Gene editing for the treatment of surgery-related fibrosis

Abstract

Compositions and methods for treating musculoskeletal fibrosis and / or scarring by eliminating intracellular signaling through specific cell surface receptors via gene editing are provided herein. In some embodiments, the compositions and methods target a TGFB1 ligand. In other embodiments, the compositions and methods target a TGFB1 receptor (TGFBR1 / TGFBR2). In some embodiments, the compositions and methods are for treating or preventing post-traumatic fibrosis and / or scarring. In some embodiments, the compositions and methods are for treating or preventing postoperative fibrosis and / or scarring. In some embodiments, the compositions and methods are for treating or preventing local pain, inflammation, degeneration, or morphological changes associated with fibrosis and / or scarring. In some embodiments, the compositions and methods are for treating fibrosis.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to International Application No. PCT / US23 / 61392, filed on January 26, 2023, and U.S. Provisional Patent Application No. 63 / 480,642, filed on January 19, 2023, the contents of which are hereby incorporated by reference in their entirety for all purposes.

[0002] Reference to Electronically Submitted Sequence Listing This application includes a sequence listing submitted electronically in XML file format, which is hereby incorporated by reference in its entirety. The XML file named 123994 - 5004 - WO50 Sequence Listing.xml was created on January 17, 2024, and is 3,360 kilobytes in size.

Background Art

[0003] Skeletal tissue is particularly prone to fibrosis and / or scarring, especially after trauma. In many cases, this trauma can be surgery. Fibrosis is a result of extracellular matrix (ECM) components accumulating at a particular site and contributes to the formation of scar tissue. See Sheets, K., et al (2022). Journal of Cellular Biochemistry. This skeletal scar tissue presents several problems such as restricted range of motion or pain, and may further promote disease and complicate the medical or surgical management of skeletal tissue and joints.

[0004] 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. Often, circulating ligands bind to specific receptors immobilized on the cell membrane, which leads to transduction of the signaling pathway from the cell surface to the interior through the activation of the receptor's cytoplasmic domain. TGFB signaling events can then potentially affect various cellular activities, which in turn lead to changes at the tissue and biological levels.

[0005] One such change is musculoskeletal fibrosis. Indeed, increased TGFB1 expression is observed in fascial fibroblasts at 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 type I collagen and fibronectin, ECM components whose aggregation can contribute to fibrosis. TGFB, a known driver of pro-inflammatory signaling, leads to a link between fibrosis and / or scarring and chronic inflammation.

[0006] Current treatments for such musculoskeletal conditions, such as conservative care, physical rehabilitation, or surgery, focus on treating the symptoms rather than addressing the underlying causes of fibrosis and / or scarring, and may even lead to further trauma. Therefore, there is a very strong need for new methods and compositions that address the causes of this condition. [Overview of the project]

[0007] This specification provides compositions and methods for silencing the signaling function of either TGFB1 or its cell receptor in animals where such silencing is required, to treat diseases, illnesses, or conditions caused by abnormal or excessive signaling through said receptor.

[0008] In some embodiments, receptor signaling is silenced by CRISPR editing of the gene encoding the TGFBR1 receptor. In some embodiments, receptor signaling is silenced by CRISPR editing of the gene encoding the TGFBR2 receptor. In some embodiments, receptor signaling is silenced by CRISPR editing of the gene encoding the TGFB1 ligand. In some embodiments, CRISPR editing results in the removal of the transmembrane domain (i.e., the generation of a soluble receptor). In some embodiments, CRISPR editing results in the removal of the cytoplasmic domain.

[0009] The ubiquity of the receptor-ligand paradigm in cell biology means that many diseases, illnesses, and conditions are caused, holistically or partially, by abnormal or excessive signaling through various cellular receptors, and a variety of approaches are employed to address this. While receptor-ligand interactions can be disrupted using small and large molecules, the issues of off-target effects and potential immunogenicity remain.

[0010] More recently, genetic approaches have been investigated to either transiently reduce (i.e., knock down) or permanently remove (i.e., gene knockout) the expression of a given ligand or receptor, for example, siRNA. However, more sophisticated genetic tools are needed to address the countless diseases, illnesses, and conditions that involve such abnormal signaling.

[0011] 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 for the treatment and prevention of fibrotic tissues such as musculoskeletal tissue, cardiac tissue, lung tissue, and renal tissue. [Brief explanation of the drawing]

[0012] Embodiments of the present disclosure will be further described with reference to the accompanying drawings. The drawings shown are not necessarily to scale and instead generally focus on illustrating the principles of the embodiments of the present disclosure.

[0013] [Figure 1A] Sequence IDs 1-198, (A-D) show the spacer sequences of CRISPR RNA (crRNA) generated by the bioinformatics method described herein for targeting human TGFB1 with CRISPR / Cas9 to produce a gene knockout, as well as additional information regarding the genomic coordinates of the binding DNA, targeted DNA strand, targeted exon, and several quality control parameters. [Figure 1B] Figures 1A, 1B, 1C, 1D, 1E, 1F, 1G, and 1H collectively show the spacer sequences of CRISPR RNA (crRNA) generated by the bioinformatics method described herein for targeting human TGFB1 with CRISPR / Cas9 (A-D) to produce gene knockouts, as well as additional information regarding the genomic coordinates of the binding DNA, targeted DNA strand, targeted exon, and several quality control parameters (E-H). [Figure 1C] Figures 1A, 1B, 1C, 1D, 1E, 1F, 1G, and 1H collectively show the spacer sequences of CRISPR RNA (crRNA) generated by the bioinformatics method described herein for targeting human TGFB1 with CRISPR / Cas9 (A-D) to produce gene knockouts, as well as additional information regarding the genomic coordinates of the binding DNA, targeted DNA strand, targeted exon, and several quality control parameters (E-H). [Figure 1D]Figures 1A, 1B, 1C, 1D, 1E, 1F, 1G, and 1H collectively show the spacer sequences of CRISPR RNA (crRNA) generated by the bioinformatics method described herein for targeting human TGFB1 with CRISPR / Cas9 (A-D) to produce gene knockouts, as well as additional information regarding the genomic coordinates of the binding DNA, targeted DNA strand, targeted exon, and several quality control parameters (E-H). [Figure 1E] Figures 1A, 1B, 1C, 1D, 1E, 1F, 1G, and 1H collectively show the spacer sequences of CRISPR RNA (crRNA) generated by the bioinformatics method described herein for targeting human TGFB1 with CRISPR / Cas9 (A-D) to produce gene knockouts, as well as additional information regarding the genomic coordinates of the binding DNA, targeted DNA strand, targeted exon, and several quality control parameters (E-H). [Figure 1F] Figures 1A, 1B, 1C, 1D, 1E, 1F, 1G, and 1H collectively show the spacer sequences of CRISPR RNA (crRNA) generated by the bioinformatics method described herein for targeting human TGFB1 with CRISPR / Cas9 (A-D) to produce gene knockouts, as well as additional information regarding the genomic coordinates of the binding DNA, targeted DNA strand, targeted exon, and several quality control parameters (E-H). [Figure 1G] Figures 1A, 1B, 1C, 1D, 1E, 1F, 1G, and 1H collectively show the spacer sequences of CRISPR RNA (crRNA) generated by the bioinformatics method described herein for targeting human TGFB1 with CRISPR / Cas9 (A-D) to produce gene knockouts, as well as additional information regarding the genomic coordinates of the binding DNA, targeted DNA strand, targeted exon, and several quality control parameters (E-H). [Figure 1H]Figures 1A, 1B, 1C, 1D, 1E, 1F, 1G, and 1H collectively show the spacer sequences of CRISPR RNA (crRNA) generated by the bioinformatics method described herein for targeting human TGFB1 with CRISPR / Cas9 (A-D) to produce gene knockouts, as well as additional information regarding the genomic coordinates of the binding DNA, targeted DNA strand, targeted exon, and several quality control parameters (E-H). [Figure 2A] Figures 2A, 2B, 2C, 2D, 2E, and 2F collectively show the spacer sequences of crRNAs generated by the bioinformatics methods described herein for sequence numbers 199-320, (A-C) targeting CRISPR / Cas9 to human TGFBR1 to generate gene knockout, soluble, or membrane-bound decoy receptors, and (D-F) additional information regarding the genomic coordinates of the binding DNA, targeted DNA strand, targeted exon, and several quality control parameters. [Figure 2B] Figures 2A, 2B, 2C, 2D, 2E, and 2F collectively show the spacer sequences of crRNAs generated by the bioinformatics methods described herein for sequence numbers 199-320, (A-C) targeting CRISPR / Cas9 to human TGFBR1 to generate gene knockout, soluble, or membrane-bound decoy receptors, and (D-F) additional information regarding the genomic coordinates of the binding DNA, targeted DNA strand, targeted exon, and several quality control parameters. [Figure 2C] Figures 2A, 2B, 2C, 2D, 2E, and 2F collectively show the spacer sequences of crRNAs generated by the bioinformatics methods described herein for sequence numbers 199-320, (A-C) targeting CRISPR / Cas9 to human TGFBR1 to generate gene knockout, soluble, or membrane-bound decoy receptors, and (D-F) additional information regarding the genomic coordinates of the binding DNA, targeted DNA strand, targeted exon, and several quality control parameters. [Figure 2D]Figures 2A, 2B, 2C, 2D, 2E, and 2F collectively show the spacer sequences of crRNAs generated by the bioinformatics methods described herein for sequence numbers 199-320, (A-C) targeting CRISPR / Cas9 to human TGFBR1 to generate gene knockout, soluble, or membrane-bound decoy receptors, and (D-F) additional information regarding the genomic coordinates of the binding DNA, targeted DNA strand, targeted exon, and several quality control parameters. [Figure 2E] Figures 2A, 2B, 2C, 2D, 2E, and 2F collectively show the spacer sequences of crRNAs generated by the bioinformatics methods described herein for sequence numbers 199-320, (A-C) targeting CRISPR / Cas9 to human TGFBR1 to generate gene knockout, soluble, or membrane-bound decoy receptors, and (D-F) additional information regarding the genomic coordinates of the binding DNA, targeted DNA strand, targeted exon, and several quality control parameters. [Figure 2F] Figures 2A, 2B, 2C, 2D, 2E, and 2F collectively show the spacer sequences of crRNAs generated by the bioinformatics methods described herein for sequence numbers 199-320, (A-C) targeting CRISPR / Cas9 to human TGFBR1 to generate gene knockout, soluble, or membrane-bound decoy receptors, and (D-F) additional information regarding the genomic coordinates of the binding DNA, targeted DNA strand, targeted exon, and several quality control parameters. [Figure 3A] Figures 3A, 3B, 3C, 3D, 3E, 3F, 3G, and 3H collectively show the spacer sequences of crRNAs generated by the bioinformatics methods described herein for targeting CRISPR / Cas9 to human TGFBR2 (Sequence IDs 321-519), (A-D) to generate gene knockout, soluble, or membrane-bound decoy receptors, as well as additional information regarding the genomic coordinates of the binding DNA, targeted DNA strand, targeted exon, and several quality control parameters (E-H). [Figure 3B]Figures 3A, 3B, 3C, 3D, 3E, 3F, 3G, and 3H collectively show the spacer sequences of crRNAs generated by the bioinformatics methods described herein for targeting CRISPR / Cas9 to human TGFBR2 (Sequence IDs 321-519), (A-D) to generate gene knockout, soluble, or membrane-bound decoy receptors, as well as additional information regarding the genomic coordinates of the binding DNA, targeted DNA strand, targeted exon, and several quality control parameters (E-H). [Figure 3C] Figures 3A, 3B, 3C, 3D, 3E, 3F, 3G, and 3H collectively show the spacer sequences of crRNAs generated by the bioinformatics methods described herein for targeting CRISPR / Cas9 to human TGFBR2 (Sequence IDs 321-519), (A-D) to generate gene knockout, soluble, or membrane-bound decoy receptors, as well as additional information regarding the genomic coordinates of the binding DNA, targeted DNA strand, targeted exon, and several quality control parameters (E-H). [Figure 3D] Figures 3A, 3B, 3C, 3D, 3E, 3F, 3G, and 3H collectively show the spacer sequences of crRNAs generated by the bioinformatics methods described herein for targeting CRISPR / Cas9 to human TGFBR2 (Sequence IDs 321-519), (A-D) to generate gene knockout, soluble, or membrane-bound decoy receptors, as well as additional information regarding the genomic coordinates of the binding DNA, targeted DNA strand, targeted exon, and several quality control parameters (E-H). [Figure 3E] Figures 3A, 3B, 3C, 3D, 3E, 3F, 3G, and 3H collectively show the spacer sequences of crRNAs generated by the bioinformatics methods described herein for targeting CRISPR / Cas9 to human TGFBR2 (Sequence IDs 321-519), (A-D) to generate gene knockout, soluble, or membrane-bound decoy receptors, as well as additional information regarding the genomic coordinates of the binding DNA, targeted DNA strand, targeted exon, and several quality control parameters (E-H). [Figure 3F]Figures 3A, 3B, 3C, 3D, 3E, 3F, 3G, and 3H collectively show the spacer sequences of crRNAs generated by the bioinformatics methods described herein that target SEQ ID NOs: 321-519, (A-D) CRISPR / Cas9 to human TGFBR2 to generate gene knockouts, soluble or membrane-bound decoy receptors, and additional information regarding (E-H) binding DNA, the targeted DNA strand, the targeted exon, and the genomic coordinates of several quality control parameters. [Figure 3G] Figures 3A, 3B, 3C, 3D, 3E, 3F, 3G, and 3H collectively show the spacer sequences of crRNAs generated by the bioinformatics methods described herein that target SEQ ID NOs: 321-519, (A-D) CRISPR / Cas9 to human TGFBR2 to generate gene knockouts, soluble or membrane-bound decoy receptors, and additional information regarding (E-H) binding DNA, the targeted DNA strand, the targeted exon, and the genomic coordinates of several quality control parameters. [Figure 3H] Figures 3A, 3B, 3C, 3D, 3E, 3F, 3G, and 3H collectively show the spacer sequences of crRNAs generated by the bioinformatics methods described herein that target SEQ ID NOs: 321-519, (A-D) CRISPR / Cas9 to human TGFBR2 to generate gene knockouts, soluble or membrane-bound decoy receptors, and additional information regarding (E-H) binding DNA, the targeted DNA strand, the targeted exon, and the genomic coordinates of several quality control parameters. [Figure 4A] (A) Schematic showing the targeting domain of TGFBR1 and the orientation of the spacer sequences of various Cas9-guide single-guide RNAs (sgRNAs) predicted to generate knockouts, and (B) a series of parameters considered for designing sgRNAs against TGFBR1, and an exemplary sgRNA-targeted protospacer DNA sequence are shown. [Figure 4B](A) Schematic showing the targeting domain of TGFBR1 and the orientation of the spacer sequences of various Cas9-guided single-guide RNAs (sgRNAs) predicted to generate knockouts, and (B) a series of parameters considered for designing sgRNAs against TGFBR1, and an exemplary sgRNA-targeting protospacer DNA sequence are shown. [Figure 5A] (A) Schematic showing the targeting domain of TGFBR1 and the orientation of the protospacer sequences targeted by various CRISPR / Cas9 predicted to generate knockouts, soluble or membrane-bound decoy receptors (ECD: extracellular domain, TMD: transmembrane domain, ICD: intracellular domain, GSM: GS-rich motif), and (B) a series of parameters considered for the design of sgRNAs against TGFBR1, and an exemplary sgRNA-targeting protospacer DNA sequence are shown. [Figure 5B] (A) Schematic showing the targeting domain of TGFBR1 and the orientation of the protospacer sequences targeted by various CRISPR / Cas9 predicted to generate knockouts, soluble or membrane-bound decoy receptors (ECD: extracellular domain, TMD: transmembrane domain, ICD: intracellular domain, GSM: GS-rich motif), and (B) a series of parameters considered for the design of sgRNAs against TGFBR1, and an exemplary sgRNA-targeting protospacer DNA sequence are shown. [Figure 6A] (A) Schematic showing the targeting domain of TGFBR2 and the orientation of the protospacer sequences targeted by various CRISPR / Cas9 predicted to generate knockouts and soluble or membrane-bound decoy receptors (ECD: extracellular domain, TMD: transmembrane domain, ICD: intracellular domain, GSM: GS-rich motif), and (B) a series of parameters considered for the design of sgRNAs against TGFBR2, and an exemplary sgRNA-targeting protospacer DNA sequence are shown. [Figure 6B](A) A schematic diagram showing the targeting domain of TGFBR2 and the orientation of various CRISPR / Cas9-targeted protospacer sequences predicted to generate knockout and soluble or membrane-bound decoy receptors (ECD: extracellular domain, TMD: transmembrane domain, ICD: intracellular domain, GSM: GS-rich motif), and (B) a set of parameters considered for the design of sgRNAs against TGFBR2, as well as exemplary sgRNA-targeted protospacer DNA sequences. [Figure 7A] This shows a summary of the efficiency of editing the human TGFBR1 gene in THP-1 cells using identified sgRNAs and either (A) wild-type Cas9 (WTCas9) or (B) high-fidelity (R691A) Cas9 (ARCas9) proteins. The bar graph shows both top-tier and all other frameshift edits contributing to the knockout (KO) score estimated by Sungart trace deconvolution. [Figure 7B] This shows a summary of the efficiency of editing the human TGFBR1 gene in THP-1 cells using identified sgRNAs and either (A) wild-type Cas9 (WTCas9) or (B) high-fidelity (R691A) Cas9 (ARCas9) proteins. The bar graph shows both top-tier and all other frameshift edits contributing to the knockout (KO) score estimated by Sungart trace deconvolution. [Figure 8A] This shows a summary of the efficiency of top-level editing of the human TGFBR1 gene in THP-1 cells using identified sgRNAs and either (A) wild-type Cas9 (WTCas9) or (B) high-fidelity (R691A) Cas9 (ARCas9) proteins. The bar graph shows both top-level and all other frameshift edits contributing to the knockout (KO) score estimated by Sungart trace deconvolution. [Figure 8B]This shows a summary of the efficiency of top-level editing of the human TGFBR1 gene in THP-1 cells using identified sgRNAs and either (A) wild-type Cas9 (WTCas9) or (B) high-fidelity (R691A) Cas9 (ARCas9) proteins. The bar graph shows both top-level and all other frameshift edits contributing to the knockout (KO) score estimated by Sungart trace deconvolution. [Figure 9A] This shows a summary of the efficiency of editing the human TGFBR2 gene in THP-1 cells using identified sgRNAs and either (A) wild-type Cas9 (WTCas9) or (B) high-fidelity (R691A) Cas9 (ARCas9) proteins. The bar graph shows both top-tier and all other frameshift edits contributing to the knockout (KO) score estimated by Sungart trace deconvolution. [Figure 9B] This shows a summary of the efficiency of editing the human TGFBR2 gene in THP-1 cells using identified sgRNAs and either (A) wild-type Cas9 (WTCas9) or (B) high-fidelity (R691A) Cas9 (ARCas9) proteins. The bar graph shows both top-tier and all other frameshift edits contributing to the knockout (KO) score estimated by Sungart trace deconvolution. [Figure 10A] Figures 10A, 10B, 10C, and 10D show the relative levels of (A, C)TGFB1 and (B, D)TIMP1 gene expression in unedited (WT) or THP-1 cells edited to knock out TGFB1 (OHTG), TGFBR1 (OHTIR), or TGFBR2 (OHTIIR) after 6 hours of attack inoculation with lipopolysaccharide (LPS) (Panels A and B) or TGF beta (Panels C and D). [Figure 10B]Figures 10A, 10B, 10C, and 10D show the relative levels of (A, C)TGFB1 and (B, D)TIMP1 gene expression in unedited (WT) or THP-1 cells edited to knock out TGFB1 (OHTG), TGFBR1 (OHTIR), or TGFBR2 (OHTIIR) after 6 hours of attack inoculation with lipopolysaccharide (LPS) (Panels A and B) or TGF beta (Panels C and D). [Figure 10C] Figures 10A, 10B, 10C, and 10D show the relative levels of (A, C)TGFB1 and (B, D)TIMP1 gene expression in unedited (WT) or THP-1 cells edited to knock out TGFB1 (OHTG), TGFBR1 (OHTIR), or TGFBR2 (OHTIIR) after 6 hours of attack inoculation with lipopolysaccharide (LPS) (Panels A and B) or TGF beta (Panels C and D). [Figure 10D] Figures 10A, 10B, 10C, and 10D show the relative levels of (A, C)TGFB1 and (B, D)TIMP1 gene expression in unedited (WT) or THP-1 cells edited to knock out TGFB1 (OHTG), TGFBR1 (OHTIR), or TGFBR2 (OHTIIR) after 6 hours of attack inoculation with lipopolysaccharide (LPS) (Panels A and B) or TGF beta (Panels C and D). [Figure 11A] Figures 11A, 11B, 11C, and 11D show the relative levels of (A) SERPINE1, (B) Col1A2, (C) FN1, and (D) CTGF gene expression in unedited (WT) or THP-1 cells edited to knock out TGFB1 (OHTG), TGFBR1 (OHTIR), or TGFBR2 (OHTIIR) after 6 hours of attack inoculation with TGF beta treatment. [Figure 11B]Figures 11A, 11B, 11C, and 11D show the relative levels of (A) SERPINE1, (B) Col1A2, (C) FN1, and (D) CTGF gene expression in unedited (WT) or THP-1 cells edited to knock out TGFB1 (OHTG), TGFBR1 (OHTIR), or TGFBR2 (OHTIIR) after 6 hours of attack inoculation with TGF beta treatment. [Figure 11C] Figures 11A, 11B, 11C, and 11D show the relative levels of (A) SERPINE1, (B) Col1A2, (C) FN1, and (D) CTGF gene expression in unedited (WT) or THP-1 cells edited to knock out TGFB1 (OHTG), TGFBR1 (OHTIR), or TGFBR2 (OHTIIR) after 6 hours of attack inoculation with TGF beta treatment. [Figure 11D] Figures 11A, 11B, 11C, and 11D show the relative levels of (A) SERPINE1, (B) Col1A2, (C) FN1, and (D) CTGF gene expression in unedited (WT) or THP-1 cells edited to knock out TGFB1 (OHTG), TGFBR1 (OHTIR), or TGFBR2 (OHTIIR) after 6 hours of attack inoculation with TGF beta treatment. [Figure 12A] Figures 12A, 12B, and 12C collectively show spacer sequences of crRNAs targeting human (A)TGFB1 (sequence numbers 520-527), (B)TGFBR1 (sequence numbers 528-552), and (C)TGFBR2 (sequence numbers 553-604) for use with sgRNA to validate in vitro editing with different delivery modes. [Figure 12B] Figures 12A, 12B, and 12C collectively show spacer sequences of crRNAs targeting human (A)TGFB1 (sequence numbers 520-527), (B)TGFBR1 (sequence numbers 528-552), and (C)TGFBR2 (sequence numbers 553-604) for use with sgRNA to validate in vitro editing with different delivery modes. [Figure 12C]Figures 12A, 12B, and 12C collectively show spacer sequences of crRNAs targeting human (A)TGFB1 (sequence numbers 520-527), (B)TGFBR1 (sequence numbers 528-552), and (C)TGFBR2 (sequence numbers 553-604) for use with sgRNA to validate in vitro editing with different delivery modes. [Modes for carrying out the invention]

