNPs are 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-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleo ... undecanoyl-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-cholesteryl hemisuccinoyl-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 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.

[0334] In some embodiments, the LNP comprises a component selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. For example, the PEG lipid may be a PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DMA, PEG-DSPE lipid, and mixtures thereof.

[0335] In some embodiments, the LNPs comprise a component 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.

[0336] In some embodiments, the composition comprises one or more liposomes that collectively comprise: (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease; and (ii) at least one guide RNA that targets a gene encoding a transmembrane receptor or a nucleic acid encoding at least one guide RNA.

[0337] In some embodiments, the one or more liposomes comprise a first plurality of liposomes encapsulating an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease and a second plurality of liposomes encapsulating at least one guide RNA or a nucleic acid encoding at least one guide RNA.

[0338] In some embodiments, the one or more liposomes comprise a plurality of liposomes encapsulating both (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease and (ii) at least one guide RNA targeting a gene encoding a transmembrane receptor or a nucleic acid encoding at least one guide RNA.

[0339] In some embodiments, the composition comprises one or more virus-like particles that collectively comprise: (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease; and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA that targets a gene encoding a transmembrane receptor.

[0340] In some embodiments, the one or more virus-like particles comprise: In some embodiments, a first plurality of virus-like particles encapsulating an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease, and a second plurality of virus-like particles encapsulating at least one guide RNA or a nucleic acid encoding at least one guide RNA.

[0341] In some embodiments, the one or more virus-like particles comprise a plurality of virus-like particles encapsulating both (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease and (ii) at least one guide RNA targeting a gene encoding a transmembrane receptor or a nucleic acid encoding at least one guide RNA.

[0342] In some embodiments, the compositions are formulated for parenteral administration.

[0343] In some embodiments, the composition is formulated for intra-articular injection into a joint of a subject.

[0344] In some embodiments, the compositions are formulated for intradiscal injection.

[0345] In some embodiments, the composition is formulated for peridiscal injection.

[0346] In some embodiments, the compositions are formulated for intraspinal injection.

[0347] In some embodiments, the disclosure provides a method for treating or preventing free oxygen radicals in a subject in need thereof by administering a therapeutically effective amount of a composition comprising (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease, and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA targeting the TGFB1 gene, the TGFBR1 gene, the TGFBR2 gene, or a combination thereof.

[0348] In some embodiments, the disclosure provides a method for treating or preventing, in a subject in need thereof, a disorder having a symptom caused at least in part by intercellular signaling mediated through the transforming growth factor beta (TGFβ) signaling pathway, the method comprising administering a therapeutically effective amount of a composition comprising (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease, and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA targeting the TGFB1 gene, the TGFBR1 gene, the TGFBR2 gene, or a combination thereof.

[0349] In some embodiments, the disorder is fibrosis.

[0350] In some embodiments, the fibrosis is post-operative fibrosis.

[0351] In some embodiments, the fibrosis is post-ligament reconstruction fibrosis.

[0352] In some embodiments, the fibrosis is post-anterior cruciate ligament (ACL) reconstruction fibrosis, post-autograft ACL reconstruction fibrosis, or post-allograft ACL reconstruction fibrosis.

[0353] In some embodiments, the fibrosis is knee arthrofibrosis.

[0354] In some embodiments, the fibrosis results from intra-articular fibrous nodules.

[0355] In some embodiments, the fibrosis is following total knee arthroplasty (TKA).

[0356] In some embodiments, the fibrosis is following total knee arthroplasty (TKA).

[0357] In some embodiments, the fibrosis is post-fracture repair fibrosis.

[0358] In some embodiments, the fibrosis is post-microdiscectomy fibrosis.

[0359] In some embodiments, the fibrosis is epidural fibrosis following microdiscectomy.

[0360] In some embodiments, the fibrosis is post-lumbar laminectomy fibrosis.

[0361] In some embodiments, the fibrosis is epidural fibrosis following lumbar laminectomy.

[0362] In some embodiments, the fibrosis is post-anterior acromioplasty fibrosis.

[0363] In some embodiments, the fibrosis is post-subacromial decompression fibrosis.

[0364] In some embodiments, the fibrosis is soft tissue fibrosis following anterior acromioplasty.

[0365] In some embodiments, the fibrosis is joint contracture following anterior acromioplasty.

[0366] In some embodiments, the fibrosis is post-arthroscopic fibrosis. EXAMPLES

[0367] VIII. Examples The embodiments encompassed herein will now be described with reference to the following examples, which are provided for purposes of illustration only, and the disclosure encompassed herein should in no way be construed as being limited to these examples, but rather to encompass any and all variations that become evident as a result of the teachings provided herein.

[0368] Example 1: Bioinformatic design of guides for disrupting TGF-beta signaling To identify candidate CRISPR-Cas guides for gene editing, an in silico and in vitro screening pipeline was developed. The pipeline was applied to identify guides that effectively edit human TGFB1 (hTGFB1), TGFBR1 (hTGFRB1), and TGFBR2 genes (hTGFRB2), thereby disrupting TGF-beta signaling in vivo to treat musculoskeletal fibrosis and / or scarring. The first step of the pipeline was to identify all possible crRNA sequences for a particular CRISPR-Cas protein in the coding portion of hTGFB1, hTGFRB1, and hTGFRB2. Many algorithms for identifying such sequences are known in the art. In general, these algorithms work by identifying a protospacer adjacent motif (PAM) sequence for a particular CRISPR-Cas protein, and then positioning a sequence according to the requirements of the particular Cas protein, typically directly 5' to the PAM site and spaced apart. For example, the S. pyogenes Cas9 (SpCas9) protein used in this example recognizes the 5'-NGG-3'PAM sequence. Therefore, all sequences directly 5' of the NGG trinucleotide are potential crRNA sequences. The crRNA sequences were identified in this manner as shown in Figure 27 along with biographical information about the crRNA sequence.

[0369] Each identified crRNA sequence (see Figures 1-3) was then assessed across three different metrics: potential off-target editing at locations in the genome other than the target gene, on-target editing efficiency, and the likelihood of editing causing frameshift mutations, as shown in Figures 4B, 5B, and 6B, using multiple algorithms for each metric. The basis of the combinatorial approach used lies in the assumption that all models have blind spots that can skew the suitability of a particular guide RNA. Weighting these scores to obtain a consensus score for each of these properties allows for a much better prediction of sgRNA suitability.

[0370] Off-target editing effects were predicted by averaging the scores generated by the MIT, CFD, and Elevation (human only) models. The MIT algorithm is also known as the Hsu-Zhang score. Hsu, PD et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nature Biotechnology 31, 827-832 (2013). The model is based on a position penalty matrix (1 × 20) generated from 15 EMX1 sgRNA libraries with mismatches to the target at each position. The CFD algorithm (cutting frequency determination) is based on a threat matrix (12 × 20) that considers both position and mismatch type as well as PAM completeness (27,897 'CD33' sgRNAs + 10,618 negative control sgRNAs). Doench, JGet al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nature Biotechnology 34, 184-191 (2016). The Elevation score uses a machine learning algorithm trained by genome-wide (GUIDE-Seq) and other aggregated off-target profiling data. Listgarten, J. et al. Prediction of off-target activities for the end-to-end design of CRISPR guide RNAs. Nature Biomedical Engineering 2, 38-47 (2018). The column labeled "Off" shows the average of the scores provided by two or three models, respectively. The column labeled "Number of off-targets" shows the number of potential off-targets with up to four mismatches calculated by CRISPOR.See Haeussler, M. et al. Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biology 17, 148 (2016).

[0371] On-target editing efficiency was predicted by averaging the scores generated by the Azimuth, DeepSpCas9, and CrisprScan models. The Azimuth model is a boosted regression tree model trained on 881 sgRNAs delivered by lentivirus (MOLM13 / NB4 / TF1 cells and additional unpublished data). Doench, JGet et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nature Biotechnology 34, 184-191 (2016). DeepSpCas9 is a deep learning model trained using editing data from 12,832 sgRNAs. Kim, HK et al. SpCas9 activity prediction by DeepSpCas9, a deep learning-based model with high generalization performance. Science Advances 5(11) (2019). CrisprScan is a linear regression model, trained using editing data from 1000 sgRNAs injected into zebrafish embryos targeting >100 genes. Moreno-Mateos, MA et al. CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo. Nature Methods 12, 982-988 (2015). The column labeled "On" indicates the average of the scores provided by the three models.