[0014] I. Introduction Compositions and methods for silencing the signaling function of one or more cell receptors in animals, thereby treating diseases, illnesses, or conditions caused by abnormal or excessive signaling through said receptors.

[0015] In some embodiments, receptor signaling is reduced or eliminated by the use of compositions and methods described herein for editing the TGFB1, TGFBR1, and / or TGFBR2 genes. In some embodiments, the editing knocks out the TGFB1, TGFBR1, and / or TGFBR2 genes, which is a gene editing such that the gene no longer codes for a functional protein. In some embodiments, the editing results in the removal of the transmembrane domain of the protein encoded by the TGFBR1 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 TGFBR1 or TGFBR2 gene, e.g., the generation of a membrane-bound receptor decoy. In some embodiments, TGFB1, TGFBR1, and / or TGFBR2 are regulated in expression by altering coding or non-coding regulatory sequences, or their epigenetic context. In some embodiments, the receptor is TGFBR1. In some embodiments, the receptor is TGFBR2. In some embodiments, receptor signaling is reduced or eliminated by using the compositions and methods described herein for editing the TGFB1 ligand.

[0016] II. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in which the present invention pertains. All patents and publications referenced herein are incorporated in their entirety by reference.

[0017] The terms "transforming growth factor beta-1" or "TGFB1" refer 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 the proteins and glycotypes that possess sequence variants, conserved amino acid substitutions, etc. Normatively, 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 the activation of other cytoplasmic signaling mediators. In some cases, and simply for disambiguation, prefixes are added to refer to proteins or genes of a specific species (h, c, e, and f refer to the human, canine, horse, and feline morphologies, respectively).

[0018] 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 horse origin (eTGFB1). In some embodiments, the TGFB1 gene targeted by the RNA-guided nuclease is of feline origin (fTGFB1).

[0019] The terms "transformed growth factor beta receptor 1" or "TGFBR1" refer 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 the proteins and glycotypes that possess sequence variants, conserved amino acid substitutions, etc. Normatively, the proteins encoded by the genes listed above are transmembrane serine / threonine kinases that, together with TGFBR2, form the innate receptors for the TGF-beta cytokines TGFB1, TGFB2, and TGFB3. Upon binding to its ligand, TGFBR1 is phosphorylated by TGFBR2, activating intracellular signaling that controls several 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 to refer to proteins or genes of a specific species (h, c, e, and f refer to human, canine, horse, and feline morphologies, respectively).

[0020] 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 guide nuclease is of feline origin (fTGFBR1).

[0021] The term "transformed 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 the proteins and glycotypes that possess sequence variants, conserved amino acid substitutions, etc. Normatively, the proteins encoded by the genes listed above are transmembrane serine / threonine kinases that, together with TGFBR2, form the innate receptors for the TGF-beta cytokines TGFB1, TGFB2, and TGFB3. Upon binding to its ligand, TGFBR1 is phosphorylated by TGFBR2, activating intracellular signaling that controls several 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 to refer to proteins or genes of a particular species (h, c, e, and f refer to the human, canine, horse, and feline morphologies, respectively).

[0022] 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).

[0023] The term “treatment” means obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in the sense of completely or partially preventing a disease or its symptoms, and / or therapeutic in the sense of partial or complete cure of a disease and / or adverse effect resulting from the disease. For example, the compositions, methods, or systems of the Disclosure may be administered as prophylactic treatment to a subject having a predisposition to a given condition (e.g., arthritis). As used herein, “treatment” extends to the treatment of any disease in mammals, particularly humans, dogs, cats, or horses, and includes (a) preventing the development of the disease in a subject that is predisposed to the disease but has not yet been diagnosed with the disease, (b) inhibiting the disease, i.e., suppressing the onset or progression of the disease, and (c) alleviating the disease, i.e., causing regression of the disease, and / or alleviating one or more symptoms of the disease.

[0024] "Therapy" also means the delivery of a drug to provide a pharmacological effect even in the absence of a disease or condition. For example, "Therapy" includes the delivery of a composition that can induce an immune response or confer immunity in the absence of a disease condition, for example, in the case of a vaccine. The compositions and methods of this disclosure are understood to be applicable to the treatment of all mammals, including human, dog, cat, horse, and cattle subjects, but are not limited thereto.

[0025] The term “therapeutically effective” refers to an amount of any composition or combination of compositions described herein that is sufficient to produce an intended use, including, but is not limited to, the treatment of a disease. The therapeutically effective dose may vary depending on the intended use (in vitro or in vivo), the subject and disease state being treated (e.g., the subject’s weight, age, or sex), the severity of the disease state, or the mode of administration. The term also applies to doses that will induce a specific response in target cells (e.g., reduction of platelet adhesion and / or cell migration). The specific dose will vary depending on the particular composition selected, the administration regimen to be followed, whether the composition is administered in combination with other compositions or compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system through which the composition is transported.

[0026] The terms “fibrosis” and “fibrous scarring” are used interchangeably and typically refer to the formation of an abnormal amount of fibrous tissue in an organ or tissue, 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, e.g., the formation between organs or different tissues. Fibrosis affects joint tissues (e.g., knee joints, shoulder joints, adhesive capsulitis, elbow joints, etc.), other musculoskeletal tissues (e.g., tendon scarring), kidney tissue (e.g., in chronic kidney disease, etc.), skin tissue (e.g., related to wound healing, e.g., post-traumatic or post-operative, keloid disorders, nephrogenic systemic fibrosis, scleroderma / systemic sclerosis in humans, "granulation tissue" in horses, etc.), lung tissue (e.g., in fibrothorax, pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, radiation-induced lung injury, progressive widespread fibrosis, scleroderma / systemic sclerosis, etc.), liver tissue (e.g., in cirrhosis, bridging fibrosis, etc.), and heart tissue. It can occur in any tissue, including but not limited to the following: (for example, 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 scars), intestinal tissue (e.g., in Crohn's disease), hands and / or fingers (e.g., in Dupuytren's contracture), lymphatic tissue (e.g., mediastinal fibrosis), bone marrow tissue (e.g., myelofibrosis), penile tissue (e.g., in Peyronie's disease), retroperitoneal soft tissue (e.g., retroperitoneal fibrosis), and musculoskeletal tissue.

[0027] In some embodiments, fibrosis results from trauma to the tissue. In some embodiments, fibrosis is associated with surgical procedures, such as postoperative fibrosis. Non-limiting examples of procedures that may induce fibrosis include ligament reconstruction, anterior cruciate ligament (ACL) reconstruction, autograft ACL reconstruction, allogeneic ACL reconstruction, fracture repair, total knee arthroplasty (TKA), and microscopic discectomy. Furthermore, fibrosis may be caused by any of the following conditions that can be induced or aggravated by surgical procedures: knee joint fibrosis, intra-articular fibrous nodules, or epidural fibrosis.

[0028] As used herein, the term “musculoskeletal trauma” refers to any injury affecting bone, muscle, ligament, nerve, or tendon. In some embodiments, the trauma is the result of surgery.

[0029] The terms “polynucleotide,” “nucleotide,” and “nucleic acid” are used interchangeably herein and refer to all forms of nucleic acids and oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Polynucleotides include genomic DNA, cDNA, and antisense DNA, as well as splicing or unsplicing 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, RNAi, miRNA, lncRNA, RNA antagonists, aptamers, and any other non-coding RNA known to those skilled in the art. Polynucleotides include native, synthetic, and intentionally 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 sequences. Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. Where present, modifications to the nucleotide structure may be imparted before or after the assembly of the polymer. As used herein, the term polynucleotide is interchangeable for double-stranded and single-stranded molecules. Unless otherwise specified or requested, any embodiment described herein that encompasses a polynucleotide includes both a double-stranded form and two complementary single-stranded forms that are known or expected to constitute a double-stranded form. Polynucleotides may be single, double, or triple-stranded, linear or circular, and may be of any length. When considering polynucleotides, the sequences or structures of particular polynucleotides may be described herein in accordance with the convention that they provide a sequence in the 5' to 3' direction.

[0030] The terms "gene" or "nucleotide sequence encoding a polypeptide" refer to a segment of DNA involved in the production of a polypeptide chain. A DNA segment may include regions before and after coding regions (leaders and trailers) involved in the transcription / translation of gene products and the regulation of transcription / translation, as well as intervening sequences (introns) between individual coding segments (exons). For example, a gene contains polynucleotides that, after transcription and translation, can encode a specific protein or polypeptide, including at least one open reading frame.

[0031] The term "extracellular domain," when referring to transmembrane cell receptors, is defined as the portion of a protein that is exposed to the extracellular environment and can engage with and / or bind to a ligand.

[0032] The terms “cytoplasmic domain” and “intracellular domain” can be used interchangeably and, when referring to transmembrane receptors, define the portion of a protein exposed to the cytoplasm. Often, these portions of a protein contain signaling domains for recruiting and associating with various intracellular factors. After engagement with a ligand via the extracellular domain, the interaction effect changes, which can lead to new associations, dissociations, or recruitments of various cytoplasmic factors that aid in signal transduction.

[0033] The term "transmembrane domain," when referring to transmembrane receptors, is defined as a 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 exhibit higher hydrophobicity than either the extracellular or cytoplasmic portion and often adopt a higher-order helical structure. While their primary role is anchoring, ligand-induced conformational changes for specific receptors have been shown to affect the transmembrane domain, which is consequently essential for subsequent intracellular signaling.

[0034] The term "receptor" refers to a protein that can bind with high affinity to another homologous protein (i.e., its ligand). This receptor-ligand interaction can be 1:1 or result in multimerization, where multiple proteins aggregate and bind to one or more ligands. Receptors are generally located on the cell surface, and as a result, they can most efficiently encounter ligands and initiate intracellular signaling.

[0035] The term "intracellular signaling" refers to cellular changes resulting from events occurring on the cell surface. Typically, a soluble ligand binds to its receptor on the cell surface, which can induce changes in the receptor, consequently affecting related intracellular factors. These factors, in turn, can influence other factors within the cell, and this cascade often continues until a particular factor can alter gene expression in the nucleus in response to a surface stimulus.

[0036] The term "gene regulatory sequence" refers to double-stranded DNA sequences in the genome that control the level of gene transcription.

[0037] The term "RNA guide nuclease" refers to an enzyme that can locate and disrupt the backbone of a DNA molecule, for example. The activity of an RNA guide nuclease is induced by a nucleic acid molecule (i.e., guide RNA). When properly oriented to form a functional ribonucleoprotein complex, the enzyme is located at a specific position within the target nucleic acid (e.g., a gene or locus) via sequence complementarity with a portion of the gRNA. Non-exclusive examples of RNA guide nucleases include Cas9, Cpf1, and Cas12.

[0038] The term "Cas9" refers to an RNA guide, a double-strand DNA binding nuclease protein, a nickase protein, or an enzymatically inactive protein, or a variant thereof, and may be used to refer to any of the native or recombinant Cas9 nuclease variants (e.g., ES-Cas9, HF-Cas9, PE-Cas9, and AR-Cas9). Cas9 may be further ligated to other functional protein domains, such as nucleoside deaminases, reverse transcriptases, epigenetic modifiers, or transcription effectors. Wild-type Cas9 nucleases have two functional nuclease domains, e.g., RuvC and HNH, which simultaneously cleave different strands of double-strand DNA, resulting in double-strand breaks. The Cas9 enzymes described herein may include an HNH or HNH-like nuclease domain and / or a RuvC or RuvC-like nuclease domain, without affecting their ability to induce double-strand breaks of genomic DNA (e.g., at target loci) when both functional domains are active. The Cas9 enzyme may contain one or more catalytic domains of a Cas9 protein derived from bacteria belonging to the group consisting of Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, and Campylobacter. In some embodiments, the two catalytic domains are derived from different bacterial species.

[0039] As used herein, “PAM” refers to a protospacer facilitation motif, which is necessary for an RNA guide nuclease to bind to a target nucleic acid. Often, PAMs are directly facilitation of a complementary sequence in the target. For example, the native Cas9 molecule recognizes a specific PAM sequence (see, for example, 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 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, the native Cas9 molecule may be modified so 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 may be modified (e.g., to lengthen the PAM recognition sequence, to improve Cas9 specificity to a high level of identity, to reduce off-target sites, and / or to increase specificity). In one embodiment, the length of the PAM recognition sequence is at least 4, 5, 6, 7, 8, 9, 10, or 15 amino acids. In some embodiments, the Cas9 molecule may be modified to excise PAM recognition.

[0040] The terms “guide RNA,” “gRNA,” or “sgRNA” may be used interchangeably and refer to RNA molecules, preferably synthetic single guide RNA molecules, that consist of a targeting spacer and a scaffold sequence. When these molecules are loaded onto a functional RNA guide nuclease, they can induce sequence-specific cleavage of a target nucleic acid.

[0041] sgRNAs can be administered or formulated, for example, as synthetic RNA or as nucleic acids containing sequences encoding gRNAs that are later expressed in target cells. As will be apparent to those skilled in the art, various tools can be used in the design and / or optimization of sgRNAs, for example, to improve the specificity and / or precision of genome editing at specific sites.

[0042] Generally, candidate sgRNAs can be designed and identified by first positioning suitable PAMs within the genome sequence. Additional calculations may then be used to predict on-target and off-target efficiencies. Available web-based tools to assist in the initial setup and modeling of candidate sgRNAs include, but are not limited to, CALITAS, SWOffinder, 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 in its entirety by reference for all purposes. Such tools are also described, for example, in PCT application publication 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 herein by reference for all purposes. Candidate sgRNAs may be further evaluated by experimental screening or other methodologies.

[0043] The term "spacer" refers to a portion of an sgRNA molecule that has complementarity with the target nucleic acid. Normatively, a spacer is a portion of CRISPR RNA (crRNA) that hybridizes to the target nucleic acid, for example, via a spacer sequence, and hybridizes with trans-activated CRISPR RNA (tracrRNA), for example, via a non-targeting sequence. However, when crRNA is used herein in conjunction with a sequence that targets a gene, such as the TNFB or TNFBR gene, the sequence referred to is the spacer sequence, as opposed to both the spacer sequence and the non-targeting sequence present in the canonical crRNA molecule. In sgRNA, crRNA and tracrRNA are combined into a single molecule.

[0044] The term "pharmaceutically acceptable" means any compound, material, composition, and / or dosage form that is within the bounds of sound medical judgment suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0045] The terms “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” are intended to include any and all solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic and absorption retardants, and inactive components. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical components is well known in the art. Unless any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical component, its use in the therapeutic compositions of the present disclosure is intended. Additional active pharmaceutical components, such as other drugs, may also be incorporated into the compositions and methods described.

[0046] The term “pharmaceutically acceptable excipients” is intended to include vehicles and carriers that can be administered co-administered with a compound to facilitate the performance of its intended function. The use of such media for pharmaceutically active substances is well known in the art. Examples of such vehicles and carriers include solutions, solvents, dispersion media, retarders, emulsions, and the like. Any other conventional carriers suitable for use with polyconjugated compounds are also within the scope of this disclosure.

[0047] As used herein, the terms "a," "an," or "the" are generally interpreted to encompass both singular and plural forms.

[0048] The terms “about” and “approximately” mean a statistically significant range of values. Such a range may be within one order of magnitude of a given value or range, preferably within 50%, more preferably within 20%, more preferably still within 10%, and even more preferably within 5%. The acceptable variation encompassed by the terms “about” or “approximately” depends on the particular system under study and is readily understandable to those skilled in the art. Furthermore, as used herein, the terms “about” and “approximately” mean that compositions, quantities, formulations, parameters, shapes, and other quantities and properties are not and do not need to be exact, but are approximations, and / or may be greater or less, reflecting tolerances, conversion factors, rounding, measurement errors, and other factors known to those skilled in the art, as desired. In general, dimensions, sizes, formulations, parameters, shapes, or other quantities or properties are “about” or “approximately,” whether explicitly stated as such. It should be noted that very different embodiments of size, shape, and dimensions may adopt the arrangements described.

[0049] As used herein, the term “substantially” can mean a majority or the 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.

[0050] When used in the original and amended forms in the appended claims, the conjunctions “including,” “essentially consisting of,” and “consisting of” define the claims with respect to which additional elements or steps of the claims not enumerated, if present, are excluded from the claims. The term “including” is intended to be inclusive or non-exclusive and does not exclude any additional unenumerated elements, methods, steps, or materials. The term “consisting of” excludes any elements, steps, or materials other than those specified in the claims, and in the latter case, impurities that are typically associated with the specified materials. The term “essentially consisting of” limits the claims to those that do not substantially affect the basic and novel characteristics of the methods and compositions of the claims, as well as any particular elements, steps, or materials. All compositions, methods, and kits described herein that embody this disclosure may be more specifically defined by any of the conjunctions “including,” “essentially consisting of,” and “consisting of” in alternative embodiments.

[0051] III. Method A.CRISPR Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure pertains. Similar or equivalent methods and materials to those described herein may be used in carrying out or testing the disclosure, but preferred methods and materials are described below. All publications, patent applications, patents, and other references referenced herein are incorporated in their entirety by reference. In addition, materials, methods, and examples are illustrative and not intended to limit the scope of this disclosure.

[0052] In one embodiment, the disclosure encompasses compositions relating to clustered and regularly arranged short palindromic sequence repeats (CRISPR) and CRISPR-related RNA guide nucleases, as well as related methods, components, and compositions (hereinafter, the CRISPR / Cas system). Such a system requires at least one isolated or non-natural RNA guide nuclease (e.g., Cas9 protein) and at least one isolated or non-natural guide RNA (e.g., sgRNA) to result in enhancement of nucleic acid sequences (e.g., genomic DNA) or their epigenetic features (e.g., DNA methylation).

[0053] In some embodiments, the CRISPR / Cas system causes a sequence alteration at a target site (e.g., insertion or deletion (collectively, indel) resulting in a loss-of-function (i.e., knockout) of the affected gene or allele); for example, a cleavage, nonsense mutation, or other loss-of-function of one or more TGFB1, TGFBR1, or TGFBR2 genes (i.e., mRNA or protein) resulting in a nucleotide substitution; for example, a cleavage, nonsense mutation, or other loss-of-function of one or more nucleotide deletions resulting in one or more TGFB1, TGFBR1, or TGFBR2 genes resulting in a cleavage, nonsense mutation, or other loss-of-function of the encoded product) Losses; for example, loss of function of encoded mRNA or protein due to single nucleotide, double nucleotide, or other frameshift deletions, or deletions resulting in immature stop codons; or insertions resulting in cleavage, nonsense mutations, or other loss of function of encoded gene products, such as one or more TGFB1, TGFBR1, or TGFBR2 genes (i.e., mRNA or protein); for example, by causing single nucleotide, double nucleotide, or other frameshift insertions, or insertions resulting in immature stop codons, resulting in modification of target genes or loci in eukaryotic cells. In some embodiments, the CRISPR / Cas system of the present disclosure provides modification of genes and / or gene-encoded products, as a result the modified product has a resulting loss of function, and becomes dominant-negative or decoy (e.g., a transmembrane receptor or soluble receptor that cannot initiate intracellular signaling). In some embodiments, the CRISPR / Cas system of the present disclosure alters the transcriptional level of one or more TGFB1, TGFBR1, or TGFBR2 genes.

[0054] In one embodiment, the CRISPR / Cas system induces changes in nucleic acid sequences via nuclease activity. For example, in the case of genomic DNA, RNA guide nucleases cleave genomic DNA when they are located at a target position within a targeted gene or locus, and recognize a specific nuclease-specific motif called a protospacer adjacency motif (PAM), through sequence complementarity with the target genomic sequence (e.g., a spacer sequence of CRISPR RNA (crRNA) or synthetic single guide RNA (sgRNA)). For general information, see Collias, D., & Beisel, CL (2021). Nature Communications, 12(1), 1-12.

[0055] Nuclease activity (i.e., cleavage) induces double-strand breaks (DSBs) in genomic DNA. The intrinsic cellular mechanisms of DSB repair, namely non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and homologous recombination, result in erroneous repair at a given target site at some computable frequency as a result of interference from the relevant components of the CRISPR / Cas system, thereby introducing substitutions or indels into genomic DNA. See, for general information, Scully, R., et al. (2019). Nature Reviews Molecular Cell Biology, 20(11), 698-714. At some frequency, these indels and / or substitutions can result in frameshifts, nonsense mutations (i.e., early stop codons), or cleavage, affecting the availability of gene products such as mRNA and / or proteins. In certain embodiments, the CRISPR / Cas system may induce homology-directed repair (HDR) mechanisms, leading to the insertion of non-random sequences at target sites, through the use of templates (e.g., HDR templates) provided to cells as part of the system along with nucleases and gRNAs. See Bloh, K., & Rivera-Torres, N. (2021). International Journal of Molecular Sciences, 22(8), 3834.

[0056] Generally, the minimum requirements for a CRISPR / Cas system depend on the nuclease (i.e., Cas protein) provided with it. To this extent, these bacterial nucleases are functionally divided into types I, III, and V, all of which are classified as class 1, and further into types II, IV, and VI, which are classified as class 2.

[0057] Class 1 CRISPR / Cas system: While the exact components, compositions, and methods for inducing alterations to target nucleic acid sequences using Class 1 CRISPR / Cas systems vary, they should at least include a nuclease (e.g., selected from at least type I and type III), e.g., 1) at least one guide RNA selected from sgRNA, or 2) a combination of crRNA and tracrRNA. These CRISPR / Cas systems are classified together as Class 1 CRISPR / Cas systems due to their similarity in requirements and modes of action within eukaryotic cells. For this purpose, 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.

[0058] Cas3 (see Table 1) is a prototype type I DNA nuclease that functions as an effector protein as part of a larger complex (the Cascade complex, which includes Cse1 and Cse2) capable of genome editing. For general information, see He, L., et al. (2020). Genes, 11(2), 208. Unlike other CRISPR / Cas systems, the type I system localizes to a DNA target without the Cas3 nuclease via the Cascade complex, which then recruits Cas3 to cleave the DNA in order to bind and locate the 3'PAM. The Cascade complex also plays a role in processing crRNA so that it can be used to guide crRNA to a target site. Due to this functionality, the 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, the type I system may be used to edit multiple targeted genes or loci from a single molecule.

[0059] Since the natural Cas3 substrate is ssDNA, its function in genome editing is thought to be that of a nicase; however, when targeted in tandem, the resulting edit is a blunt-end cleavage of the opposing strand to approximate the blunt-end cleavage of endonucleases such as Cas9. See Pickar-Oliver, A., & Gersbach, CA (2019). Nature Reviews Molecular Cell Biology, 20(8), 490-507.

[0060] Similar to type I nucleases, type III systems rely on a protein complex to achieve nucleic acid cleavage. In particular, Cas10 possesses nuclease activity for cleaving ssDNA in prokaryotes. (See Tamulaitis, G., Trends in Microbiology, 25(1), 49-61.) Interestingly, this archaeal-derived CRISPR / Cas system exhibits bispecificity, targeting both ssDNA and ssRNA. Apart from this variation, the system functions very similarly to type I, in that crRNA targets the effector complex (similar to a cascade) in a sequence-dependent manner. Similarly, the effector complex processes the crRNA before association. This bifunctional nature of the nuclease potentially makes its use in genome editing more powerful, as both genomic DNA and, in some cases, mRNA with the same sequence can be targeted to silence specific target genes.

[0061] Class 2 CRISPR / Cas system: While the exact components, compositions, and methods for inducing alterations to target nucleic acid sequences using Class 2 CRISPR / Cas systems vary, they should at least 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 are classified together as Class 2 CRISPR / Cas systems due to their similarity in requirements and modes of action within eukaryotic cells. For this purpose, 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.

[0062] Type II nucleases are the best-characterized CRISPR / Cas systems, particularly the canonical genome editing nuclease Cas9 (see Table 1). Multiple Cas9 proteins have been isolated from various bacterial species. The main difference among these nucleases lies in the required recognition site (PAM) within the targeted dsDNA. After association with the gRNA molecule, the spacer sequence orients the nuclease to the correct position, while protein recognition of the PAM induces a cleavage event near that site, resulting in blunt DSBs.

[0063] In addition to naturally occurring Cas9 proteins, several engineered variants have also been reported. These range from Cas9 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, resulting in them inducing only single-strand breaks (i.e., Cas9 nickase). See Frock, R. et al. (2015). Nature Biotechnology, 33(2), 179-186. These have also been shown to be less prone to errors in editing. Such mitigation of off-target effects is crucial when selecting desired insertions (i.e., knock-in mutations in which a desired nucleotide sequence is introduced into a target nucleic acid molecule) rather than deletions. Indeed, less off-target effect can aid preferred DNA repair mechanisms (most often HDR for knock-in mutations). For general information, please refer to Naeem, M., et al. (2020). Cells, 9(7), 1608.

[0064] In some embodiments, the CRISPR-related endonuclease is Cas9 endonuclease. In some embodiments, the Cas9 endonuclease includes the amino acid substitutions K848A, K1003A, and R1060A. In some embodiments, the Cas9 endonuclease includes the amino acid substitution R691A. In some embodiments, the Cas9 endonuclease includes the amino acid substitutions N497A, R661A, Q695A, and Q926A. In some embodiments, the Cas9 endonuclease includes the amino acid substitutions N692A, M694A, Q695A, and H698A. In some embodiments, the Cas9 endonuclease includes the amino acid substitutions R63A and Q768A. In some embodiments, the Cas9 endonuclease includes the amino acid substitutions M495V, Y515N, K526E, and R661Q. In some embodiments, Cas9 endonuclease includes the K526E or K526N amino acid substitution. In some embodiments, Cas9 endonuclease includes the F539S, M763I, and K890N amino acid substitutions. In some embodiments, Cas9 endonuclease includes the E1007L amino acid substitution. In some embodiments, Cas9 endonuclease includes the N690C, T769I, G915M, and N980K amino acid substitutions. In some embodiments, Cas9 endonuclease includes the E1007L amino acid substitution.

[0065] Additional exemplary engineered variants (e.g., mutants, chimeras) of the canonical Cas protein include (each of these, and all of them incorporated herein by reference): WO2015 / 035162A2, WO2019 / 126716A1, WO2019 / 126774A1, WO2014 / 093694A1, WO2014 / 150624A1, US2019 / 0225955A1, U.S. Patent No. 11427818, U.S. Patent No. 11242542, U.S. Patent No. 11098297, U.S. Patent No. 10876100, U.S. Patent No. 10767193, U.S. Patent No. 10494621, and U.S. Patent No. 10100291.

[0066] To avoid any doubt, the Cas9 (spCas9) of Streptococcus pyogenes collectively refers to one of the groups consisting of espCas9 (also referred to herein as ESCas9 or esCas9), HFCas9, PECas9, and ARCas9.

[0067] Similar to the canonical Cas9 system, type V nucleases require only synthetic sgRNAs with targeting domains 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 alternating cleavage on the dsDNA substrate distal to the PAM, compared to, for example, the blunt cleavage of Cas9 adjacent 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 acting on dsDNA as a quaternary structure that simultaneously nicks each strand. See Zetsche B, et al. Cell. 2015;163(3):759-771, and 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 very little tolerance to mismatches. See Yang H, et al. Cell. 2016;167(7):1814-1828.e12. [Table 1]

[0068] For general information, please refer to 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.