[0372] The likelihood of a putative guide generating a frameshift mutation was predicted by averaging the scores generated by the Lindel and InDelphi models. Lindel is a machine learning model trained using profile data of 1.16 million independent mutation events induced by CRISPR / Cas9-mediated cleavage and non-homologous end joining-mediated double-strand break repair of 6872 synthetic target sequences introduced into human cell lines via lentiviral infection. Chen, W. et al. Massively parallel profiling and predictive modeling of the outcomes of CRISPR / Cas9-mediated double-strand break repair. Nucleic Acids Research 47, 7989-8003 (2019). InDelphi is a machine learning model trained on indels generated by 1872 sgRNAs. Shen, MW et al. Predictable and precise template-free CRISPR editing of pathogenic variants. Nature 563, 646-651 (2018). The column labeled "Frameshift" shows the average of the scores provided by the two models. The column labeled "Accuracy" shows the frequency distribution of indels estimated by inDelphi. High accuracy is close to 100, representing sgRNAs characterized by one or a very low number of repair outcomes.

[0373] Candidate crRNA sequences were then evaluated for the presence of Graf motifs, TT or GCC, present in the four PAM-proximal bases of the crRNA sequence, as shown, for example, in Figures 26-42. Graf, R. et al. sgRNA Sequence Motifs Blocking Efficient CRISPR / Cas9-Mediated Gene Editing. Cell Reports, 26(5), 1098-103 (2019). Graf et al. reported that TT and GCC motifs are hallmarks of inefficient sgRNAs. When possible, crRNAs with Graf motifs, especially the GCC motif, were avoided. In contrast to the TT motif, the GCC motif remains important when the sgRNA is synthesized de novo rather than by transcription.

[0374] Three consensus scores were then calculated for each crRNA sequence, for example as shown in Figures 1-3. The "On-Off" score represents the average of the "On" and "Off" scores. The "Off-FS" score represents the average of the "Off" and "Frameshift" scores. The overall score represents the average of the "On", "Off", and "Frameshift" scores. Furthermore, for humans only, Bioscore of VBC was used to predict whether possible in-frame mutations may disrupt protein function, which are more likely to occur in conserved genomic DNA sequences that code for important protein domains. Thus, Bioscore is based on protein domain annotation, phylogenetic conservation, amino acid identity, and exon size. Michlits, G. et al. Multilayered VBC score predicts sgRNAs that efficiently generate loss-of-function alleles. Nature Methods 17, 708-716 (2020). The final criteria for selecting candidates are primarily based on overall score (>70 in most cases), a relatively low count of potential off-targets (<200 off-targets in most cases), absence of Graf motifs (where possible), and knockout or, in the case of hTGFBR1 and hTGFBR2, genomic cleavage site within the coding sequence to produce a truncated protein with reduced functionality (e.g., a decoy receptor).

[0375] Finally, the domain that each crRNA guide was editing was determined based on its nucleotide position in the gene (Figure 4A, 5A, 6A). The domain positions are used to predict whether the edits would result in a wild-type-like protein (editing sequences encoding the most C-terminus of the receptor, which is unlikely to disrupt receptor-mediated signaling), a complete knockout of any functional protein (editing sequences encoding the N-terminus and / or essential functional domains), and, if applicable, a soluble decoy receptor (editing sequences encoding the transmembrane domain of the receptor) or a membrane-bound decoy receptor (editing sequences encoding the intracellular signaling domain of the receptor).

[0376] Indeed, this in silico analysis established that several candidate sgRNAs were predicted to produce dominant negative (DN) TGF-β receptors, thereby resulting in genome editing that affects intracellular pro-inflammatory signaling. These prediction studies provided a high-throughput method for validating candidates without the need for time- and resource-intensive brute force methods, and demonstrated the feasibility of predicting the induction of gene knockouts (or dominant negative TGF-β receptors) as a unique approach to treat musculoskeletal fibrosis and / or scarring.

[0377] Example 2: Validation of TGFB1 editing in human cells After designing and analyzing a number of sgRNAs predicted to edit TGFB1 and result in gene knockout by the bioinformatics methods described in Example 1 (see FIG. 1), the next step was to evaluate their ability to act in vitro. Of particular interest is whether a single favorable edit is observed.

[0378] To test this, 80 pmoles of sgRNA candidates were introduced into THP-1 human monocytes via electroporation as part of a ribonucleoprotein (i.e., preassembled with 25 pmoles of Cas9 protein for at least 5 min at room temperature). Briefly, approximately 400,000 cells resuspended in 20 μl of SG buffer (Lonza) were added to the preassembled Cas9 ribonucleoprotein complex, transferred to a 16-well Nucleocuvette, and electroporated with pulse code FF-100 using a 4D Nucleofector (Lonza). After keeping the electroporated cells in SG buffer for approximately 10 min, they were transferred to a 12-well tissue culture plate containing complete culture medium. After 2–5 days in culture, cells were dissociated for genomic DNA extraction using the DNeasy Blood & Tissue Kit (Qiagen). The sgRNA target region was amplified by PCR, and then editing efficiency was estimated from Sanger sequencing traces using Inference of CRISPR Edits (ICE v1.2). See Conant, D. et al. (2022). The CRISPR Journal, 5(1), 123-130.

[0379] Multiple sgRNA candidates were able to induce mutations in the hTGFB1 gene with high efficiency, with some showing frequencies above 50%, and only one candidate, OHTG03 (SEQ ID NO: 682), showed the ability to introduce the same edits into a high percentage of cells when paired with either wild-type (WT) Cas9 or high-fidelity AR-Cas9 (IDT, Catalog No. IDT, Catalog No. 1081060, Vakulskas, CA et al. (2018). Nature Medicine, 24(8):1216-1224) (Figure 7A, B). The use of AR-Cas9 is expected to improve editing specificity (i.e., reduce off-target editing) compared to WT-Cas9. These results generally confirmed the in-silico analysis described in Example 1.

[0380] Example 3: Validation of TGFBR1 editing in human cells After designing and analyzing a number of sgRNAs predicted to edit the TGFBR1 receptor to produce either a knockout or a (soluble or membrane-bound) dominant negative decoy by the bioinformatics methods described in Example 1 (see FIG. 2), the next step was to evaluate their ability to act in vitro. Of particular interest was whether a single favorable on-target edit was observed in any of the candidates.

[0381] To test this, sgRNA candidates were complexed with Cas9 nuclease to form ribonucleoprotein (RNP) complexes, and human THP-1 monocytes were electroporated to introduce the RNPs into the cells as described above. Editing efficiency was then measured via Sanger sequencing. Multiple candidates were able to induce mutations in the hTGFBR1 gene with high efficiency, some showing frequencies above 50% with WT-Cas9 (Figure 8A), and this efficiency was universally reduced with the use of AR-Cas9 (Figure 8B). These results generally confirmed the in silico analysis and suggested that several sgRNA candidates, including OHTIR04, were able to efficiently edit TGFBR1 with some frequency in human cells.

[0382] Example 4: Validation of TGFBR2 editing in human cells After designing and analyzing a number of sgRNAs predicted to edit the TGFBR2 receptor to produce either a knockout or a (soluble or membrane-bound) dominant negative decoy by the bioinformatics methods described in Example 1 (see FIG. 3), the next step was to evaluate their ability to act in vitro. Of particular interest was whether a single favorable edit was observed in any of the candidates.

[0383] To test this, sgRNA candidates were complexed with Cas9 nuclease to form ribonucleoprotein (RNP) complexes, and human THP-1 monocytes were electroporated to introduce RNPs into the cells as described above. Editing efficiency was then measured via Sanger sequencing. Multiple candidates were able to induce mutations in the hTGFBR2 gene with high efficiency, with some showing frequencies close to or above 50% with WT-Cas9 (Figure 9A), and this efficiency was universally reduced with the use of AR-Cas9 (Figure 9B). Notably, pairing AR-Cas9 with OHTIIR11 (SEQ ID NO: 1065) resulted in an increase in editing dominance (i.e., the proportion of editing that was composed of the desired on-target editing), albeit with lower editing efficiency than WT-Cas9. These results generally confirmed the in silico analysis and suggested that several sgRNA candidates, including OHTIIR11, were able to efficiently edit TGFBR2 with some frequency in human cells.

[0384] Example 5: Editing of TGFBR1 or TGFBR2 renders human monocytes unresponsive to TGF-beta stimulation After designing and validating a number of guides targeting the TGF-beta pathway, the next step involved functional testing of the edited cells. As part of this test, approximately 400,000 edited human THP-1 monocytes were treated with either 1 μg / ml LPS or 20 ng / μl recombinant TGF-beta for 6 hours. Reverse transcription of total cellular RNA followed by quantitative PCR (RT-qPCR) was performed to verify whether either human LPS or TGF-beta could stimulate the transcription of TGFB1 as previously reported. See generally Hall, MC, et al. (2003). Journal of Biological Chemistry, 278(12), 10304-10313; Xiang Yin, et al 2017, J Atheroscler Thromb, 2017, 24:55-67. doi:10.5551 / jat.35204. LPS treatment caused a modest increase in TGFB1 transcription in control cells (WT-Cas9 injection only) (Figure 10A). However, TGFB1 transcription levels were reduced by as much as 5-fold for TGFBR1-edited cells compared to unedited controls, while TGFBR2-edited cells showed a 2-fold reduction.

[0385] As an additional test, the same cellular RNA was assayed for induction of TIMP1, which is canonically upregulated by TGF-beta signaling via SMAD proteins (see generally Hall, MC, et al. (2003). Journal of Biological Chemistry, 278(12), 10304-10313.). In LPS-treated cells, robust induction of TIMP1 was observed in unedited controls compared to untreated cells (Figure 10B). TGFBR1-edited monocytes demonstrated a 1-fold decrease in TIMP1 induction compared to unedited controls. There was no change for TGFBR2-edited cells.

[0386] The results were even more striking after treatment with the natural ligand. In response to TGF-beta treatment, TGFB1 induction was more robust in cells injected with only unedited WTCas9, and editing to either TGFBR1 or TGFBR2 reduced this response by 3-fold (Figure 10C). These results were similar to those observed for TIMP1 induction after TGF-beta treatment (Figure 10D). These results collectively indicated that the edited cells did not respond to TGF-beta treatment, as their TGFB1 levels remained well below the control. Taken together, these results demonstrated that both the positive feedback (TGFB1 induction) and downstream signaling (TIMP1 induction) of TGF-beta were impaired in cells with edited TGFBR1 and TGFBR2.

[0387] Example 6: Validation of TGFB1 editing in human cells After designing and analyzing a number of sgRNA candidates predicted to edit the human TGFB1 gene and produce either gene knockouts (see FIG. 11A), the next step is to evaluate their ability to act in vitro. Of particular interest is whether favorable (i.e., on-target) editing is observed in edited cells.

[0388] To test this, sgRNA candidates (SEQ ID NOs: 1-198) are introduced into human THP-1 monocytes via electroporation as part of a ribonucleoprotein, as described previously in Example 2.

[0389] The results of these studies are expected to further confirm the robustness of the in silico analysis and identify favorable candidates that can actually induce mutations in the TGFB1 gene with high efficacy. The use of high-fidelity AR-Cas9 is further expected to improve editing specificity (i.e., reduce off-target editing) compared to WT-Cas9.

[0390] Example 7: In vitro delivery of TGFB1-edited guide RNA to human cells via lipid nanoparticles Additional experiments evaluate the suitability of using lipid nanoparticles (LNPs) to deliver mRNA to human cells. LNPs containing sgRNA (SEQ ID NOs: 1-198) and WT- or AR-Cas9 mRNA are exposed to THP-1 monocytes for 8-24 hours under typical cell culture conditions. Editing is verified using Sanger sequencing as described in Example 2. After confirmation of editing, various functional assays are performed as described in Example 5.

[0391] Example 8: Adeno-associated virus-mediated in vitro delivery of TGFB1-edited guide RNA to human cells Since all validation experiments are performed via RNA-dependent nucleases and electroporation of cell cultures to introduce sgRNAs, additional experiments will evaluate the suitability of using adeno-associated virus (AAV) vectors to deliver sgRNAs to human cells. [Table 2]

[0392] Exemplary AAV vectors are shown in Table 2. After preparation according to the manufacturer's specifications to incorporate one or more of the sgRNAs containing SEQ ID NOs: 1-198 into the viral vector, the vector is incubated with approximately 500,000 U2OS cells stably transfected with Cas9 (MOI=0.1-10) (see, e.g., Rojas-Fernandez, A., et al. (2015). Scientific Reports, 5(1), 1-6.). Two to seven days after infection, editing is verified using Sanger sequencing as described in Example 2. After confirmation of editing, various functional assays are performed as described in Example 5.

[0393] Example 9: Validation of TGFBR1 editing in human cells After designing and analyzing a number of sgRNA candidates predicted to edit human transforming growth factor beta receptor 1 protein to produce either a knockout or a dominant negative receptor (see FIG. 11B), the next step is to evaluate their ability to act in vitro. Of particular interest is whether favorable (i.e., on-target) editing is observed in the edited cells.

[0394] To test this, sgRNA candidates (SEQ ID NOs: 199-320) are introduced into human THP-1 monocytes via electroporation as part of a ribonucleoprotein, as previously described in Example 2.

[0395] The results of these studies are expected to further confirm the robustness of the in silico analysis and identify several favorable candidates that can indeed induce mutations in the TGFBR1 gene with high efficacy. It is further expected that the use of high-fidelity AR-Cas9 will improve editing specificity (i.e., reduce off-target editing) compared to WT-Cas9.

[0396] Example 10: In vitro delivery of TGFBR1-edited guide RNA to human cells via lipid nanoparticles Since all validation experiments are performed via electroporation of cell cultures to introduce RNA-dependent nucleases and sgRNA, additional experiments evaluate the suitability of using lipid nanoparticles (LNPs) to deliver mRNA to human cells. LNPs containing either sgRNA (SEQ ID NOs: 199-320) or Cas9 mRNA were incubated with THP-1 monocytes, followed by validation of editing as described in Example 7. After validation of editing, various functional assays are performed as described in Example 5.

[0397] Example 11: Adeno-associated virus-mediated in vitro delivery of TGFBR1-edited guide RNA to human cells Since all validation experiments are performed via RNA-dependent nucleases and electroporation of cell cultures to introduce sgRNAs, additional experiments evaluate the suitability of using adeno-associated virus (AAV) vectors to deliver sgRNAs to human cells. Exemplary AAV vectors are shown in Table 2. After preparation according to the manufacturer's specifications such that one or more of the sgRNAs containing SEQ ID NOs: 199-320 are integrated into the viral vector, the vector is introduced into human U2OS cells and editing is verified as described above in Example 8. After confirmation of editing, various functional assays described in Example 5 are performed.

[0398] Example 12: Validation of TGFBR2 editing in human cells After designing and analyzing a number of sgRNA candidates predicted to edit the human transforming growth factor beta receptor 2 protein to produce either a knockout or a dominant negative receptor (see FIG. 11C), the next step is to evaluate their ability to act in vitro. Of particular interest is whether favorable (i.e., on-target) editing is observed in the edited cells.

[0399] To test this, the sgRNA candidates (SEQ ID NOs: 321-519) are introduced into human THP-1 monocytes via electroporation as part of a ribonucleoprotein, as previously described in Example 2.

[0400] The results of these studies are expected to further confirm the robustness of the in silico analysis and identify several favorable candidates that can indeed induce mutations in the TGFBR2 gene with high efficacy. It is further expected that the use of high-fidelity AR-Cas9 will improve editing specificity (i.e., reduce off-target editing) compared to WT-Cas9.

[0401] Example 13: In vitro delivery of TGFBR2-edited guide RNA to human cells via lipid nanoparticles Since all validation experiments are performed via electroporation of cell cultures to introduce RNA-dependent nucleases and sgRNA, additional experiments evaluate the suitability of using lipid nanoparticles (LNPs) to deliver mRNA to human cells. LNPs containing either sgRNA (SEQ ID NOs: 321-519) or Cas9 mRNA were incubated with THP-1 monocytes, followed by validation of editing as described in Example 7. After validation of editing, various functional assays are performed as described in Example 5.