[0069] In one embodiment, the CRISPR / Cas system of the present disclosure comprises at least one RNA guide nuclease (e.g., Cas protein) derived 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 derived from the Deltaproteobacteria or Planctomycetes bacterial species.

[0070] Several embodiments of this disclosure provide strategies, methods, compositions, and therapeutic modalities for modifying a targeted sequence within a gene locus (e.g., modifying the sequence of wild-type and / or mutant sequences in cells or mammals) by insertion or deletion of one or more nucleotides mediated by an RNA guide nuclease and one or more guide RNAs (gRNAs) that result in loss of function of a targeted gene product. In some embodiments, loss of function results in "knockout" of the gene of interest (i.e., production of a "knockout") by removing gene expression. In some embodiments, 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, loss of function results in the expression of a gene product having different properties (e.g., different binding affinity or different cellular localization).

[0071] In certain embodiments, the target gene is selected from TGFB1, TGFBR1, TGFBR2, and combinations thereof. In some embodiments, any region of the target gene (e.g., promoter region, 5' untranslated region, 3' untranslated region, exon, intron, or exon / intron boundary) is targeted by an RNA-guided nuclease to modify the gene, its expression, or both. In some embodiments, non-coding regions of the target gene (e.g., enhancer region, promoter region, intron, 5'UTR, 3'UTR, polyadenylation signal) are targeted to modify the gene, its expression, or both. CRISPR guide RNA:

[0072] In one embodiment, the CRISPR / Cas system of the present disclosure further provides a gRNA molecule (e.g., an isolated or non-native RNA molecule) that interacts with an RNA guide nuclease. In certain embodiments, the gRNA is an sgRNA comprising a spacer sequence containing a nucleotide sequence complementary to a sequence in the target nucleic acid. In some embodiments, the sgRNA further comprises an RNA scaffold (a chimeric fusion of the 3' end of a crRNA and the 5' end of a tracrRNA) that interacts with the RNA guide nuclease, so that the spacer sequence is positioned to scan the target nucleic acid for complementarity. In some embodiments, the system further optionally comprises an oligonucleotide-HDR template having homology to either side of the target site. See Bloh, K., & Rivera-Torres, N, at 3836.

[0073] In one embodiment, the RNA guide nuclease and sgRNA are configured to orient the associated nuclease such that a cleavage event (e.g., a double-strand or single-strand break) occurs in close proximity to the complementary sequence in the targeted nucleic acid, thereby promoting modification of the nucleic acid sequence. In some embodiments, the spacer sequence is 20 nucleotides long. In some embodiments, the spacer sequence is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long.

[0074] In some embodiments, the spacer sequence orients the RNA guide 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 targeted nucleic acid. The cleavage of double-stranded or single-stranded DNA may be located upstream or downstream of the complementary sequence in the target 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.

[0075] In certain embodiments, a second gRNA molecule containing a second spacer sequence orients the second RNA guide nuclease such that the cleavage event occurs in close proximity to the complementary sequence in the targeted nucleic acid, thereby promoting modification of the nucleic acid sequence. In some embodiments, the first and second gRNAs promote cleavage events within a single targeted gene. In some embodiments, the first and second gRNAs promote cleavage events within different targeted genes. In some embodiments, the second spacer sequence is 20 nucleotides long. In some embodiments, the second spacer sequence is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long.

[0076] In some embodiments, a second spacer sequence orients the RNA guide 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 targeted nucleic acid. The cleavage of double-stranded or single-stranded DNA may be located upstream or downstream of the complementary sequence in the target 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.

[0077] In some embodiments, the targeting domains of the first and second gRNAs are configured such that the cleavage event is located independently 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 for each gRNA molecule. In some embodiments, the first and second gRNA molecules modify the target nucleic acid sequence simultaneously. In some embodiments, the first and second gRNA molecules modify the target nucleic acid sequence sequentially.

[0078] In some embodiments, a single-strand break is accompanied by a second single-strand break positioned by spacer sequences in the first and second gRNAs, respectively. For example, the spacer sequences may orient the relevant RNA guide nucleases so that the break events (e.g., two single-strand breaks) 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 and second spacer sequences are configured to orient the relevant RNA guide nucleases such that, for example, two single-strand breaks occur on opposing strands of genomic DNA at the same location or within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides of each other, thereby essentially approximating a double-strand break.

[0079] In some embodiments, the nucleic acid encoding one or more spacer sequences is selected from any sequence disclosed in Table 2. In some embodiments, the nucleic acid encoding one or more spacer sequences is selected from any sequence disclosed in Table 3. In some embodiments, the nucleic acid encoding one or more spacer sequences is selected from any sequence 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, one or more sgRNAs are encoded by nucleic acids selected from any sequence numbers 1 to 519.

[0080] In certain embodiments, the nucleic acid may include (a) a sequence encoding a first sgRNA containing a spacer sequence complementary to the sequence in the targeting gene, (b) a sequence encoding a second sgRNA containing a spacer sequence complementary to the sequence in the second targeting gene, and (c) a sequence encoding an RNA guide nuclease (e.g., Cas9). Optionally, (d) and (e) are sequences encoding a third 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 three or more sgRNAs are used, any combination of (a), (b), (c), (d), and (e) may be encoded within a single or separate nucleic acid molecule.

[0081] In one embodiment, a nucleic acid molecule (i.e., one encoding (a), (b), (c), (d), or (e)) is delivered to a target cell (i.e., any combination of the RNA guide nuclease encoding (c) and at least one gRNA molecule encoding (a), (b), (d), or (e) comes into contact with 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).

[0082] In some embodiments, contacting target cells involves delivering the RNA guide nuclease of (c) as a protein along with at least one nucleic acid molecule selected from (a), (b), (d), and (e). In some embodiments, contacting target cells involves delivering the encoded RNA guide nuclease of (c) as DNA along with at least one nucleic acid molecule selected from (a), (b), (d), and (e). In some embodiments, contacting target cells involves delivering the encoded RNA guide nuclease of (c) as mRNA along with at least one nucleic acid molecule selected from (a), (b), (d), and (e).

[0083] In certain embodiments, CRISPR components are delivered to target cells via nanoparticles. Exemplary nanoparticles that can be used with all CRISPR / Cas systems disclosed herein include at least lipid nanoparticles or liposomes, hydrogel nanoparticles, metal-organic nanoparticles, gold nanoparticles, and magnetic nanoparticles. For general information, see Xu, CF, et al. (2021). Advanced Drug Delivery Reviews, 168, 3-29.

[0084] B.TALEN In one embodiment, the present disclosure intends to use methods, components, and compositions relating to nucleic acid sequences (e.g., transcription activator-like effector nucleases (TALENs) for enhancing targeted genes).

[0085] TALE is an abbreviation for "Transcription Activator-Like Effector" proteins, including TALEN ("Transcription Activator-Like Effector Nucleases"). Methods using the TALE system for gene editing may also be referred to herein as the TALE method. TALE is a native protein derived from plant pathogenic bacteria of the genus Xanthomonas, and contains a DNA-binding domain composed of a series of 33-35 amino acid repeat domains, each recognizing a single base pair. TALE specificity is determined by two hypervariable amino acids known as repeat variable duo (RVDs). Modular TALE repeats are ligated together to recognize a continuous DNA sequence. Specific RVDs within the DNA-binding domain recognize bases within the target locus and provide structural features that assemble a predictable DNA-binding domain. The DNA-binding domain of TALE is fused to the catalytic domain of an IIS-type FokI endonuclease to create a targetable TALE nuclease. To induce site-directed mutation, two individual TALEN arms, separated by a 14-20 base pair spacer region, bring the FokI monomer into close proximity, dimerize, and produce a targeted double-strand break.

[0086] Several large-scale, systematic studies utilizing various assembly methods have demonstrated 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 this disclosure are described in U.S. Patent Publications 2011 / 0201118A1, 2013 / 0117869A1, 2013 / 0315884A1, 2015 / 0203871A1, and 2016 / 0120906A1, which are incorporated herein by reference.

[0087] Non-limiting examples of genes that can be silenced or inhibited by permanent gene editing via TALE include TGFB1, TGFBR1, TGFBR2, and combinations thereof. Non-limiting examples of genes that can be enhanced by permanent gene editing via TALE so that the resulting products function as decoys or dominant-negatives include TGFB1, TGFBR1, TGFBR2, and combinations thereof. Non-limiting examples of genes that can be strengthened by permanent gene editing via TALE include TGFB1, TGFBR1, TGFBR2, and combinations thereof. In one embodiment, the present disclosure provides compositions for upregulating protein receptors (wild-type or gene-edited), including those that bind to anti-inflammatory cytokines via TALE.

[0088] Examples of systems, methods, and compositions for modifying the expression of a target gene sequence by the TALE method, and which may be used in accordance with embodiments of this disclosure, are described in U.S. Patent No. 8,586,526, which is incorporated herein by reference.

[0089] C. zinc finger nuclease (ZFN) In one embodiment, the present disclosure intends to describe the use of methods, components, and compositions relating to zinc finger nucleases (ZFNs) for enhancing nucleic acid sequences (e.g., targeted genes).

[0090] 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 the 3 bp of the main groove of DNA with varying levels of selectivity. Zinc fingers have two protein domains. The first domain is a DNA-binding domain containing eukaryotic transcription factors and the zinc finger. The second domain is a nuclease domain containing FokI restriction enzymes and involved in the catalytic cleavage of DNA.

[0091] 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 to their intended target sites, even a pair of 3-finger ZFNs recognizing a total of 18 base pairs can theoretically target a single locus in the mammalian genome. One way to generate novel zinc finger arrays is to combine zinc finger "modules" with known lower specificity. The most common modular assembly process involves combining three distinct zinc fingers, each capable of recognizing a 3-base pair DNA sequence, to generate a 3-finger array capable of recognizing a 9-base pair target site. Alternatively, selection-based approaches such as oligomerization pool engineering (OPEN) can be used to select novel zinc finger arrays from a randomized library that considers context-dependent interactions between adjacent fingers. Manipulated zinc fingers are commercially available, and Sangamo Biosciences (Richmond, CA, USA) is collaborating with Sigma-Aldrich (St. Louis, MO, USA) to develop a proprietary platform (CompoZr®) for zinc finger constructs.

[0092] Non-limiting examples of genes that can be silenced or inhibited by permanent gene editing via zinc finger methods include TGFB1, TGFBR1, TGFBR2, and combinations thereof. Non-limiting examples of genes that can be enhanced by permanent gene editing via zinc finger methods so that the resulting products function as decoys or dominant-negatives include TGFB1, TGFBR1, TGFBR2, and combinations thereof. Non-limiting examples of genes that can be strengthened by permanent gene editing via zinc finger methods include TGFB1, TGFBR1, TGFBR2, and combinations thereof. In one embodiment, the present disclosure provides compositions for upregulating protein receptors (wild-type or gene-edited), including those that bind to anti-inflammatory cytokines via zinc finger methods.

[0093] Examples of systems, methods, and compositions for modifying the expression of a target gene sequence by the zinc finger method, and which may be used in accordance with embodiments of this disclosure, are U.S. Patents No. 6,534,261, 6,607,882, 6,746,838, 6,794,136, 6,824,978, 6,866,997, and 6,933,113. This is described in Patent 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.

[0094] Other examples of systems, methods, and compositions for modifying the expression of a target gene sequence by the zinc finger method, and which may be used in accordance with embodiments of this disclosure, are described in Beene, et al., Mol. Therapy, 2015, 23 1380-1390, which disclosure is incorporated herein by reference.

[0095] IV. Fibrosis / Scarring A. Introduction Musculoskeletal tissue is particularly susceptible to fibrosis and / or scarring after trauma. Often, this trauma is surgical. Indeed, the formation of musculoskeletal scar tissue presents several problems, including limited range of motion, pain, and complication of subsequent surgical access to the injured site. While we do not wish to be constrained by any particular theory, increased TGFB1 expression has been observed, among other factors, particularly in fascial fibroblasts at the site of such musculoskeletal trauma. See Ihn, H. (2019). Allergology International, 68(4), 437-439. This expression pattern correlated with higher expression of type I collagen and fibronectin, contributing factors to the fibrotic process.

[0096] In one embodiment, the compositions and methods described herein are for the treatment of excessive fibrosis and / or scarring. In some embodiments, the scarring is the result of a disease (e.g., hepatic fibrosis) or trauma (e.g., tendon injury). In some embodiments, the fibrosis and / or scarring is the result of surgery. In some embodiments, the surgery is ligament reconstruction. In some embodiments, the surgery is anterior cruciate ligament (ACL) reconstruction. In some embodiments, the surgery is autologous ACL reconstruction. In some embodiments, the surgery is allogeneic ACL reconstruction. In some embodiments, the surgery is fracture repair. In some embodiments, the surgery is total knee arthroplasty (TKA). In some embodiments, the surgery is microscopic discectomy.

[0097] In some embodiments, fibrosis and / or scarring are the result of a condition that can be induced or aggravated by surgery. In some embodiments, the condition contributing to musculoskeletal fibrosis and / or scarring is knee joint fibrosis. In some embodiments, the condition contributing to musculoskeletal fibrosis and / or scarring is intra-articular fibrous nodules. In some embodiments, the condition contributing to musculoskeletal fibrosis and / or scarring is epidural fibrosis.

[0098] B. Fibrosis In one embodiment, the disclosure encompasses therapies for fibrosis. In some embodiments, a CRISPR gene editing system for the therapy of fibrosis comprises one or more sgRNAs that target TGFB1. In some embodiments, a CRISPR gene editing system for the therapy of fibrosis comprises one or more sgRNAs that target hTGFB1. In some embodiments, a CRISPR gene editing system for the therapy of fibrosis comprises one or more sgRNAs that target cTGFB1. In some embodiments, a CRISPR gene editing system for the therapy of fibrosis comprises one or more sgRNAs that target eTGFB1. In some embodiments, a CRISPR gene editing system for the therapy of fibrosis comprises one or more sgRNAs that target fTGFB1.

[0099] In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 1 of hTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 2 of hTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 3 of hTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 4 of hTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 5 of hTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 6 of hTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 7 of hTGFB1.

[0100] In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 1 of cTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 2 of cTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 3 of cTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 4 of cTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 5 of cTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 6 of cTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 7 of cTGFB1.

[0101] In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 1 of eTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 2 of eTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 3 of eTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 4 of eTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 5 of eTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 6 of eTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 7 of eTGFB1.

[0102] In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 1 of fTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 2 of fTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 3 of fTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 4 of fTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 5 of fTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 6 of fTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 7 of fTGFB1.

[0103] In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target TGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target cTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target fTGFBR1.

[0104] In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 1 of hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 2 of hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 3 of hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 4 of hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 5 of hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 6 of hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 7 of hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 8 of hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 9 of hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 10 of hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 11 of hTGFBR1.

[0105] In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 1 of cTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 2 of cTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 3 of cTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 4 of cTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 5 of cTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 6 of cTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 7 of cTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 8 of cTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 9 of cTGFBR1.

[0106] In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 1 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 2 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 3 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 4 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 5 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 6 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 7 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 8 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 9 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 10 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 11 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 12 of eTGFBR1.

[0107] In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 1 of fTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 2 of fTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 3 of fTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 4 of fTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 5 of fTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 6 of fTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 7 of fTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 8 of fTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 9 of fTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 10 of fTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 11 of fTGFBR1.

[0108] In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target TGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target hTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target cTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target eTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target fTGFBR2.

[0109] In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 1 of hTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 2 of hTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 3 of hTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 4 of hTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 5 of hTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 6 of hTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 7 of hTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 8 of hTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 9 of hTGFBR2.

[0110] In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 1 of cTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 2 of cTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 3 of cTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 4 of cTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 5 of cTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 6 of cTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 7 of cTGFBR2. In some embodiments, a CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 8 of cTGFBR2.

[0111] In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 1 of eTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 2 of eTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 3 of eTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 4 of eTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 5 of eTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 6 of eTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 7 of eTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 8 of eTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 9 of eTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 10 of eTGFBR2.

[0112] In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 1 of fTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 2 of fTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 3 of fTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 4 of fTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 5 of fTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 6 of fTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 7 of fTGFBR2. In some embodiments, a CRISPR gene editing system for the treatment of fibrosis includes one or more sgRNAs that target exon 8 of fTGFBR2.