[0402] Example 14: Adeno-associated virus-mediated in vitro delivery of TGFBR2-edited guide RNA to human cells Since all validation experiments are performed via RNA-dependent nucleases and electroporation of cell cultures to introduce sgRNAs, additional experiments evaluate the suitability of using adeno-associated virus (AAV) vectors to deliver sgRNAs to human cells. Exemplary AAV vectors are shown in Table 2. After preparation according to the manufacturer's specifications such that one or more of the sgRNAs containing SEQ ID NOs: 321-519 are integrated into the viral vector, the vector is introduced into human U2OS cells and editing is verified as described above in Example 8. After confirmation of editing, various functional assays described in Example 5 are performed.

[0403] Example 15: Post-operative treatment of arthrofibrosis Additional experiments are performed to demonstrate the efficacy of in vivo arthrofibrosis treatment in a postoperative setting. Briefly, the composition of pre-assembled RNP complexes containing sgRNAs directed to rabbit TGFB1, TGFBR1, and TGFBR2 genes and Cas9 enzyme is prepared as described in Example 2. These RNPs are injected intra-articularly into rabbits exhibiting arthrofibrosis.

[0404] Briefly, two cohorts of rabbits undergo contracture formation surgery with knee immobilization followed by remobilization surgery at 8 weeks. At the time of remobilization, one cohort undergoes capsular release while the other does not. Each cohort is then divided into subcohorts. For each cohort, one subcohort receives an injection of the RNP prepared above while the other subcohort does not. Evaluation for clinical symptoms of arthrofibrosis is then performed at several time points after injection, for example, at 10, 16, and 24 weeks, and the amputated limb is evaluated for fibrosis at the end of the study. An example of a study using this rabbit model of arthrofibrosis is described in Trousdale et al. Bone Joint Res., 2022, 11(1):32-39, doi:10.1302 / 2046-3758.111.BJR-2021-0546.R1.

[0405] Example 16: Editing TGFB1, TGFBR1, or TGFBR2 renders human monocytes unresponsive to driving the expression of multiple TGF-beta responsive gene products Additional functional studies were performed on edited and control THP-1 monocytes, adding TGF-beta for 6 hours, followed by collection of total cellular RNA for RT-qPCR analysis, as described above in Example 5. While SERPINE1 showed strong induction as a result of TGF-beta treatment compared to unedited vehicle control cells (Figure 11A), all edited cells, especially TGFBR1- and TGFBR2-edited monocytes, experienced strongly reduced induction. Similarly, Col1A2 was induced by TGF-beta treatment, with all edited monocytes showing an approximately two-fold reduction in induction (Figure 11B). In particular, more robust induction was observed for FN1 (Figure 11C) and CTGF (Figure 11D). Thus, it should be noted that TGFBR1- and TGFBR2-edited monocytes showed equivalent FN1 and CTGF levels to unedited vehicle controls, although the relative induction is less clear in edited cells. The results suggested that these cells were unable to induce RNA above background levels even after 6 hours of TGF-beta treatment. Collectively, these results indicated that following exposure to TGF-beta, several additional TGF-beta responsive gene products showed little or no expression above background in TGFBR1 and TGFBR2 edited cells, while TGFB1 edited cells also showed strongly reduced induction of these TGF-beta responsive gene products.

[0406] The above examples are provided to give those of ordinary skill in the art a complete disclosure and description of how to make and use the embodiments of the disclosed compositions, systems, and methods, and are not intended to limit the scope of what the inventors regard as their invention. Modifications of the above modes for implementing the embodiments of the disclosure that are obvious to those of ordinary skill in the art are intended to be within the scope of the following claims. All patents and publications discussed in this specification are indicative of the level of skill of those skilled in the art to which this disclosure pertains.

[0407] All headings and section names are used for clarity and reference only and are not to be construed as limiting in any manner For example, those skilled in the art will recognize the utility of combining various aspects from different headings and sections as appropriate in accordance with the spirit and scope of the disclosure described herein.

[0408] It will be understood that the methods described herein are not limited to the specific methodology, protocols, subjects, and sequencing techniques described herein, and thus may vary. Also, the terms used herein are merely for the purpose of describing certain embodiments, and are not intended to limit the scope of the methods and compositions described herein, which will be limited only by the scope of the appended claims. Although several embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will be made by those skilled in the art without departing from the present disclosure in the future. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be used in the implementation of the present disclosure. The following claims are intended to encompass the scope of the present disclosure, and methods and structures within the scope of these claims, and their equivalents.

[0409] The above examples are provided to give those of ordinary skill in the art a complete disclosure and description of how to make and use the embodiments of the disclosed compositions, systems, and methods, and are not intended to limit the scope of what the inventors regard as their invention. Modifications of the above modes for implementing the embodiments of the disclosure that are obvious to those of ordinary skill in the art are intended to be within the scope of the following claims. All patents and publications discussed in this specification are indicative of the level of skill of those skilled in the art to which this disclosure pertains.

[0410] All headings and section names are used for clarity and reference only and are not to be construed as limiting in any manner For example, those skilled in the art will recognize the utility of combining various aspects from different headings and sections as appropriate in accordance with the spirit and scope of the disclosure described herein.

[0411] It will be understood that the methods described herein are not limited to the specific methodology, protocols, subjects, and sequencing techniques described herein, and thus may vary. Also, the terms used herein are merely for the purpose of describing certain embodiments, and are not intended to limit the scope of the methods and compositions described herein, which will be limited only by the scope of the appended claims. Although several embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will be made by those skilled in the art without departing from the present disclosure in the future. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be used in the implementation of the present disclosure. The following claims are intended to encompass the scope of the present disclosure, and methods and structures within the scope of these claims, and their equivalents.

[0412] Some aspects are described with reference to exemplary applications for illustration. Unless otherwise indicated, any embodiment may be combined with any other embodiment. It should be understood that numerous specific details, relationships, and methods are set forth to provide a complete understanding of the features described herein. However, one of ordinary skill in the art will readily recognize that the features described herein may be implemented without one or more of the specific details, or with other methods. The features described herein are not limited by the illustrated order of acts or events, as some acts may occur in different orders and / or simultaneously with other acts or events. Furthermore, not all illustrated acts or events are required to implement a methodology in accordance with the features described herein.

[0413] Although some embodiments are shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the present disclosure be limited by the specific examples provided herein. Although the present disclosure has been described with reference to the above specification, the description and illustration of the embodiments herein are not intended to be construed in a limited sense. Numerous modifications, changes, and substitutions will be made by those skilled in the art in the future without departing from the present disclosure.

[0414] Furthermore, it should be understood that all aspects of the present disclosure are not limited to the specific depictions, configurations, or relative proportions set forth herein, which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be used in practicing the present disclosure. It is therefore contemplated that the present disclosure should encompass any such alternatives, modifications, variations, or equivalents. It is intended that the following claims be encompassed by the scope of the present disclosure, and that methods and structures within the scope of these claims, and their equivalents, are thereby encompassed.

[0415] All publications, patents, and patent applications herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict between a term in this specification and a term in an incorporated reference, the term in this specification shall control.

Claims

1. 1. A pharmaceutical composition for treating or preventing a disorder having a symptom caused at least in part by intercellular signaling mediated through the transforming growth factor beta (TGFβ) signaling pathway, comprising: (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease; (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA targeting the TGFB1 gene, the TGFBR1 gene, the TGFBR2 gene, or a combination thereof.

2. The pharmaceutical composition of claim 1 , wherein the disorder is fibrosis.

3. The pharmaceutical composition of claim 2 , wherein the fibrosis is postoperative fibrosis.

4. The pharmaceutical composition according to claim 2 , wherein the fibrosis is fibrosis following ligament reconstruction surgery.

5. 5. The pharmaceutical composition of claim 4, wherein the fibrosis is fibrosis after anterior cruciate ligament (ACL) reconstruction, fibrosis after autograft ACL reconstruction, or fibrosis after allograft ACL reconstruction.

6. The pharmaceutical composition according to claim 2 , wherein the fibrosis is knee arthrofibrosis.

7. The pharmaceutical composition of claim 2 , wherein the fibrosis results from intra-articular fibrous nodules.

8. The pharmaceutical composition of claim 2, wherein the fibrosis is after total knee arthroplasty (TKA).

9. The pharmaceutical composition of claim 2, wherein the fibrosis is after total knee arthroplasty (TKA).

10. The pharmaceutical composition of claim 2 , wherein the fibrosis is post-fracture repair fibrosis.

11. The pharmaceutical composition according to claim 2 , wherein the fibrosis is post-microdiscectomy fibrosis.

12. The pharmaceutical composition according to claim 2, wherein the fibrosis is epidural fibrosis following microdiscectomy.

13. The pharmaceutical composition according to claim 2 , wherein the fibrosis is post-lumbar laminectomy fibrosis.

14. The pharmaceutical composition according to claim 2, wherein the fibrosis is epidural fibrosis following lumbar laminectomy.

15. The pharmaceutical composition of claim 2 , wherein the fibrosis is post-anterior acromioplasty fibrosis.

16. The pharmaceutical composition according to claim 2 , wherein the fibrosis is post-subacromial decompression fibrosis.

17. The pharmaceutical composition of claim 2 , wherein the fibrosis is soft tissue fibrosis following anterior acromioplasty.

18. The pharmaceutical composition according to claim 2 , wherein the fibrosis is joint contracture following anterior acromioplasty.

19. The pharmaceutical composition according to claim 2 , wherein the fibrosis is post-arthroscopic fibrosis.

20. The pharmaceutical composition according to claim 2 , wherein the fibrosis is arthrofibrosis following arthroscopic surgery.

21. The pharmaceutical composition of claim 2 , wherein the fibrosis is trabecular meshwork fibrosis.

22. The pharmaceutical composition of claim 21 , wherein the trabecular meshwork fibrosis is post-glaucoma surgery fibrosis.

23. The pharmaceutical composition of claim 2 , wherein the fibrosis is epidural fibrosis.

24. The pharmaceutical composition of claim 2 , wherein the fibrosis is atrial fibrosis, cardiac fibrosis, myocardial fibrosis, and / or post-open heart surgery fibrosis.

25. The pharmaceutical composition according to claim 2 , wherein the fibrosis is post-liposuction fibrosis.

26. The pharmaceutical composition of claim 2, wherein the fibrosis is idiopathic pulmonary fibrosis (IPF).

27. The pharmaceutical composition of claim 2 , wherein the fibrosis is fibrosis in the knee, shoulder, or elbow joint.

28. The pharmaceutical composition of claim 1 , wherein the disorder comprises fibrosis in kidney tissue.

29. 29. The pharmaceutical composition of claim 28, wherein the disorder is chronic kidney disease.

30. The pharmaceutical composition of claim 1 , wherein the disorder comprises fibrosis in skin tissue.

31. 31. The pharmaceutical composition of claim 30, wherein the disorder is scarring after wound healing, a keloid disorder, nephrogenic systemic fibrosis, or scleroderma / systemic sclerosis.

32. The pharmaceutical composition of claim 1 , wherein the disorder comprises fibrosis in lung tissue.

33. 33. The pharmaceutical composition of claim 32, wherein the disorder is fibrothorax, pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, radiation-induced lung injury, progressive diffuse fibrosis, or scleroderma / systemic sclerosis.

34. The pharmaceutical composition of claim 1 , wherein the disorder comprises fibrosis in liver tissue.

35. 35. The pharmaceutical composition of claim 34, wherein the disorder is cirrhosis or bridging fibrosis.

36. The method of claim 1 , wherein the disorder comprises fibrosis in cardiac tissue.

37. 37. The pharmaceutical composition of claim 36, wherein the fibrosis is interstitial fibrosis.

38. 37. The pharmaceutical composition of claim 36, wherein the disorder is congestive heart failure or hypertension.

39. 37. The pharmaceutical composition of claim 36, wherein the fibrosis is replacement fibrosis.

40. 37. The pharmaceutical composition of claim 36, wherein the disorder is myocardial infarction.

41. The pharmaceutical composition of claim 1 , wherein the disorder comprises fibrosis in brain tissue.

42. 42. The pharmaceutical composition of claim 41, wherein the fibrosis is a glial scar.

43. The method of claim 1 , wherein the disorder comprises fibrosis in intestinal tissue.

44. 44. The pharmaceutical composition of claim 43, wherein the disorder is Crohn's disease.

45. The pharmaceutical composition of claim 1 , wherein the disorder comprises fibrosis in the hand or finger.

46. 46. ​​The pharmaceutical composition of claim 45, wherein the disorder is Dupuytren's contracture.

47. The pharmaceutical composition of claim 1 , wherein the disorder comprises fibrosis in lymphatic tissue.

48. 48. The pharmaceutical composition of claim 47, wherein the fibrosis is mediastinal fibrosis.

49. The pharmaceutical composition of claim 1 , wherein the disorder comprises fibrosis in bone marrow tissue.

50. 50. The pharmaceutical composition of claim 49, wherein the fibrosis is myelofibrosis.

51. 2. The pharmaceutical composition of claim 1, wherein the disorder comprises fibrosis in penile tissue.

52. 52. The pharmaceutical composition of claim 51, wherein the disorder is Peyronie's disease.

53. The pharmaceutical composition of claim 1 , wherein the disorder comprises fibrosis in the soft tissue of the retroperitoneum.

54. 50. The pharmaceutical composition of claim 49, wherein the fibrosis is retroperitoneal fibrosis.

55. The pharmaceutical composition of claim 1 , wherein the disorder comprises fibrosis in musculoskeletal tissue.

56. The pharmaceutical composition of claim 1 , wherein the disorder is a musculoskeletal disorder.

57. 33. The pharmaceutical composition of claim 32, wherein the musculoskeletal disorder is a genetic musculoskeletal disease.

58. 33. The pharmaceutical composition of claim 32, wherein the musculoskeletal disorder is a muscular dystrophy.

59. 33. The pharmaceutical composition of claim 32, wherein the musculoskeletal disorder is selected from Duchenne muscular dystrophy (DMD), myotonic dystrophy (DM), facioscapulohumeral muscular dystrophy (FSHD), and limb-girdle muscular dystrophy (LGMD).

60. The pharmaceutical composition of claim 32, wherein the musculoskeletal disorder is type II collagenopathy.

61. 33. The pharmaceutical composition of claim 32, wherein the musculoskeletal disorder is selected from achondroplasia type II, hypochondroplasia, Stickler syndrome, and Czech dysplasia.

62. 33. The pharmaceutical composition of claim 32, wherein the musculoskeletal disorder is osteogenesis imperfecta (OI).

63. 33. The pharmaceutical composition of claim 32, wherein the musculoskeletal disorder is an autoimmune musculoskeletal disease.

64. 33. The pharmaceutical composition of claim 32, wherein the musculoskeletal disorder is an autoimmune musculoskeletal disease with a polygenic susceptibility trait.

65. 33. The pharmaceutical composition of claim 32, wherein the musculoskeletal disorder is spondyloarthropathy (SA).

66. 33. The pharmaceutical composition of claim 32, wherein the musculoskeletal disorder is ankylosing spondylitis.

67. 33. The pharmaceutical composition of claim 32, wherein the musculoskeletal disorder is rheumatoid arthritis.

68. 33. The pharmaceutical composition of claim 32, wherein the musculoskeletal disorder is autoimmune osteoarthritis.

69. 33. The pharmaceutical composition of claim 32, wherein the musculoskeletal disorder is osteoarthritis (OA).

70. 33. The pharmaceutical composition of claim 32, wherein the musculoskeletal disorder is Kamuracci-Engelmann disease.

71. 33. The pharmaceutical composition of claim 32, wherein the musculoskeletal disorder is Sjogren's syndrome.

72. 33. The pharmaceutical composition of claim 32, wherein the musculoskeletal disorder is a mechanical musculoskeletal injury.

73. 33. The pharmaceutical composition of claim 32, wherein the musculoskeletal disorder is selected from chronic muscle injury, tendon rupture, post-traumatic osteoarthritis, and post-traumatic arthrofibrosis.

74. 74. The pharmaceutical composition of any one of claims 1 to 73, wherein said at least one guide RNA or a nucleic acid encoding at least one guide RNA targets a mammalian TGFB1 gene.

75. 75. The pharmaceutical composition of claim 74, wherein the mammalian TGFB1 gene is a canine, equine, or feline TGFB1 gene.

76. 75. The pharmaceutical composition of claim 74, wherein the mammalian TGFB1 gene is a human TGFB1 gene.

77. 77. The pharmaceutical composition of claim 76, wherein the at least one guide RNA comprises a crRNA sequence selected from the group consisting of the sequences depicted in Figures 1A-1D (SEQ ID NOs: 1-198).