[0113] C. Musculoskeletal scarring In one embodiment, the disclosure encompasses therapies for musculoskeletal scarring. In some embodiments, a CRISPR gene editing system for the therapy of musculoskeletal scarring comprises one or more sgRNAs that target TGFB1. In some embodiments, a CRISPR gene editing system for the therapy of musculoskeletal scarring comprises one or more sgRNAs that target hTGFB1. In some embodiments, a CRISPR gene editing system for the therapy of musculoskeletal scarring comprises one or more sgRNAs that target cTGFB1. In some embodiments, a CRISPR gene editing system for the therapy of musculoskeletal scarring comprises one or more sgRNAs that target eTGFB1. In some embodiments, a CRISPR gene editing system for the therapy of musculoskeletal scarring comprises one or more sgRNAs that target fTGFB1.

[0114] In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 1 of hTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 2 of hTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 3 of hTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 4 of hTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 5 of hTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 6 of hTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 7 of hTGFB1.

[0115] In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 1 of cTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 2 of cTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 3 of cTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 4 of cTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 5 of cTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 6 of cTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 7 of cTGFB1.

[0116] In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 1 of eTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 2 of eTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 3 of eTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 4 of eTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 5 of eTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 6 of eTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 7 of eTGFB1.

[0117] In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 1 of fTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 2 of fTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 3 of fTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 4 of fTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 5 of fTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 6 of fTGFB1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 7 of fTGFB1.

[0118] In some embodiments, a CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target TGFBR1. In some embodiments, a CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target hTGFBR1. In some embodiments, a CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target cTGFBR1. In some embodiments, a CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target eTGFBR1. In some embodiments, a CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target fTGFBR1.

[0119] In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 1 of hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 2 of hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 3 of hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 4 of hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 5 of hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 6 of hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 7 of hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 8 of hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 9 of hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 10 of hTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 11 of hTGFBR1.

[0120] In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 1 of cTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 2 of cTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 3 of cTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 4 of cTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 5 of cTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 6 of cTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 7 of cTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 8 of cTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 9 of cTGFBR1.

[0121] In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 1 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 2 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 3 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 4 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 5 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 6 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 7 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 8 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 9 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 10 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 11 of eTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 12 of eTGFBR1.

[0122] In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 1 of fTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 2 of fTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 3 of fTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 4 of fTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 5 of fTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 6 of fTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 7 of fTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 8 of fTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 9 of fTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 10 of fTGFBR1. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 11 of fTGFBR1.

[0123] In some embodiments, a CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target TGFBR2. In some embodiments, a CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target hTGFBR2. In some embodiments, a CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target cTGFBR2. In some embodiments, a CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target eTGFBR2. In some embodiments, a CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target fTGFBR2.

[0124] In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 1 of hTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 2 of hTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 3 of hTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 4 of hTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 5 of hTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 6 of hTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 7 of hTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 8 of hTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 9 of hTGFBR2.

[0125] In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 1 of cTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 2 of cTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 3 of cTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 4 of cTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 5 of cTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 6 of cTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 7 of cTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 8 of cTGFBR2.

[0126] In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 1 of eTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 2 of eTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 3 of eTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 4 of eTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 5 of eTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 6 of eTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 7 of eTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 8 of eTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 9 of eTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 10 of eTGFBR2.

[0127] In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 1 of fTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 2 of fTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 3 of fTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 4 of fTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 5 of fTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 6 of fTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 7 of fTGFBR2. In some embodiments, the CRISPR gene editing system for the treatment of musculoskeletal scarring includes one or more sgRNAs that target exon 8 of fTGFBR2.

[0128] V. Delivery A. Viral vectors In one embodiment, the disclosure comprises a method for delivering a CRISPR gene editing system that targets 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 includes a recombinant virus selected from retroviruses, adenoviruses, adeno-associated viruses, lentiviruses, and herpes simplex virus-1. In some embodiments, one of the more viral vectors includes recombinant adeno-associated virus (AAV). In some embodiments, the recombinant AAV is serotype 5 (AAV5). In some embodiments, the recombinant AAV is serotype 6 (AAV6). In some embodiments, one or more viral vectors include a first viral vector containing a first nucleic acid from one or more nucleic acids encoding a Cas protein, and a second viral vector containing a second nucleic acid from one or more nucleic acids encoding at least one guide RNA. In some embodiments, one or more viral vectors include a viral vector containing a single nucleic acid, the single nucleic acid encoding a Cas9 protein and at least one guide RNA.

[0129] 1. Adeno-associated virus (AAV) A useful viral vector system for nucleic acid delivery is adeno-associated virus (AAV). Adeno-associated viruses are naturally occurring deficient viruses that require another virus, such as an adenovirus or herpesvirus, as a helper virus for efficient replication and a productive life cycle. (For a review, see Muzyczka et al., Curr. Topics in Micro. and Immunol. 158:97-129 (1992). Adeno-associated viruses are also one of the few viruses that integrate their DNA into non-dividing cells, exhibiting high-frequency stable integration (see, for example, 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 AAV of only 300 base pairs can be packaged and integrated. The space for exogenous DNA is limited to approximately 4.5 kb. Tratschin et al.) AAV vectors, such as those described in al., Mol.Cell.Biol.5:3251-3260 (1985), can be used to introduce DNA into cells. Various nucleic acids have been introduced into different cell types using AAV vectors (see, for example, Hermonat et al., Proc.Natl.Acad.Sci.USA81:6466-6470 (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 stable and effective in vivo, easily produced, FDA-approved, and tested in multiple clinical trials, and can be packaged into adeno-associated virus (AAV) vectors, opens new avenues for therapeutic gene editing.

[0130] According to certain embodiments, a CRISPR gene editing system that targets 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, which is a recombinant AAV genome. In some embodiments, the AAV vector is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10 vectors.

[0131] In some embodiments, the AAV vector includes a serotype selected from the group consisting of AAV1, AAV1(Y705+731F+T492V), AAV2(Y444+500+730F+T491V), AAV3(Y705+731F), AAV4, AAV5, AAV5(Y436+693+719F), AAV6, AAV6(VP3 variant Y705F / Y731F / T492V), AAV-7m8, AAV8, AAV8(Y733F), AAV9, AAV9(VP3 variant Y731F), AAV10(Y733F), AAV-ShH10, and AAV-DJ / 8. In some embodiments, the AAV vector contains a serotype selected from the group consisting of AAV1, AAV5, AAV6, AAV6(Y705F / Y731F / T492V), AAV8, AAV9, and AAV9(Y731F).

[0132] In one embodiment, the use of a 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 disorders, conditions, and diseases, including but not limited to Loeys-Dietz syndrome, osteoarthritis, Marfan syndrome, aortic aneurysms (e.g., familial thoracic aortic aneurysms), craniofacial malformations, and combinations thereof. In other embodiments, the use of the system treats neoplastic disorders, conditions, and diseases, including pancreatic cancer, multiple spontaneously resolving 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.

[0133] 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 virus-1 (HIV-1), human immunodeficiency virus-2 (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Jambrana disease virus (JDV), equine infectious anemia virus (EIAV), and canine arthritis encephalitis virus (CAEV).

[0134] Lentiviral transduction systems are known in the art and are described, for example, in Levine, et al., Proc. Nat'l Acad. Sci. 2006, 103, 17372-77, Zufferey, et al., Nat. Biotechnol. 1997, 15, 871-75, Dull, et al., J. Virology 1998, 72, 8463-71, and U.S. Patent No. 6,627,442, each of which disclosures are incorporated herein by reference.

[0135] In one embodiment, the use of a 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 disorders, conditions, and diseases, including but not limited to Loeys-Dietz syndrome, osteoarthritis, Marfan syndrome, aortic aneurysms (e.g., familial thoracic aortic aneurysms), craniofacial malformations, and combinations thereof. In other embodiments, the use of the system treats neoplastic disorders, conditions, and diseases, including pancreatic cancer, multiple spontaneously resolving 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.

[0136] B. Lipid nanoparticles (LNPs) In some embodiments, the CRISPR gene editing system is delivered by nanoparticles. While we do not wish to be bound by any particular theory, in certain embodiments, nucleic acids, when present in nanoparticles, are resistant to nuclease degradation in aqueous solution. In other embodiments, proteins are protected from protease degradation. In some embodiments, proteins and nucleic acids encapsulated by nanoparticles can penetrate the cell plasma membrane.

[0137] Lipid nanoparticles containing nucleic acids and methods for preparing the same are at least as specified in WO2017 / 019935, WO2017 / 049074, WO2017 / 201346, WO2017 / 218704, WO2018 / 006052, WO2018 / 013525, WO2018 / 089540, WO2018 / 119115, WO2018 / 126084, WO2018 / 157009, WO2018 / 170336, WO2018 / 222890, and 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, US Patents No. 8,058,069, No. 8,492,359, No. 8,822,668, No. 9,364,435, No. 9,404,127, No. 9,504,651, No. 9,593,077, No. 9,738,593, No. 9,868,691, No. 9,868,692, No. 9,95 These are disclosed in Patent 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 entirety for all purposes.

[0138] Lipid nanoparticle composition In some embodiments, the maximum dimensions of the nanoparticle composition are less than or equal to 1 micrometer (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) when measured by, for example, dynamic light scattering (DLS), transmission electron microscopy, scanning electron microscopy, or another method. The nanoparticle composition includes, for example, lipid nanoparticles (LNPs), liposomes, lipid vesicles, and lipoplexes. In some embodiments, the nanoparticle composition is a vesicle containing one or more lipid bilayers. In certain embodiments, the nanoparticle composition includes two or more concentric bilayers separated by aqueous compartments. The lipid bilayers may be functionalized and / or crosslinked with each other. The lipid bilayers may contain one or more ligands, proteins, or channels. In various embodiments, the lipid nanoparticles described herein have wavelengths of approximately 30 nm to 150 nm, approximately 40 nm to 150 nm, approximately 50 nm to 150 nm, approximately 60 nm to 130 nm, approximately 70 nm to 110 nm, approximately 70 nm to 100 nm, approximately 80 nm to 100 nm, approximately 90 nm to 100 nm, approximately 70 nm to 90 nm, approximately 80 nm to 90 nm, and approximately 70 nm to 80 It has an average diameter of approximately 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 is substantially nontoxic.

[0139] In certain embodiments, the lipid nanoparticles described herein comprise one or more components, including lipid components and (optionally) structural components. The lipid components comprise lipids selected from ionic lipids and / or cationic lipids (i.e., lipids that may have a positive or partially positive charge at physiological pH), neutral lipids (e.g., phospholipids or sphingolipids), and polymer conjugate lipids (e.g., PEGylated lipids). In some embodiments, the lipid components comprise a single ionic lipid. In other embodiments, the lipid components comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 ionic lipids. In some embodiments, the lipid components comprise a single neutral lipid. In other embodiments, the lipid components comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 neutral lipids. In some embodiments, the lipid components comprise a single polymer conjugate lipid. In other embodiments, the lipid components include 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 polymer-conjugate lipids. In some embodiments, the structural components include a single structural lipid. In other embodiments, the structural components include 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 structural lipids. In some embodiments, the lipid components include at least one cationic lipid, at least one neutral lipid, and at least one polymer-conjugate lipid. This disclosure intends that the lipid components may include any combination of the aforementioned components.

[0140] Ionic / cationic lipids In some embodiments, the lipid components include ionic lipids. In some embodiments, the ionic lipids are anionic. In other embodiments, the ionic lipids are cationic lipids. In some embodiments, the lipid components are not limited to these, but include 3-(didodecylamino)-N1,N1,4-tridedecyl-1-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridedecyl-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), and 1,2-dilinoleyloxy-N,N-dimethyl Minopropane (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(octadeca-9,12-dien-1-yloxy) The material contains cationic lipids selected from the group consisting of [ooxy]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 cyclic amine groups, and mixtures thereof.

[0141] Non-exclusive and non-limiting examples of cationic lipids include the following: [ka] [ka] [ka] [ka]

[0142] Neutral lipids / phospholipids In some embodiments, the lipid components are not limited to these, but include 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), and 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-difitanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 The material further comprises neutral lipids including phospholipids selected from the group consisting of 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.

[0143] Polymer-conjugated lipids In some embodiments, the lipid components further include polymer conjugate lipids, which are not limited to these, but include PEGylated lipids 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 lipids are PEG-c-DOMG, PEG-DMG, PEG 2000 -c-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DMA, or PEG-DSPE lipids may also be used.

[0144] Non-exclusive and non-limiting examples of PEG lipids include the following: [ka] [ka] [ka]

[0145] Structural lipids / sterols In some embodiments, the LNP further comprises structural components. For general information, see Patel, S., et al. (2020). Nature Communications, 11(1), 1-13. In some embodiments, the structural components include sterols, but are not limited to, cholesterol, fecosterol, stigmasterol, stigmamanol, sitosterol, β-sitosterol, lupeol, betulin, ursolic acid, oleanolic acid, campesterol, fucosterol, brassicasterol, ergosterol, 9,11-dehydroergosterol, tomatidine, tomatine, α-tocopherol, and mixtures thereof. In other embodiments, the structural lipids include cholesterol and corticosteroids (e.g., prednisolone, dexamethasone, prednisone, and hydrocortisone), or combinations thereof.

[0146] Non-exclusive and non-limiting examples of structural lipids include the following: [ka]

[0147] formulation Nanoparticle compositions may comprise lipid components and one or more additional components, such as therapeutic and / or prophylactic agents. Nanoparticle compositions may be designed for one or more specific uses or targets. The elements of a nanoparticle composition may be selected based on a specific use or target, and / or on the efficacy, toxicity, cost, ease of use, availability, or other characteristics of one or more elements. Similarly, specific formulations of nanoparticle compositions may be selected for a specific use or target, for example, according to the efficacy and toxicity of a particular combination of elements.

[0148] The lipid components of the nanoparticle composition may include, for example, cationic lipids, phospholipids (unsaturated lipids, such as DOPE or DSPC), PEG lipids, and structural lipids. The elements of the lipid components may be provided in specific fractions.

[0149] In some embodiments, the lipid components of the nanoparticle composition include ionic lipids, phospholipids, PEG lipids, and structural lipids. In certain embodiments, the lipid components of the nanoparticle composition include about 30 mol% to about 60 mol% of ionic lipids, about 0 mol% to about 30 mol% of phospholipids, about 0 mol% to about 10 mol% of PEG lipids, and about 17.5 mol% to about 50 mol% of structural 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% of ionic lipid compounds, about 5 mol% to about 25 mol% of phospholipids, about 0 mol% to about 10 mol% of PEG lipids, and about 30 mol% to about 40 mol% of structural lipids. In certain embodiments, the lipid components include about 50 mol% of such compounds, about 10 mol% of phospholipids, about 38.5 mol% of structural lipids, and about 1.5 mol% of PEG lipids. In another embodiment, the lipid composition comprises about 40 mol% of the compound, about 20 mol% of phospholipids, about 38.5 mol% of structural lipids, and about 1.5 mol% of PEG lipids. In some embodiments, the phospholipids may be DOPE or DSPC. In other embodiments, the PEG lipids may be PEG-DMG, and / or the structural lipids may be cholesterol.

[0150] In some embodiments, the ionic lipid contains about 20 to about 60 mol% of lipid components. In other embodiments, the ionic lipid contains about 35 to about 55 mol% of lipid components. In various embodiments, the ionic lipid contains about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, or 60 mol% of lipid components.

[0151] In some embodiments, the neutral lipids contain about 0 to about 30 mol% of lipid components. In other embodiments, the neutral lipids contain about 5 to about 25 mol% of lipid components. In various embodiments, the neutral lipids contain 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 lipid components.

[0152] In some embodiments, the polymer conjugate lipid contains about 0 to about 15 mol% of lipid components. In other embodiments, the polymer conjugate lipid contains about 0.5 to about 10 mol% of lipid components. In various embodiments, the polymer conjugate lipid contains 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 lipid components.

[0153] In some embodiments, the structural components include about 17.5 mol% to about 50 mol% of lipid components. In other embodiments, the structural components include about 30 to about 40 mol% of lipid components. In various embodiments, the structural components include 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 lipid components.

[0154] Alternatively, structural components can be expressed as a ratio to lipid components. In some embodiments, structural components are in a ratio of approximately 1:1 with lipid components (sterols:lipids). In other embodiments, structural components are in a ratio of approximately 1:5 with lipid components (sterols:lipids). In various embodiments, structural components are in a ratio of approximately 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 lipid components (sterols:lipids).

[0155] Nanoparticle compositions may be designed for one or more specific uses or targets. For example, a nanoparticle composition 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 body of a mammal. The physiological and chemical properties of the nanoparticle composition may be modified to increase selectivity for specific bodily targets. For example, particle size may be adjusted based on the window sizes of different organs. The therapeutic and / or prophylactic agents contained in the nanoparticle composition may also be selected based on the desired delivery target. For example, the therapeutic and / or prophylactic agents may be selected for specific indications, conditions, diseases, or disorders, and / or for delivery to specific cells, tissues, organs, or systems or groups thereof (e.g., local or specific delivery). In certain embodiments, the nanoparticle composition may contain mRNA encoding a target polypeptide that can be translated in cells to produce the target polypeptide. Such compositions may be designed to be specifically delivered to specific organs. In some embodiments, the composition may be designed to be specifically delivered to mammalian joints. In some embodiments, the nanoparticles are bound to or complexed with a targeting moiety to improve delivery to specific target cells and / or tissues. For example, in some embodiments, nanoparticles are bound to or complexed with a drug that binds to cell surface markers of target cells and / or tissues.