78. 77. The pharmaceutical composition of claim 76, wherein the at least one guide RNA comprises a crRNA sequence selected from the group consisting of the sequences depicted in FIG. 12A (SEQ ID NOs: 520-527).

79. 77. The pharmaceutical composition of claim 76, wherein the at least one guide RNA comprises a crRNA sequence of OHTG03 (SEQ ID NO: 522) or OHTG04 (SEQ ID NO: 523).

80. 74. The pharmaceutical composition of any one of claims 1 to 73, wherein the at least one guide RNA or the nucleic acid encoding at least one guide RNA targets a mammalian TGFBR1 gene.

81. 81. The pharmaceutical composition of claim 80, wherein the mammalian TGFBR1 gene is a canine, equine, or feline TGFBR1 gene.

82. 81. The pharmaceutical composition of claim 80, wherein the mammalian TGFBR1 gene is a human TGFBR1 gene.

83. 83. The pharmaceutical composition of claim 82, wherein the at least one guide RNA comprises a crRNA sequence selected from the group consisting of the sequences depicted in Figures 2A-2C (SEQ ID NOs: 199-320).

84. 83. The pharmaceutical composition of claim 82, wherein the at least one guide RNA comprises a crRNA sequence selected from the group consisting of the sequences depicted in FIG. 12B (SEQ ID NOs: 528-552).

85. 83. The pharmaceutical composition of claim 82, wherein the at least one guide RNA comprises a crRNA sequence selected from the group consisting of the sequences depicted in FIG. 5B.

86. 83. The pharmaceutical composition of claim 82, wherein the at least one guide RNA comprises a crRNA sequence of OHTIR04 (SEQ ID NO: 532) or OHTIR08 (SEQ ID NO: 539).

87. 74. The pharmaceutical composition of any one of claims 1 to 73, wherein the at least one guide RNA or the nucleic acid encoding at least one guide RNA targets a mammalian TGFBR2 gene.

88. 88. The pharmaceutical composition of claim 87, wherein the mammalian TGFBR2 gene is a canine, equine, or feline TGFBR2 gene.

89. 88. The pharmaceutical composition of claim 87, wherein the mammalian TGFBR2 gene is a human TGFBR2 gene.

90. 90. The pharmaceutical composition of claim 89, wherein the at least one guide RNA comprises a crRNA sequence selected from the group consisting of the sequences depicted in Figures 3A-3D (SEQ ID NOs: 321-519).

91. 90. The pharmaceutical composition of claim 89, wherein the at least one guide RNA comprises a crRNA sequence selected from the group consisting of the sequences depicted in FIG. 12C (SEQ ID NOs: 553-604).

92. 90. The pharmaceutical composition of claim 89, wherein the at least one guide RNA comprises a crRNA sequence selected from the group consisting of the sequences depicted in FIG. 6B.

93. 90. The pharmaceutical composition of claim 89, wherein the at least one guide RNA comprises a crRNA sequence of OHTIIR04 (SEQ ID NO:563).

94. The pharmaceutical composition of any one of claims 1 to 93, wherein the RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease is the RNA-guided nuclease.

95. The pharmaceutical composition according to any one of claims 1 to 93, wherein the RNA-guided nuclease or the nucleic acid encoding the RNA-guided nuclease is a DNA encoding the RNA-guided nuclease.

96. The pharmaceutical composition according to any one of claims 1 to 93, wherein the RNA-guided nuclease or the nucleic acid encoding the RNA-guided nuclease is an mRNA encoding the RNA-guided nuclease.

97. The pharmaceutical composition of any one of claims 1 to 96, wherein the RNA-guided nuclease is a Cas protein.

98. 98. The pharmaceutical composition of claim 97, wherein the Cas protein is a Cas9 protein.

99. 98. The pharmaceutical composition of claim 97, wherein the Cas9 protein is a S. pyogenes Cas9 polypeptide.

100. 98. The pharmaceutical composition of claim 97, wherein the Cas9 protein is selected from the group consisting of esCas9, hfCas9, peCas9, and ARCas9.

101. The pharmaceutical composition of any one of claims 1 to 100, wherein the at least one guide RNA or the nucleic acid encoding at least one guide RNA is the at least one guide RNA.

102. The pharmaceutical composition of any one of claims 1 to 100, wherein the at least one guide RNA or the nucleic acid encoding at least one guide RNA is a DNA encoding the at least one guide RNA.

103. 101. The pharmaceutical composition of any one of claims 1 to 100, comprising a nucleic acid encoding both the RNA-guided nuclease and the at least one guide RNA.

104. The pharmaceutical composition of any one of claims 1 to 103, wherein the at least one guide RNA is a single guide RNA (sgRNA).

105. 105. The pharmaceutical composition of any one of claims 1 to 104, wherein the composition comprises one or more viral vectors collectively comprising (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease, and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA targeting a gene encoding a transmembrane receptor.

106. 106. The pharmaceutical composition of claim 105, wherein one of more viral vectors comprises a recombinant virus selected from a retrovirus, an adenovirus, an adeno-associated virus, a lentivirus, and herpes simplex virus-1.

107. The pharmaceutical composition of claim 105, wherein one of the more viral vectors comprises a recombinant adeno-associated virus (AAV).

108. The pharmaceutical composition of claim 107, wherein the recombinant AAV is of serotype 5 (AAV5).

109. The pharmaceutical composition of claim 107, wherein the recombinant AAV is of serotype 6 (AAV6).

110. 105. The pharmaceutical composition of any one of claims 1 to 104, wherein the composition comprises one or more lipid nanoparticles (LNPs) collectively comprising (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease, and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA that targets a gene encoding a transmembrane receptor.

111. The one or more LNPs a first plurality of LNPs encapsulating the RNA-guided nuclease or a nucleic acid encoding the RNA-guided nuclease; and a second plurality of LNPs encapsulating the at least one guide RNA or a nucleic acid encoding the at least one guide RNA.

112. The pharmaceutical composition of claim 110, wherein the one or more LNPs comprise a plurality of LNPs encapsulating both (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease and (ii) at least one guide RNA targeting a gene encoding a transmembrane receptor or a nucleic acid encoding at least one guide RNA.

113. The one or more LNPs may be 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-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 (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 113. The pharmaceutical composition of any one of claims 110-112, comprising a component 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(2S)), lipids comprising a cyclic amine group, and mixtures thereof.

114. The LNPs may be 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 1-Oleoyl-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 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.

115. The pharmaceutical composition of any one of claims 110-114, wherein the LNP comprises a component selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. For example, the PEG lipid may be a PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DMA, PEG-DSPE lipid, and mixtures thereof.

116. 116. The composition of any one of claims 110-115, wherein the LNPs comprise a component 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.

117. 105. The pharmaceutical composition of any one of claims 1 to 104, wherein the composition comprises one or more liposomes collectively comprising (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease, and (ii) at least one guide RNA targeting a gene encoding a transmembrane receptor or a nucleic acid encoding at least one guide RNA.

118. the one or more liposomes a first plurality of liposomes encapsulating the RNA-guided nuclease or a nucleic acid encoding the RNA-guided nuclease; and a second plurality of liposomes encapsulating the at least one guide RNA or a nucleic acid encoding the at least one guide RNA.

119. The pharmaceutical composition of claim 117, wherein the one or more liposomes comprise a plurality of liposomes encapsulating both (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease and (ii) at least one guide RNA targeting a gene encoding a transmembrane receptor or a nucleic acid encoding at least one guide RNA.

120. 105. The pharmaceutical composition of any one of claims 1 to 104, wherein the composition comprises one or more virus-like particles collectively comprising (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease, and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA targeting a gene encoding a transmembrane receptor.

121. the one or more virus-like particles being a first plurality of virus-like particles encapsulating the RNA-guided nuclease or a nucleic acid encoding the RNA-guided nuclease; and a second plurality of virus-like particles encapsulating the at least one guide RNA or a nucleic acid encoding the at least one guide RNA.

122. The pharmaceutical composition of claim 120, wherein the one or more virus-like particles comprise a plurality of virus-like particles encapsulating both (i) the RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA targeting a gene encoding a transmembrane receptor.

123. The pharmaceutical composition of any one of claims 1 to 122, wherein the composition is formulated for parenteral administration.

124. 123. The pharmaceutical composition of any one of claims 1 to 122, wherein the composition is formulated for intra-articular injection into a joint of a subject.

125. The pharmaceutical composition of any one of claims 1 to 122, wherein the composition is formulated for intradiscal injection.

126. The pharmaceutical composition of any one of claims 1 to 122, wherein the composition is formulated for peridiscal injection.

127. The pharmaceutical composition of any one of claims 1 to 122, wherein the composition is formulated for intraspinal injection.

128. 1. A method for treating or preventing, in a subject in need thereof, a disorder having a symptom caused at least in part by intercellular signaling mediated through the transforming growth factor beta (TGFβ) signaling pathway, comprising: administering a therapeutically effective amount of a composition, said composition comprising: (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease; (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA targeting the TGFB1 gene, the TGFBR1 gene, the TGFBR2 gene, and combinations thereof.

129. 129. The method of claim 128, wherein the disorder is fibrosis.

130. 130. The method of claim 129, wherein the fibrosis is post-operative fibrosis.

131. 130. The method of claim 129, wherein the fibrosis is post-ligament reconstruction fibrosis.

132. 132. The method of claim 131, wherein the fibrosis is post-anterior cruciate ligament (ACL) reconstruction fibrosis, post-autograft ACL reconstruction fibrosis, or post-allograft ACL reconstruction fibrosis.

133. 130. The method of claim 129, wherein the fibrosis is knee arthrofibrosis.

134. 130. The method of claim 129, wherein the fibrosis results from intra-articular fibrous nodules.

135. 130. The method of claim 129, wherein the fibrosis is following total knee arthroplasty (TKA).

136. The pharmaceutical composition of claim 2 , wherein the fibrosis is post-fracture repair fibrosis.

137. 130. The method of claim 129, wherein the fibrosis is post-TKA knee arthrofibrosis.

138. 130. The method of claim 129, wherein the fibrosis is post-microdiscectomy fibrosis.

139. 130. The method of claim 129, wherein the fibrosis is epidural fibrosis following microdiscectomy.

140. 130. The method of claim 129, wherein the fibrosis is post-lumbar laminectomy fibrosis.

141. 130. The method of claim 129, wherein the fibrosis is epidural fibrosis following lumbar laminectomy.

142. 130. The method of claim 129, wherein the fibrosis is post-anterior acromioplasty fibrosis.

143. 130. The method of claim 129, wherein the fibrosis is post-subacromial decompression fibrosis.

144. 130. The method of claim 129, wherein the fibrosis is soft tissue fibrosis following anterior acromioplasty.

145. 130. The method of claim 129, wherein the fibrosis is joint contracture following anterior acromioplasty.

146. 130. The method of claim 129, wherein the fibrosis is post-arthroscopic fibrosis.

147. 130. The method of claim 129, wherein the fibrosis is post-arthroscopic arthrofibrosis.

148. 130. The method of claim 129, wherein the fibrosis is trabecular meshwork fibrosis.

149. The method of claim 148, wherein the trabecular meshwork fibrosis is post-glaucoma surgery fibrosis.

150. 130. The method of claim 129, wherein the fibrosis is epidural fibrosis.

151. 130. The method of claim 129, wherein the fibrosis is atrial fibrosis, cardiac fibrosis, myocardial fibrosis, and / or post-open chest surgery fibrosis.

152. 130. The method of claim 129, wherein the fibrosis is post-liposuction fibrosis.

153. 130. The method of claim 129, wherein the fibrosis is idiopathic pulmonary fibrosis (IPF).

154. 129. The method of claim 128, wherein the fibrosis is fibrosis in the knee, shoulder, or elbow joint.

155. 129. The method of claim 128, wherein the disorder comprises fibrosis in kidney tissue.

156. 129. The method of claim 128, wherein the disorder is chronic kidney disease.

157. 129. The method of claim 128, wherein the disorder comprises fibrosis in skin tissue.

158. 129. The method of claim 128, wherein the disorder is scarring after wound healing, a keloid disorder, nephrogenic systemic fibrosis, or scleroderma / systemic sclerosis.

159. 129. The method of claim 128, wherein the disorder comprises fibrosis in lung tissue.

160. 129. The method of claim 128, wherein the disorder is fibrothorax, pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, radiation-induced lung injury, progressive diffuse fibrosis, or scleroderma / systemic sclerosis.

161. 129. The method of claim 128, wherein the disorder comprises fibrosis in liver tissue.

162. 129. The method of claim 128, wherein the disorder is cirrhosis or bridging fibrosis.

163. 129. The method of claim 128, wherein the disorder comprises fibrosis in cardiac tissue.

164. 129. The method of claim 128, wherein the fibrosis is interstitial fibrosis.

165. 129. The method of claim 128, wherein the disorder is congestive heart failure or hypertension.

166. 129. The method of claim 128, wherein the fibrosis is replacement fibrosis.

167. 129. The method of claim 128, wherein the disorder is myocardial infarction.

168. 129. The method of claim 128, wherein the disorder comprises fibrosis in brain tissue.

169. 129. The method of claim 128, wherein the disorder is a glial scar.

170. 129. The method of claim 128, wherein the disorder comprises fibrosis in intestinal tissue.

171. 129. The method of claim 128, wherein the disorder is Crohn's disease.

172. 129. The method of claim 128, wherein the disorder comprises fibrosis in the hand or finger.

173. 129. The method of claim 128, wherein the disorder is Dupuytren's contracture.

174. 129. The method of claim 128, wherein the disorder comprises fibrosis in lymphatic tissue.

175. 129. The method of claim 128, wherein the fibrosis is mediastinal fibrosis.

176. 129. The method of claim 128, wherein the disorder comprises fibrosis in bone marrow tissue.

177. 129. The method of claim 128, wherein the fibrosis is myelofibrosis.

178. 129. The method of claim 128, wherein the disorder comprises fibrosis in penile tissue.

179. 129. The method of claim 128, wherein the disorder is Peyronie's disease.

180. 129. The method of claim 128, wherein the disorder comprises fibrosis in the soft tissue of the retroperitoneum.

181. 129. The method of claim 128, wherein the fibrosis is retroperitoneal fibrosis.

182. 129. The method of claim 128, wherein the disorder comprises fibrosis in musculoskeletal tissue.

183. 129. The method of claim 128, wherein the disorder is a musculoskeletal disorder.

184. 184. The method of claim 183, wherein the musculoskeletal disorder is a genetic musculoskeletal disease.

185. 184. The method of claim 183, wherein the musculoskeletal disorder is a muscular dystrophy.

186. 184. The method of claim 183, wherein the musculoskeletal disorder is selected from Duchenne muscular dystrophy (DMD), myotonic dystrophy (DM), facioscapulohumeral muscular dystrophy (FSHD), and limb-girdle muscular dystrophy (LGMD).

187. The method of claim 183, wherein the musculoskeletal disorder is type II collagenopathy.

188. 184. The method of claim 183, wherein the musculoskeletal disorder is selected from achondroplasia type II, hypochondroplasia, Stickler syndrome, and Czech dysplasia.

189. 184. The method of claim 183, wherein the musculoskeletal disorder is osteogenesis imperfecta (OI).

190. 184. The method of claim 183, wherein the musculoskeletal disorder is an autoimmune musculoskeletal disease.

191. The method of claim 183, wherein the musculoskeletal disorder is an autoimmune musculoskeletal disease with a polygenic susceptibility trait.

192. 184. The method of claim 183, wherein the musculoskeletal disorder is spondyloarthropathy (SA).

193. 184. The method of claim 183, wherein the musculoskeletal disorder is ankylosing spondylitis.

194. 184. The method of claim 183, wherein the musculoskeletal disorder is rheumatoid arthritis.

195. 184. The method of claim 183, wherein the musculoskeletal disorder is autoimmune osteoarthritis.

196. 184. The method of claim 183, wherein the musculoskeletal disorder is osteoarthritis (OA).

197. 184. The method of claim 183, wherein the musculoskeletal disorder is Kamuracci-Engelmann disease.

198. 184. The method of claim 183, wherein the musculoskeletal disorder is Sjogren's syndrome.

199. 184. The method of claim 183, wherein the musculoskeletal disorder is a mechanical musculoskeletal injury.

200. 184. The method of claim 183, wherein the musculoskeletal disorder is selected from chronic muscle injury, tendon rupture, post-traumatic osteoarthritis, and post-traumatic arthrofibrosis.

201. 201. The method of any one of claims 128-200, wherein said at least one guide RNA or a nucleic acid encoding at least one guide RNA targets a mammalian TGFB1 gene.