[0156] The amount of therapeutic and / or prophylactic agent in a nanoparticle composition may depend on the size, composition, desired target and / or application, or other properties of the nanoparticle composition, as well as the properties of the therapeutic and / or prophylactic agent. For example, the amount of RNA useful in a nanoparticle composition may depend on the size, sequence, and other properties of the RNA. The relative amounts of therapeutic and / or prophylactic agent and other elements (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, for example, 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 components to therapeutic and / or prophylactic agents may be 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., ultraviolet-visible spectroscopy).

[0157] In some embodiments, the therapeutic and / or prophylactic agent comprises nucleic acid components. In some embodiments, the nucleic acid components include RNA, but are not limited to, RNA selected from the group consisting of messenger RNA (mRNA), CRISPR RNA (crRNA), tracrRNA, single-stranded RNA (sgRNA), short interference RNA (siRNA), antisense oligonucleotides (ASOs), and mixtures thereof. In other embodiments, the nucleic acid components include DNA, but are not limited to, DNA selected from the group consisting of linear DNA, plasmid DNA, antisense oligonucleotides, and mixtures thereof.

[0158] In some embodiments, the nanoparticle composition comprises one or more RNAs, and the one or more RNAs, lipids, and their amounts may be selected to provide a specific N:P ratio. The N:P ratio of the composition refers to the molar ratio of nitrogen atoms in one or more lipids to the number of phosphate groups in RNA. Generally, a lower N:P ratio is preferred. The one or more RNAs, lipids, and their amounts may be selected to provide an N:P ratio of about 2:1 to about 30:1, for example, 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 may 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 may be approximately 5.0:1, 5.5:1, 5.67:1, 6.0:1, 6.5:1, or 7.0:1.

[0159] In some embodiments, nucleic acid components consist of modified nucleic acids. For example, RNA may be modified RNA. That is, RNA may contain one or more non-natural nucleic acid bases, nucleosides, nucleotides, or linkers. A “modified” species may also be referred to herein as a “modified” species. A species may be chemically, structurally, or functionally modified or altered. For example, a modified nucleic acid base species may contain one or more non-natural substitutions.

[0160] In certain embodiments, the Disclosure includes a method for treating a condition or disorder of the back or spine. In other embodiments, the Disclosure includes a method for treating a discogenic disorder. In some embodiments, the Disclosure includes a method for providing treatment to a subject requiring treatment of local pain, inflammation, or morphological changes associated with a condition or disorder of the back or spine, the method comprising administering a therapeutically effective amount of a CRISPR-Cas composition encapsulated in or associated with lipid nanoparticles (LNPs), the composition comprising one or more non-natural polynucleotides encoding the Cas9 protein and at least one sgRNA. In some embodiments, the LNPs are administered subarachnoidally. In other embodiments, the LNPs are administered epidurally. In some embodiments, the LNPs are administered peridiscally. In some embodiments, the LNPs are administered perivertebrally.

[0161] physical properties The properties of a nanoparticle composition may depend on its constituent components. For example, a nanoparticle composition containing cholesterol as a structural lipid may have different properties than a nanoparticle composition containing a different structural lipid. Similarly, the properties of a nanoparticle composition may depend on the absolute or relative amounts of its constituent components. For example, a nanoparticle composition containing a higher molar fraction than that of a phospholipid may have different properties than a nanoparticle composition containing a lower molar fraction than that of a phospholipid. The properties may also vary depending on the method and conditions of preparation of the nanoparticle composition.

[0162] Nanoparticle compositions can be characterized by various methods. For example, the morphology and size distribution of the nanoparticle composition may be examined using a microscope (e.g., transmission electron microscopy or scanning electron microscopy). Zeta potential may be measured using dynamic light scattering or potentiometric measurements (e.g., potentiometric titration). Particle size may be determined using dynamic light scattering. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can also be used to measure multiple properties of the nanoparticle composition, such as particle size, polydispersity index, and zeta potential.

[0163] The average size of the nanoparticle composition can range from 10 nm to 1 micrometer and is measured, for example, by dynamic light scattering (DLS). For example, the average size may range from approximately 40 nm to approximately 150 nm, for example, approximately 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 may 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 a particular embodiment, the average size of the nanoparticle composition may be about 70 nm to about 100 nm. In a particular embodiment, the average size may be about 80 nm. In another embodiment, the average size may be about 100 nm.

[0164] Nanoparticle compositions can be relatively homogeneous. The polydispersity index may be used to indicate the homogeneity of a nanoparticle composition, for example, the particle size distribution of the nanoparticle composition. A small polydispersity index (e.g., less than 0.3) generally indicates a narrow particle size distribution. Nanoparticle compositions may have a polydispersity index of about 0 to about 0.25, for example, 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 a nanoparticle composition may be about 0.10 to about 0.20.

[0165] The zeta potential of a nanoparticle composition can be used to indicate the electrodynamic potential of the composition. For example, the zeta potential can explain the surface charge of a nanoparticle composition. Relatively low-charge, positive or negative nanoparticle compositions are generally preferred because higher-charge species may interact with cells, tissues, and other elements in the body, which is undesirable. In some embodiments, the zeta potential of the nanoparticle composition may be approximately -10mV to approximately +20mV, approximately -10mV to approximately +15mV, approximately -10mV to approximately +10mV, approximately -10mV to approximately +5mV, approximately -10mV to approximately 0mV, approximately -10mV to approximately -5mV, approximately -5mV to approximately +20mV, approximately -5mV to approximately +15mV, approximately -5mV to approximately +10mV, approximately -5mV to approximately +5mV, approximately -5mV to approximately 0mV, approximately 0mV to approximately +20mV, approximately 0mV to approximately +15mV, approximately 0mV to approximately +10mV, approximately 0mV to approximately +5mV, approximately +5mV to approximately +20mV, approximately +5mV to approximately +15mV, or approximately +5mV to approximately +10mV.

[0166] The encapsulation efficiency of the therapeutic and / or prophylactic agent is described as the amount of the therapeutic and / or prophylactic agent encapsulated in the nanoparticle composition after preparation, or otherwise associated with the nanoparticle composition, relative to the initial amount provided. High encapsulation efficiency is desirable (e.g., close to 100%). Encapsulation efficiency can be measured, for example, by comparing the amount of the therapeutic and / or prophylactic agent in a solution containing the nanoparticle composition before and after decomposition of the nanoparticle composition with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free therapeutic and / or prophylactic agent (e.g., RNA) in the solution. For the nanoparticle compositions described herein, the encapsulation efficiency of the therapeutic and / or prophylactic agent may 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%.

[0167] The nanoparticle composition may optionally include one or more coatings. For example, the nanoparticle composition may be formulated as a capsule, film, or tablet having a coating. Capsules, films, or tablets containing the compositions described herein may have any useful size, tensile strength, hardness, or density.

[0168] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure pertains. Similar or equivalent methods and materials to those described herein may be used in carrying out or testing the disclosure, but preferred methods and materials are described below. All publications, patent applications, patents, and other references referenced herein are incorporated in their entirety by reference. In addition, materials, methods, and examples are illustrative and not intended to limit the scope of this disclosure.

[0169] 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 comprises guide RNA. In some embodiments, the one or more RNA-containing compositions comprises mRNA. In some embodiments, the one or more RNA-containing compositions comprises RNPs (e.g., Cas9 and guide RNA). In some embodiments, the one or more nanoparticles are lipid nanoparticles (LNPs).

[0170] In some embodiments, the CRISPR gene editing system includes (i) an RNA guide nuclease or a nucleic acid encoding an RNA guide nuclease, and (ii) one or more LNPs that collectively encapsulate at least one guide RNA or a nucleic acid encoding at least one guide RNA. In some embodiments, one or more LNPs include a first group of LNPs that encapsulate an RNA guide nuclease or a nucleic acid encoding an RNA guide nuclease, and a second group of LNPs that encapsulate at least one guide RNA or a nucleic acid encoding at least one guide RNA.

[0171] In some embodiments, one or more LNPs are 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethaneamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethaneamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), and 1,2-dilinoleyloxy-N,N-dimethylaminopropyl Pan(DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl 2-(dimethylamino)butanoate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropyl Pan(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 The material contains components selected from the group consisting of -diene-1-yloxy]propan-1-amine(octyl-CLinDMA(2R)), (2S)-2-({8-[(3.beta.)-cholest-5-ene-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z-,12Z)-octadeca-9,12-diene-1-yloxy]propan-1-amine(octyl-CLinDMA(2S)), lipids containing cyclic amine groups, and mixtures thereof.

[0172] In some embodiments, one or more LNPs are 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-difitanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 The material contains components selected from the group consisting of 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.

[0173] In some embodiments, one or more LNPs include components 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 lipids may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DMA, PEG-DSPE lipids, and mixtures thereof.

[0174] In some embodiments, one or more LNPs include components selected from the group consisting of cholesterol, fecosterol, stigmasterol, stigmanol, sitosterol, β-sitosterol, lupeol, betulin, ursolic acid, oleanolic acid, campesterol, fucosterol, brassicasterol, ergosterol, 9,11-dehydroergosterol, tomatidine, tomatine, α-tocopherol, and mixtures thereof.

[0175] In one embodiment, the use of a 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 disorders, conditions, and diseases, including but not limited to Loeys-Dietz syndrome, osteoarthritis, Marfan syndrome, aortic aneurysms (e.g., familial thoracic aortic aneurysms), craniofacial malformations, and combinations thereof. In other embodiments, the use of the system treats neoplastic disorders, conditions, and diseases, including pancreatic cancer, multiple spontaneously resolving 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.

[0176] Exemplary LNP system In some embodiments, the pharmaceutical compositions described herein are formulated using an LNP system selected from those listed in the following table. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11] [Table 2-12] [Table 2-13]

[0177] In some embodiments of any one LNP system LNP001 to LNP240, the LNP system contains steroid components in a molar ratio of about 36 to about 46, or about 38 to about 42. In some embodiments of any one LNP system LNP001 to LNP240, the LNP system contains steroid components in a molar ratio of about 36±0.5, about 37±0.5, about 38±0.5, about 39±0.5, about 40±0.5, about 41±0.5, about 42±0.5, about 43±0.5, about 44±0.5, about 45±0.5, or about ±0.5. In some embodiments, the LNP system contains steroid components in a molar ratio of about 37, about 38, about 39, about 40, about 41, or about 42. In some embodiments of any one LNP system LNP001 to LNP240, the steroid components include cholesterol. In some embodiments of any one LNP system LNP001 to LNP240, the steroid component comprises cholesterol and one or more additional steroids, wherein the cholesterol constitutes about 60% (w / w) or more, about 65% (w / w) or more, about 70% (w / w) or more, about 75% (w / w) or more, or about 80% (w / w) or more of the steroid component. In some embodiments, the steroid component comprises dexamethasone. In some embodiments, the steroid component comprises modified cholesterol, e.g., hydroxy-modified or alkyl-modified. In some embodiments, the steroid component comprises any other steroid disclosed herein. In some embodiments, the LNP system comprises PEGylated lipid components in a molar ratio of about 1.5 to about 2.5. In some embodiments, the PEGylated lipid component comprises PEG2000 lipids. In some embodiments, the PEGylated lipid component comprises one or more DMG-PEG and / or DMG-C-PEG. In some embodiments, the PEGylated lipid components include one or more of DMG-PEG2000, DMG-C-PEG2000, DSG-PEG2000, C14-PEG2000, C16-PEG2000, and / or C18-PEG2000.

[0178] The LNPs described herein typically comprise biodegradable ionic lipids, helper phospholipids, cholesterol (with the potential addition of dexamethasone or other steroids), and PEG2000 components. The molar lipid ratios described herein are ionic lipids:helper lipids:cholesterol[:dexamethasone]:PEG.

[0179] Modification of Molar Lipid Ratio: While we do not wish to be bound by any particular theory, when altering the molar lipid ratio of ionic lipids, the amount of cholesterol or helper lipids is either added to or reduced. Typically, while the ionic lipids are maintained at the set ratio, the helper lipids, cholesterol, and PEG content change. When the amount of PEG is changed, the amount of cholesterol, and sometimes helper lipids, is then adjusted, primarily. When the helper lipids are changed, the amount of cholesterol combined with them is adjusted. Dexamethasone, or another similar steroid, in the LNP structure directly replaces a portion of the cholesterol due to its similar chemical structure / function in the LNP.

[0180] Typical ionic lipids have molar ratios of 42-50, e.g., 44; 44-50, e.g., 50; SM-102 (lipid H): 44-50, e.g., 50; ALC-0315: 44-50, e.g., 50; LP01 (LP0000001): 42-50, e.g., 45. Helper lipids may have molar ratios of 9-11, e.g., 10. Examples of helper lipids include DSPC, DOPE, DOTMA, and DPPC. PEG2000 lipids have a typical molar ratio of approximately 1.5-2.5. These include, but are not limited to, DMG-PEG, DMG-C-PEG, DMG-PEG2000, DMG-C-PEG2000, DSG-PEG2000, and C14,16,18-PEG2000. The cholesterol molar-lipid ratio is typically about 36 to about 46. Dexamethasone or similar steroids complement part of the cholesterol molar-lipid ratio, for example, a 9:1 cholesterol:dexamethasone (C:D) ratio, though not limited to these. The molar-lipid ratio range for cholesterol can be about 8 to about 10, though not limited to these, and for dexamethasone, about 0.1 to about 2, though not limited to these. Modification or substitution of cholesterol, for example, hydroxyl or alkyl modifications (for example, to improve mRNA delivery), or substitution of potential therapeutic parts such as anti-inflammatory steroids, can be considered, although we do not wish to be bound by any particular theory. In some embodiments, the molar N / P ratio can be 1 to 8. While we do not wish to be bound by any particular theory, the ideal range for N / P to reduce the inflammatory response of LNPs in vivo is considered to be 1-5, although N / P ratios of 6-8 are being tested.

[0181] In some embodiments, the LNP formulations described herein include LP01:DSPC:cholesterol:DMG-PEG2000, LP01:DSPC:cholesterol:dexamethasone:DMG-PEG2000, MC3:DSPC:cholesterol:DMG-PEG2000, MC3:DSPC:cholesterol:dexamethasone:DMG-PEG2000, MC3:DSPC:cholesterol:DMG-C-PEG2000, MC3:DSPC:cholesterol:dexamethasone:DMG-C-PEG2000, SM-102:DSPC:cholesterol:DMG-PEG2000, SM-102:DSPC:cholesterol:dexamethasone:DMG-PEG2000, ALC-0315:DSPC:cholesterol:DMG-PEG2000, and ALC-0315:DSPC:cholesterol:dexamethasone:DMG-PEG2000. In some embodiments, the molar ratios of the formulations described herein include 50:10:38.5:1.5; 45:9:44:2.

[0182] Biophysical assay: LNPs encapsulate nucleic acids, meet size determination criteria, are homogeneous, and are stable after freeze-thaw cycles. Encapsulated payloads: GFP mRNA, luciferase mRNA, and our CRISPR / cas9 therapeutic agents (sgRNA and cas9 mRNA).

[0183] [Table 3]

[0184] Additional notes regarding LNP components: Ionic lipids (titrateable charge): These contain tertiary amines, which are positively charged at acidic pH and neutral at physiological pH. They are protonated after endosomal uptake into the cytosol. For details on different types of ionic lipids, please refer to the following.

[0185] Helper phospholipids: Anionic endosomal phospholipids that interact with protonated ionic lipids to form conical ion pairs that enhance cell membrane fusion and disruption, endosomal escape, and cargo release into the cell cytosol.

[0186] Cholesterol: Enhances circulation by maintaining the integrity of nanoparticle membranes, assisting in nucleic acid encapsulation, and reducing surface-bound proteins.

[0187] Dexamethasone or similar steroids: Provides an anti-inflammatory component to LNP, which reduces immunogenicity and increases transfection rates, especially in vivo.

[0188] PEGylated lipids (PEG2000): Improve circulating half-life, reduce LNP aggregation, minimize interactions with serum proteins such as opsonins, and enhance in vivo stability. Furthermore, they assist in particle stability, particle size determination, and in vivo distribution.

[0189] Ionic lipids: [ka]

[0190] Cationic lipids, such as DODMA, DOTMA, and DOTAP, contain a quaternary amine group, which permanently keeps the cationic lipid positively charged. Their sole use had previously been proven to have poor circulation and increased toxicity in vivo, leading to the development of ionic lipids.

[0191] Ionic lipids are characterized by the substitution of quaternary amines with tertiary amines, which allows ionic lipids to be pH-dependent; that is, they are neutrally charged at physiological pH and positively charged (protonated) at acidic pH.

[0192] This increases the circulating half-life in vivo and reduces toxicity.

[0193] The pKa of ionic lipids drives performance in vivo. A pKa of 6–6.7 has been shown to be optimal for RNA therapeutic delivery. However, the relative pKa of all component combinations within LNPs affects LNP transfection, as tertiary amines, quaternary amino acids, and hydroxyl groups from ionic lipids, helper lipids, and cholesterol all alter their relative pKa due to the proximity of their head groups. This, in turn, affects the overall surface charge.

[0194] pH dependence enables efficient encapsulation of RNA in acidic buffers and, once taken up by cells, assists in RNA release.

[0195] Furthermore, these lipid pairs form an inverted hexagonal HII phase, which assists in membrane disruption, endosomal escape, and subsequent release of the payload into the cell's cytosol.

[0196] The lipid packing theory by Albertson et al., 2022, shows the relationship between amphiphilic compounds and their self-assembled shapes. This proposed mechanism (also known as the molecular shape hypothesis) suggests that ionic lipids mediate endosomal disruption. https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC9250827 /

[0197] MC3, ALC-0135, and SM-102 all contain tertiary amines (which make them ionic), lack stereocenters, and have ester linkers. ALC-0135 and SM-102 are improved with MC3 to incorporate additional ester bonds (to aid biodegradability and reduce bioaccumulation).

[0198] Unsaturation of the linear tail increases delivery efficiency and fluidity in ionic lipids, i.e., this allows bilayer lipids to form a non-bilayer phase that increases membrane disruption and payload release. Additional studies have investigated branching of the tail of ionic lipids, which further increased potency in delivering mRNA therapeutics due to increased protonation of ionic lipids at the cross-section of the lipid tail and endosomal pH. This modification also resulted in an increase in conical structures that facilitate membrane disruption, endosomal escape, and payload release into the cytosol (Albertson et al., 2022).

[0199] LP01 incorporates increased ester bonds and tail branching, which has been shown to be an efficient and safe delivery system for in vivo gene editing in animal models. LP01 has less hepatic bioaccumulation and lower safety risks.

[0200] Exemplary LP01 LNP formulation In some embodiments, a composition is provided comprising lipid nanoparticles (LNPs) comprising (i) an RNA guide nuclease or a nucleic acid encoding an RNA guide nuclease, and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA that targets a gene, wherein the LNPs comprise LP01.

[0201] In some embodiments, LNP consists of the following structural components: LP01 ionic lipids, neutral / helper lipid components, steroid system components, and PEGylated lipid components. In some such embodiments of LP01-based LNP, the neutral / helper lipid components include DSPC. In some such embodiments of LP01-based LNP, the steroid system components include cholesterol. In some such embodiments of LP01-based LNP, the PEGylated lipid components include PEGylated myristoyl diglyceride (DMG-PEG, 1,2-dimiristoyl-sn-glycero-3-methoxypolyethylene glycol). In some embodiments, DMG-PEG is DMG-PEG2000. In some such embodiments of LP01-based LNP, the steroid system components include cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0202] In some embodiments, the structural components of LNP are present in the following relative molar amounts (provided in parentheses after the component): LP01 (40-50), neutral / helper lipid components (6-12), steroid system components (39-49), and PEGylated lipid components (1-3). In some such embodiments, the neutral / helper lipid components include DSPC. In some such embodiments of LP01-based LNP, the steroid system components include cholesterol. In some such embodiments of LP01-based LNP, the PEGylated lipid components include PEGylated myristoyl diglycerides (DMG-PEG, 1,2-dimiristoyl-sn-glycero-3-methoxypolyethylene glycol). In some embodiments, DMG-PEG is DMG-PEG2000. In some such embodiments of LP01-based LNP, the steroid system components include cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0203] In some embodiments, the structural components of LNP are present in the following relative molar amounts (provided in parentheses after the component): LP01 (41-49), neutral / helper lipid components (6.5-11.5), steroid system components (40-48), and PEGylated lipid components (1.25-2.75). In some such embodiments, the neutral / helper lipid components include DSPC. In some such embodiments of LP01-based LNP, the steroid system components include cholesterol. In some such embodiments of LP01-based LNP, the PEGylated lipid components include PEGylated myristoyl diglycerides (DMG-PEG, 1,2-dimiristoyl-sn-glycero-3-methoxypolyethylene glycol). In some embodiments, DMG-PEG is DMG-PEG2000. In some such embodiments of LP01-based LNP, the steroid system components include cholesterol and glucocorticoid steroids. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0204] In some embodiments, the structural components of LNP are present in the following relative molar amounts (provided in parentheses after the component): LP01 (42-48), neutral / helper lipid components (7-11), steroid system components (41-47), and PEGylated lipid components (1.5-2.5). In some such embodiments, the neutral / helper lipid components include DSPC. In some such embodiments of LP01-based LNP, the steroid system components include cholesterol. In some such embodiments of LP01-based LNP, the PEGylated lipid components include PEGylated myristoyl diglycerides (DMG-PEG, 1,2-dimiristoyl-sn-glycero-3-methoxypolyethylene glycol). In some embodiments, DMG-PEG is DMG-PEG2000. In some such embodiments of LP01-based LNP, the steroid system components include cholesterol and glucocorticoid steroids. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0205] In some embodiments, the structural components of LNP are present in the following relative molar amounts (provided in parentheses after the component): LP01 (43-47), neutral / helper lipid components (7.5-10.5), steroid system components (42-46), and PEGylated lipid components (1.5-2.5). In some such embodiments, the neutral / helper lipid components include DSPC. In some such embodiments of LP01-based LNP, the steroid system components include cholesterol. In some such embodiments of LP01-based LNP, the PEGylated lipid components include PEGylated myristoyl diglycerides (DMG-PEG, 1,2-dimiristoyl-sn-glycero-3-methoxypolyethylene glycol). In some embodiments, DMG-PEG is DMG-PEG2000. In some such embodiments of LP01-based LNP, the steroid system components include cholesterol and glucocorticoid steroids. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0206] In some embodiments, the structural components of LNP are present in the following relative molar amounts (provided in parentheses after the component): LP01 (44-46), neutral / helper lipid components (8-10), steroid system components (43-45), and PEGylated lipid components (1.5-2.5). In some such embodiments, the neutral / helper lipid components include DSPC. In some such embodiments of LP01-based LNP, the steroid system components include cholesterol. In some such embodiments of LP01-based LNP, the PEGylated lipid components include PEGylated myristoyl diglycerides (DMG-PEG, 1,2-dimiristoyl-sn-glycero-3-methoxypolyethylene glycol). In some embodiments, DMG-PEG is DMG-PEG2000. In some such embodiments of LP01-based LNP, the steroid system components include cholesterol and glucocorticoid steroids. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0207] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parentheses after the component): LP01 (45), neutral / helper lipid components (9), steroid system components (44), and PEGylated lipid components (2). In some such embodiments, the neutral / helper lipid components include DSPC. In some such embodiments of the LP01-based LNP, the steroid system components include cholesterol. In some such embodiments of the LP01-based LNP, the PEGylated lipid components include PEGylated myristoyl diglyceride (DMG-PEG, 1,2-dimiristoyl-sn-glycero-3-methoxypolyethylene glycol). In some embodiments, DMG-PEG is DMG-PEG2000. In some such embodiments of the LP01-based LNP, the steroid system components include cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0208] Exemplary SM-102 LNP preparation In some embodiments, a composition is provided comprising lipid nanoparticles (LNPs) comprising (i) an RNA guide nuclease or a nucleic acid encoding an RNA guide nuclease, and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA that targets a gene, wherein the LNPs include SM-102.

[0209] In some embodiments, LNP consists of the following structural components: SM-102 ionic lipids, neutral / helper lipid components, steroid system components, and PEGylated lipid components. In some such embodiments of SM-102-based LNP, the neutral / helper lipid components include DSPC. In some such embodiments of SM-102-based LNP, the steroid system components include cholesterol. In some such embodiments of SM-102-based LNP, the PEGylated lipid components include PEGylated myristoyl diglycerides (DMG-PEG, 1,2-dimiristoyl-sn-glycero-3-methoxypolyethylene glycol). In some embodiments, DMG-PEG is DMG-PEG2000. In some such embodiments of SM-102-based LNP, the steroid system components include cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0210] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parentheses after the component): SM-102 (45-55), neutral / helper lipid components (7-13), steroid system components (33.5-43.5), and PEGylated lipid components (0.5-2.5). In some such embodiments, the neutral / helper lipid components include DSPC. In some such embodiments of the SM-102-based LNP, the steroid system components include cholesterol. In some such embodiments of the SM-102-based LNP, the PEGylated lipid components include PEGylated myristoyl diglycerides (DMG-PEG, 1,2-dimiristoyl-sn-glycero-3-methoxypolyethylene glycol). In some embodiments, DMG-PEG is DMG-PEG2000. In some such embodiments of the SM-102-based LNP, the steroid system components include cholesterol and glucocorticoid steroids. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0211] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parentheses after the component): SM-102 (46-54), neutral / helper lipid components (7.5-12.5), steroid system components (34.5-42.5), and PEGylated lipid components (0.75-2.25). In some such embodiments, the neutral / helper lipid components include DSPC. In some such embodiments of the SM-102-based LNP, the steroid system components include cholesterol. In some such embodiments of the SM-102-based LNP, the PEGylated lipid components include PEGylated myristoyl diglycerides (DMG-PEG, 1,2-dimiristoyl-sn-glycero-3-methoxypolyethylene glycol). In some embodiments, DMG-PEG is DMG-PEG2000. In some such embodiments of the SM-102-based LNP, the steroid system components include cholesterol and glucocorticoid steroids. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0212] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parentheses after the component): SM-102 (47-53), neutral / helper lipid components (8-12), steroid system components (35.5-41.5), and PEGylated lipid components (1-2). In some such embodiments, the neutral / helper lipid components include DSPC. In some such embodiments of the SM-102-based LNP, the steroid system components include cholesterol. In some such embodiments of the SM-102-based LNP, the PEGylated lipid components include PEGylated myristoyl diglycerides (DMG-PEG, 1,2-dimiristoyl-sn-glycero-3-methoxypolyethylene glycol). In some embodiments, DMG-PEG is DMG-PEG2000. In some such embodiments of the SM-102-based LNP, the steroid system components include cholesterol and glucocorticoid steroids. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0213] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parentheses after the component): SM-102 (48-52), neutral / helper lipid components (8.5-11.5), steroid system components (36.5-40.5), and PEGylated lipid components (1-2). In some such embodiments, the neutral / helper lipid components include DSPC. In some such embodiments of the SM-102-based LNP, the steroid system components include cholesterol. In some such embodiments of the SM-102-based LNP, the PEGylated lipid components include PEGylated myristoyl diglycerides (DMG-PEG, 1,2-dimiristoyl-sn-glycero-3-methoxypolyethylene glycol). In some embodiments, DMG-PEG is DMG-PEG2000. In some such embodiments of the SM-102-based LNP, the steroid system components include cholesterol and glucocorticoid steroids. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0214] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parentheses after the component): SM-102 (49-51), neutral / helper lipid components (9-11), steroid system components (37.5-39.5), and PEGylated lipid components (1-2). In some such embodiments, the neutral / helper lipid components include DSPC. In some such embodiments of the SM-102-based LNP, the steroid system components include cholesterol. In some such embodiments of the SM-102-based LNP, the PEGylated lipid components include PEGylated myristoyl diglycerides (DMG-PEG, 1,2-dimiristoyl-sn-glycero-3-methoxypolyethylene glycol). In some embodiments, DMG-PEG is DMG-PEG2000. In some such embodiments of the SM-102-based LNP, the steroid system components include cholesterol and glucocorticoid steroids. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0215] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parentheses after the component): SM-102 (50), neutral / helper lipid component (10), steroid system component (38.5), and PEGylated lipid component (1.5). In some such embodiments, the neutral / helper lipid component includes DSPC. In some such embodiments of the SM-102-based LNP, the steroid system component includes cholesterol. In some such embodiments of the SM-102-based LNP, the PEGylated lipid component includes PEGylated myristoyl diglyceride (DMG-PEG, 1,2-dimiristoyl-sn-glycero-3-methoxypolyethylene glycol). In some embodiments, DMG-PEG is DMG-PEG2000. In some such embodiments of the SM-102-based LNP, the steroid system component includes cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0216] Exemplary ALC0315 LNP formulation In some embodiments, a composition is provided comprising lipid nanoparticles (LNPs) comprising (i) an RNA guide nuclease or a nucleic acid encoding an RNA guide nuclease, and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA that targets a gene, wherein the LNPs comprise ALC0315.

[0217] In some embodiments, the LNP comprises the following structural components: ALC0315 ionic lipids, neutral / helper lipid components, steroid system components, and PEGylated lipid components. In some such embodiments of the ALC0315-based LNP, the neutral / helper lipid components include DSPC. In some such embodiments of the ALC0315-based LNP, the steroid system components include cholesterol. In some such embodiments of the ALC0315-based LNP, the PEGylated lipid components include PEGylated myristoyl diglyceride (DMG-PEG, 1,2-dimiristoyl-sn-glycero-3-methoxypolyethylene glycol). In some embodiments, DMG-PEG is DMG-PEG2000. In some such embodiments of the ALC0315-based LNP, the steroid system components include cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0218] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parentheses after the component): ALC0315 (45-55), neutral / helper lipid components (7-13), steroid system components (33.5-43.5), and PEGylated lipid components (0.5-2.5). In some such embodiments, the neutral / helper lipid components include DSPC. In some such embodiments of the ALC0315-based LNP, the steroid system components include cholesterol. In some such embodiments of the ALC0315-based LNP, the PEGylated lipid components include PEGylated myristoyl diglycerides (DMG-PEG, 1,2-dimiristoyl-sn-glycero-3-methoxypolyethylene glycol). In some embodiments, DMG-PEG is DMG-PEG2000. In some such embodiments of the ALC0315-based LNP, the steroid system components include cholesterol and glucocorticoid steroids. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0219] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parentheses after the component): ALC0315 (46-54), neutral / helper lipid components (7.5-12.5), steroid system components (34.5-42.5), and PEGylated lipid components (0.75-2.25). In some such embodiments, the neutral / helper lipid components include DSPC. In some such embodiments of the ALC0315-based LNP, the steroid system components include cholesterol. In some such embodiments of the ALC0315-based LNP, the PEGylated lipid components include PEGylated myristoyl diglycerides (DMG-PEG, 1,2-dimiristoyl-sn-glycero-3-methoxypolyethylene glycol). In some embodiments, DMG-PEG is DMG-PEG2000. In some such embodiments of the ALC0315-based LNP, the steroid system components include cholesterol and glucocorticoid steroids. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0220] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parentheses after the component): ALC0315 (47-53), neutral / helper lipid components (8-12), steroid system components (35.5-41.5), and PEGylated lipid components (1-2). In some such embodiments, the neutral / helper lipid components include DSPC. In some such embodiments of the ALC0315-based LNP, the steroid system components include cholesterol. In some such embodiments of the ALC0315-based LNP, the PEGylated lipid components include PEGylated myristoyl diglycerides (DMG-PEG, 1,2-dimiristoyl-sn-glycero-3-methoxypolyethylene glycol). In some embodiments, DMG-PEG is DMG-PEG2000. In some such embodiments of the ALC0315-based LNP, the steroid system components include cholesterol and glucocorticoid steroids. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0221] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parentheses after the component): ALC0315 (48 - 52), neutral / helper lipid component (8.5 - 11.5), steroid system component (36.5 - 40.5), and PEGylated lipid component (1 - 2). In some such embodiments, the neutral / helper lipid component comprises DSPC. In some such embodiments of ALC0315-based LNPs, the steroid system component comprises cholesterol. In some such embodiments of ALC0315-based LNPs, the PEGylated lipid component comprises PEGylated myristoyl diglyceride (DMG-PEG, 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol). In some embodiments, the DMG-PEG is DMG-PEG2000. In some such embodiments of ALC0315-based LNPs, the steroid system component comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0222] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parentheses after the component): ALC0315 (49-51), neutral / helper lipid components (9-11), steroid system components (37.5-39.5), and PEGylated lipid components (1-2). In some such embodiments, the neutral / helper lipid components include DSPC. In some such embodiments of the ALC0315-based LNP, the steroid system components include cholesterol. In some such embodiments of the ALC0315-based LNP, the PEGylated lipid components include PEGylated myristoyl diglycerides (DMG-PEG, 1,2-dimiristoyl-sn-glycero-3-methoxypolyethylene glycol). In some embodiments, DMG-PEG is DMG-PEG2000. In some such embodiments of the ALC0315-based LNP, the steroid system components include cholesterol and glucocorticoid steroids. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0223] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parentheses after the component): ALC0315 (50), neutral / helper lipid component (10), steroid system component (38.5), and PEGylated lipid component (1.5). In some such embodiments, the neutral / helper lipid component includes DSPC. In some such embodiments of ALC0315-based LNP, the steroid system component includes cholesterol. In some such embodiments of ALC0315-based LNP, the PEGylated lipid component includes PEGylated myristoyl diglyceride (DMG-PEG, 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol). In some embodiments, the DMG-PEG is DMG-PEG2000. In some such embodiments of ALC0315-based LNP, the steroid system component includes cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0224] C. Virus-like particles In one aspect, the disclosure includes means for delivering a CRISPR gene editing system to mammalian cells via virus-like particles (VLPs). In some embodiments, the CRISPR gene editing system is delivered by VLPs. Without wishing to be bound by any particular theory, in certain embodiments, the nucleic acid is resistant to nuclease degradation in aqueous solution when present in the particle. In other embodiments, the protein is protected from proteolytic degradation while present in the particle. In some embodiments, the proteins and nucleic acids encapsulated by VLPs can penetrate the cell plasma membrane.