202. 202. The method of claim 201, wherein the mammalian TGFB1 gene is a canine, equine, or feline TGFB1 gene.

203. 202. The method of claim 201, wherein the mammalian TGFB1 gene is a human TGFB1 gene.

204. 204. The method of claim 203, wherein said at least one guide RNA comprises a crRNA sequence selected from the group consisting of the sequences depicted in Figures 1A-1D (SEQ ID NOs: 1-198).

205. 204. The method of Claim 203, wherein said at least one guide RNA comprises a crRNA sequence selected from the group consisting of the sequences depicted in Figure 12A (SEQ ID NOs:520-527).

206. 204. The method of claim 203, wherein the at least one guide RNA comprises a crRNA sequence of OHTG03 or OHTG04.

207. 201. The method of any one of claims 128-200, wherein said at least one guide RNA or a nucleic acid encoding at least one guide RNA targets a mammalian TGFBR1 gene.

208. 208. The method of claim 207, wherein the mammalian TGFBR1 gene is a canine, equine, or feline TGFBR1 gene.

209. 208. The method of claim 207, wherein the mammalian TGFBR1 gene is a human TGFBR1 gene.

210. 210. The method of Claim 209, wherein said at least one guide RNA comprises a crRNA sequence selected from the group consisting of the sequences depicted in Figures 2A-2C (SEQ ID NOs: 199-320).

211. 210. The method of Claim 209, wherein said at least one guide RNA comprises a crRNA sequence selected from the group consisting of the sequences depicted in Figure 12B (SEQ ID NOs:528-552).

212. 210. The method of Claim 209, wherein said at least one guide RNA comprises a crRNA sequence selected from the group consisting of the sequences depicted in Figure 5B.

213. 210. The method of claim 209, wherein the at least one guide RNA comprises a crRNA sequence of OHTIR04 or OHTIR08.

214. 201. The method of any one of claims 128-200, wherein said at least one guide RNA or a nucleic acid encoding at least one guide RNA targets a mammalian TGFBR2 gene.

215. 215. The method of claim 214, wherein the mammalian TGFBR1 gene is a canine, equine, or feline TGFBR1 gene.

216. 215. The method of claim 214, wherein the mammalian TGFBR1 gene is a human TGFBR1 gene.

217. 217. The method of Claim 216, wherein said at least one guide RNA comprises a crRNA sequence selected from the group consisting of the sequences depicted in Figures 3A-3D (SEQ ID NOs:321-519).

218. 217. The method of Claim 216, wherein said at least one guide RNA comprises a crRNA sequence selected from the group consisting of the sequences depicted in Figure 12C (SEQ ID NOs:553-604).

219. 217. The method of Claim 216, wherein said at least one guide RNA comprises a crRNA sequence selected from the group consisting of the sequences depicted in Figure 6B.

220. 217. The method of claim 216, wherein the at least one guide RNA comprises a crRNA sequence of OHTIIR04.

221. 221. The method of any one of claims 128 to 220, wherein the RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease is the RNA-guided nuclease.

222. The method of any one of claims 128 to 220, wherein the RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease is a DNA encoding the RNA-guided nuclease.

223. The method of any one of claims 128 to 220, wherein the RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease is an mRNA encoding the RNA-guided nuclease.

224. The method of any one of claims 128 to 223, wherein the RNA-guided nuclease is a Cas protein.

225. 225. The method of claim 224, wherein the Cas protein is a Cas9 protein.

226. 226. The method of claim 225, wherein the Cas9 protein is a S. pyogenes Cas9 polypeptide.

227. 226. The method of claim 225, wherein the Cas9 protein is selected from the group consisting of esCas9, hfCas9, peCas9, and ARCas9.

228. 228. The method of any one of Claims 128-227, wherein the at least one guide RNA or a nucleic acid encoding at least one guide RNA is the at least one guide RNA.

229. The method of any one of Claims 128-227, wherein the at least one guide RNA or the nucleic acid encoding at least one guide RNA is a DNA encoding the at least one guide RNA.

230. 228. The method of any one of Claims 128-227, comprising a nucleic acid encoding both the RNA-guided nuclease and the at least one guide RNA.

231. 231. The method of any one of Claims 128-230, wherein the at least one guide RNA is a single guide RNA (sgRNA).

232. 232. The method of any one of Claims 128-231, wherein the composition comprises one or more viral vectors collectively comprising (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease, and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA targeting a gene encoding a transmembrane receptor.

233. 233. The method of claim 232, wherein one of more viral vectors comprises a recombinant virus selected from a retrovirus, an adenovirus, an adeno-associated virus, a lentivirus, and herpes simplex virus-1.

234. 233. The method of claim 232, wherein one of the more viral vectors comprises a recombinant adeno-associated virus (AAV).

235. The method of claim 234, wherein the recombinant AAV is of serotype 5 (AAV5).

236. The method of claim 234, wherein the recombinant AAV is of serotype 6 (AAV6).

237. 232. The method of any one of claims 128-231, wherein the composition comprises one or more lipid nanoparticles (LNPs) collectively comprising (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease, and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA that targets a gene encoding a transmembrane receptor.

238. The one or more LNPs a first plurality of LNPs encapsulating the RNA-guided nuclease or a nucleic acid encoding the RNA-guided nuclease; and a second plurality of LNPs encapsulating the at least one guide RNA or a nucleic acid encoding the at least one guide RNA.

239. The method of claim 237, wherein the one or more LNPs comprise a plurality of LNPs encapsulating both (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA targeting a gene encoding a transmembrane receptor.

240. The one or more LNPs may be 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-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 (DODMA), 2-({8- (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)-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), 240. The method of any one of claims 237-239, comprising a component 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(2S)), lipids comprising a cyclic amine group, and mixtures thereof.

241. The LNPs may be 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 1-Oleoyl-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 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.

242. 242. The method of any one of claims 237-241, wherein the LNP comprises a component selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. For example, the PEG lipid may be a PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DMA, PEG-DSPE lipid, and mixtures thereof.

243. 243. The method of any one of claims 237-242, wherein the LNPs comprise a component 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.

244. 232. The method of any one of Claims 128-231, wherein the composition comprises one or more liposomes collectively comprising (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease, and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA targeting a gene encoding a transmembrane receptor.

245. the one or more liposomes a first plurality of liposomes encapsulating the RNA-guided nuclease or a nucleic acid encoding the RNA-guided nuclease; and a second plurality of liposomes encapsulating the at least one guide RNA or a nucleic acid encoding the at least one guide RNA.

246. 245. The method of Claim 244, wherein the one or more liposomes comprise a plurality of liposomes encapsulating both (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease and (ii) at least one guide RNA targeting a gene encoding a transmembrane receptor or a nucleic acid encoding at least one guide RNA.

247. 232. The method of any one of claims 128-231, wherein the composition comprises one or more virus-like particles collectively comprising (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease, and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA targeting a gene encoding a transmembrane receptor.

248. the one or more virus-like particles being a first plurality of virus-like particles encapsulating the RNA-guided nuclease or a nucleic acid encoding the RNA-guided nuclease; and a second plurality of virus-like particles encapsulating the at least one guide RNA or a nucleic acid encoding the at least one guide RNA.

249. 248. The method of Claim 247, wherein said one or more virus-like particles comprise a plurality of virus-like particles encapsulating both (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease, and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA targeting a gene encoding a transmembrane receptor.

250. 250. The method of any one of claims 128-249, wherein said administering comprises parenteral administration.

251. 250. The method of any one of claims 128-249, wherein the administering comprises an intra-articular injection into a joint of the subject.

252. 250. The method of any one of claims 128-249, wherein the administering comprises intradiscal injection.

253. 250. The method of any one of claims 128-249, wherein the administering comprises a peridiscal injection.

254. 250. The method of any one of claims 128-249, wherein the administering comprises intraspinal injection.

255. 250. The method of any one of claims 128-249, wherein the administering is during surgery.

256. The method of claim 255, wherein the composition is applied onto a surgical site.

257. 256. The method of claim 255, wherein the composition is sprayed onto the surgical site.

258. The method of claim 255, wherein the composition is coated on a dressing that is applied to a surgical site.

259. 256. The method of claim 255, wherein the composition is coated onto a stent that is implanted during surgery.

260. The method of claim 255, wherein the composition is dip coated onto the implant.

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