[0225] 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 comprises guide RNA. In some embodiments, the one or more RNA-containing compositions comprises mRNA. In some embodiments, the one or more RNA-containing compositions comprises RNPs (e.g., Cas9 and guide RNA).

[0226] In some embodiments, the CRISPR gene editing system comprises (i) an RNA guide nuclease or a nucleic acid encoding an RNA guide nuclease, and (ii) one or more virus-like particles that collectively encapsulate at least one guide RNA or a nucleic acid encoding at least one guide RNA. In some embodiments, the one or more virus-like particles comprises a first group of virus-like particles that encapsulate an RNA guide nuclease or a nucleic acid encoding an RNA guide nuclease, and a second group of virus-like particles that encapsulate at least one guide RNA or a nucleic acid encoding at least one guide RNA.

[0227] In one embodiment, the use of a 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 disorders, conditions, and diseases, including but not limited to Loeys-Dietz syndrome, osteoarthritis, Marfan syndrome, aortic aneurysms (e.g., familial thoracic aortic aneurysms), craniofacial malformations, and combinations thereof. In other embodiments, the use of the system treats neoplastic disorders, conditions, and diseases, including pancreatic cancer, multiple spontaneously resolving 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.

[0228] D. Other delivery methods 1. Liposomes In some embodiments, nucleic acids (e.g., Cas9 or gRNA) encoding a CRISPR gene editing system that targets genes selected from TGFB1, TGFBR1, TGFBR2, and combinations thereof are encapsulated in positively charged liposomes (e.g., lipofectin) on their surface, which may be tagged with antibodies against cell surface antigens of the target cells. These delivery vehicles may also be used to deliver the Cas9 protein / gRNA complex.

[0229] 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 comprises guide RNA. In some embodiments, the one or more RNA-containing compositions comprises mRNA. In some embodiments, the one or more RNA-containing compositions comprises RNPs (e.g., Cas9 and guide RNA).

[0230] In some embodiments, the composition comprises (i) an RNA guide nuclease or a nucleic acid encoding an RNA guide nuclease, and (ii) one or more liposomes that collectively encapsulate 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 group of liposomes that encapsulate an RNA guide nuclease or a nucleic acid encoding an RNA guide nuclease, and a second group of liposomes that encapsulate at least one guide RNA or a nucleic acid encoding at least one guide RNA.

[0231] In one embodiment, the use of a 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 disorders, conditions, and diseases, including but not limited to Loeys-Dietz syndrome, osteoarthritis, Marfan syndrome, aortic aneurysms (e.g., familial thoracic aortic aneurysms), craniofacial malformations, and combinations thereof. In other embodiments, the use of the system treats neoplastic disorders, conditions, and diseases, including pancreatic cancer, multiple spontaneously resolving 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.

[0232] 2. Lipid nanocrystals (LNCs) In one embodiment, the disclosure comprises means for delivering a CRISPR gene editing system to mammalian cells via lipid nanocrystals (LNCs). In some embodiments, the CRISPR gene editing system is delivered by LNCs. While we do not wish to be bound by any particular theory, in some embodiments, nucleic acids are resistant to nuclease degradation in aqueous solution 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 the cell plasma membrane.

[0233] 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 comprises guide RNA. In some embodiments, the one or more RNA-containing compositions comprises mRNA. In some embodiments, the one or more RNA-containing compositions comprises RNPs (e.g., Cas9 and guide RNA). In some embodiments, the one or more nanocrystals are lipid nanocrystals (LNCs).

[0234] In some embodiments, the CRISPR gene editing system includes (i) an RNA guide nuclease or a nucleic acid encoding an RNA guide nuclease, and (ii) one or more LNCs that collectively encapsulate at least one guide RNA or a nucleic acid encoding at least one guide RNA. In some embodiments, one or more LNCs include a first group of LNCs that encapsulate an RNA guide nuclease or a nucleic acid encoding an RNA guide nuclease, and a second group of LNCs that encapsulate at least one guide RNA or a nucleic acid encoding at least one guide RNA.

[0235] In one embodiment, the use of a 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 disorders, conditions, and diseases, including but not limited to Loeys-Dietz syndrome, osteoarthritis, Marfan syndrome, aortic aneurysms (e.g., familial thoracic aortic aneurysms), craniofacial malformations, and combinations thereof. In other embodiments, the use of the system treats neoplastic disorders, conditions, and diseases, including pancreatic cancer, multiple spontaneously resolving 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.

[0236] VI. Pharmaceutical Compositions In one embodiment, the disclosure encompasses a pharmaceutical composition comprising a CRISPR gene editing system for the treatment of mammals in need of treatment. In some embodiments, the CRISPR gene editing system targets genes selected from TGFB1, TGFBR1, TGFBR2, and combinations thereof. In some embodiments, the mammals are selected from humans, dogs, horses, and cats.

[0237] A.TGFB1 In some embodiments, a pharmaceutical composition comprising a 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. In some embodiments, the CRISPR gene editing system targeting TGFB1 is delivered to mammalian cells via polymer nanoparticles. For a review of polymer nanoparticles, see Zielinska A, et al., "Polymeric Nanoparticles: Production, Characterization, Toxicology and Ecotoxicology", Molecules, 25(16):3731 (2020), the disclosure thereof is incorporated herein by reference in its entirety for all purposes. In some embodiments, a CRISPR gene editing system targeting TGFB1 is delivered to mammalian cells via exosomes.For a review on exosomes, please refer to Dimik M, et al., "The exosome: a review of current therapeutic roles and capabilities in human reproduction," Drug Deliv. Transl. Res., 13(2):473-502 (2023), the disclosures thereof being incorporated herein by reference in their entirety for all purposes.

[0238] In various embodiments, a pharmaceutical composition comprising a CRISPR gene editing system targeting the TGFB1 gene is used in therapeutic methods in mammals requiring treatment. In some embodiments, the method treats fibrosis and / or scarring. In some embodiments, the method treats one or more musculoskeletal disorders, conditions, and diseases, including but not limited to Loeys-Dietz syndrome, osteoarthritis, Marfan syndrome, aortic aneurysms (e.g., familial thoracic aortic aneurysms), craniofacial malformations, and combinations thereof. In other embodiments, the method treats neoplastic disorders, conditions, and diseases, including pancreatic cancer, multiple spontaneously resolving 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.

[0239] 1. Treatment of musculoskeletal disorders by targeting the transforming growth factor beta gene. In one embodiment, a method and pharmaceutical composition for treating musculoskeletal disorders using a CRISPR pharmaceutical are provided. In one embodiment, a method and pharmaceutical composition for treating musculoskeletal disorders using a CRISPR pharmaceutical composition that targets the transforming growth factor beta gene are provided. In some embodiments, the CRISPR pharmaceutical composition comprises (i) an RNA guide nuclease or a nucleic acid encoding an RNA guide 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, wherein the gRNA specifically binds to a target sequence adjacent to the protospacer-adjacent motif (PAM) sequence of the RNA guide nuclease. In some embodiments, the CRISPR pharmaceutical composition comprises (i) mRNA encoding an RNA guide nuclease, and (ii) at least one gRNA that targets the transforming growth factor beta gene. In some embodiments, the RNA guide nuclease and the mRNA encoding at least one gRNA are packaged in lipid nanoparticles (LNPs). In some embodiments, the musculoskeletal disorder is Loeys-Dietz syndrome, osteoarthritis, Marfan syndrome, aortic aneurysm (familial thoracic aneurysm 3), or craniofacial malformation, and is treated with a CRISPR pharmaceutical composition that targets the TGFB1 gene.

[0240] In some embodiments, the transforming growth factor beta receptor gene is the TGFB1 gene. In some embodiments, the TGFB1 gene is the human TGFB1 gene, and the guide RNA includes a spacer sequence selected from SEQ ID NOs: 1-198.

[0241] 2. Treatment of fibrosis and / or scarring by targeting the transforming growth factor beta gene In one aspect, methods and pharmaceutical compositions for treating fibrosis and / or scarring using CRISPR pharmaceuticals are provided. In one aspect, methods and pharmaceutical compositions for treating fibrosis and / or scarring using a CRISPR pharmaceutical composition that targets the transforming growth factor beta gene are provided. In some embodiments, the CRISPR pharmaceutical composition comprises (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease, and (ii) at least one guide RNA (gRNA) that targets the transforming growth factor beta gene or a nucleic acid encoding at least one gRNA, wherein 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 an 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 caused by knee joint fibrosis, intra-articular fibrous nodules, or epidural fibrosis and is treated with a CRISPR pharmaceutical composition that targets the TGFB1 gene. In some embodiments, the CRISPR pharmaceutical composition described herein is a postoperative treatment for preventing or reducing fibrosis and / or scarring. For example, in some embodiments, the surgery is selected from ligament reconstruction, anterior cruciate ligament (ACL) reconstruction, autologous ACL reconstruction, allograft ACL reconstruction, tendon repair, fracture repair, total knee arthroplasty (TKA), microscopic discectomy, and thereafter, the fibrosis and / or scarring is treated with a CRISPR pharmaceutical composition that targets the TGFB1 gene. In some embodiments, the fibrosis and / or scarring is a result of a condition that can be induced or exacerbated by surgery.

[0242] In some embodiments, the transforming growth factor beta receptor gene is the TGFB1 gene. In some embodiments, the TGFB1 gene is the human TGFB1 gene and the guide RNA comprises a spacer sequence selected from SEQ ID NOs: 1-198.

[0243] B. TGFBR1 In some embodiments, a pharmaceutical composition comprising a 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. In some embodiments, the CRISPR gene editing system targeting TGFBR1 is delivered to mammalian cells via polymer nanoparticles. In some embodiments, the CRISPR gene editing system targeting TGFBR1 is delivered to mammalian cells via exosomes.

[0244] In various embodiments, pharmaceutical compositions comprising a CRISPR gene editing system targeting the TGFBR1 gene are used in therapeutic methods in mammals requiring treatment. In some embodiments, the method treats fibrosis and / or scarring. In some embodiments, the method treats one or more musculoskeletal disorders, conditions, and diseases, including but not limited to Loeys-Dietz syndrome, osteoarthritis, tendon injury, Marfan syndrome, aortic aneurysm (e.g., familial thoracic triaortic aneurysm), craniofacial malformations, and combinations thereof. In other embodiments, the method treats neoplastic disorders, conditions, and diseases, including pancreatic cancer, multiple spontaneously resolving 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.

[0245] 1. Treatment of musculoskeletal disorders by targeting the transforming growth factor beta receptor 1 gene. In one embodiment, a method and pharmaceutical composition for treating musculoskeletal disorders using a CRISPR pharmaceutical are provided. In one embodiment, a method and pharmaceutical composition for treating musculoskeletal disorders using a CRISPR pharmaceutical composition that targets the transforming growth factor beta receptor gene are provided. In some embodiments, the CRISPR pharmaceutical composition comprises (i) an RNA guide nuclease or a nucleic acid encoding an RNA guide 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 1 gene, wherein the gRNA specifically binds to a target sequence adjacent to the protospacer adjacent motif (PAM) sequence of the RNA guide nuclease. In some embodiments, the CRISPR pharmaceutical composition comprises (i) mRNA encoding an RNA guide nuclease, and (ii) at least one gRNA that targets the transforming growth factor beta receptor gene. In some embodiments, the RNA guide nuclease and the mRNA encoding at least one gRNA are packaged in lipid nanoparticles (LNPs). In some embodiments, the musculoskeletal disorder is Loeys-Dietz syndrome, osteoarthritis, Marfan syndrome, aortic aneurysm (familial thoracic aneurysm 3), or craniofacial malformation, and is treated with a CRISPR pharmaceutical composition that targets the TGFBR1 or TGFBR2 gene.

[0246] In some embodiments, the transforming growth factor beta receptor gene is the TGFBR1 gene. In some embodiments, the TGFBR1 gene is the human TGFBR1 gene, and the guide RNA includes a spacer sequence selected from SEQ ID NOs. 199-320. In some embodiments, editing of the TGFBR1 gene results in the knockout of the TGFBR1 gene. In some such embodiments, the TGFBR1 gene is the human TGFBR1 gene, and the guide RNA includes a spacer 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 the human TGFBR1 gene, and the guide RNA includes a spacer sequence selected from SEQ ID NOs. 229-308 and SEQ ID NOs. 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 the human TGFBR1 gene, and the guide RNA includes a spacer sequence selected from SEQ ID NOs. 309-318.

[0247] 2. Treatment of fibrosis by targeting the transforming growth factor beta receptor 1 gene In one embodiment, a method and pharmaceutical composition for treating fibrosis and / or scarring using a CRISPR pharmaceutical is provided. In one embodiment, a method and pharmaceutical composition for treating fibrosis and / or scarring using a CRISPR pharmaceutical composition that targets the transforming growth factor beta receptor gene is provided. In some embodiments, the CRISPR pharmaceutical composition comprises (i) an RNA guide nuclease or a nucleic acid encoding an RNA guide 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, wherein the gRNA specifically binds to a target sequence adjacent to the protospacer-adjacent motif (PAM) sequence of the RNA guide nuclease. In some embodiments, the CRISPR pharmaceutical composition comprises (i) an mRNA encoding an RNA guide nuclease, and (ii) at least one gRNA that targets the transforming growth factor beta receptor gene. In some embodiments, fibrosis and / or scarring is caused by 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 compositions described herein are postoperative treatments for preventing or reducing fibrosis and / or scarring. For example, in some embodiments, the surgical procedure is selected from ligament reconstruction, anterior cruciate ligament (ACL) reconstruction, autologous ACL reconstruction, allogeneic ACL reconstruction, tendon repair, fracture repair, total knee arthroplasty (TKA), or microscopic discectomy, and subsequently, fibrosis and / or scarring is treated with a CRISPR pharmaceutical composition targeting the TGFBR1 or TGFBR2 gene. In some embodiments, fibrosis and / or scarring is a consequence of a condition that may be induced or aggravated by surgical procedures.

[0248] In some embodiments, the transforming growth factor beta receptor gene is the TGFBR1 gene. In some embodiments, the TGFBR1 gene is the human TGFBR1 gene, and the guide RNA includes a spacer sequence selected from SEQ ID NOs. 199-320. In some embodiments, editing of the TGFBR1 gene results in the knockout of the TGFBR1 gene. In some such embodiments, the TGFBR1 gene is the human TGFBR1 gene, and the guide RNA includes a spacer 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 the human TGFBR1 gene, and the guide RNA includes a spacer sequence selected from SEQ ID NOs. 229-308 and SEQ ID NOs. 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 the human TGFBR1 gene, and the guide RNA includes a spacer sequence selected from SEQ ID NOs. 309-318.

[0249] C.TGFBR2 In some embodiments, a pharmaceutical composition comprising a CRISPR gene editing system targets TGFBR2. In some embodiments, the CRISPR gene editing system targeting TGFBR2 is delivered to mammalian cells via a viral vector. In some embodiments, the CRISPR gene editing system targeting TGFBR2 is delivered to mammalian cells via an AAV vector. In some embodiments, the CRISPR gene editing system targeting TGFBR2 is delivered to mammalian cells via a lentiviral vector. In some embodiments, the CRISPR gene editing system targeting TGFBR2 is delivered to mammalian cells via lipid nanoparticles. In some embodiments, the CRISPR gene editing system targeting TGFBR2 is delivered to mammalian cells via virus-like particles. In some embodiments, the CRISPR gene editing system targeting TGFBR2 is delivered to mammalian cells via liposomes. In some embodiments, the CRISPR gene editing system targeting TGFBR2 is delivered to mammalian cells via lipid nanocrystals. In some embodiments, the CRISPR gene editing system targeting TGFBR2 is delivered to mammalian cells via polymer nanoparticles. In some embodiments, the CRISPR gene editing system targeting TGFBR2 is delivered to mammalian cells via exosomes.

[0250] In various embodiments, pharmaceutical compositions comprising a CRISPR gene editing system targeting the TGFBR2 gene are used in therapeutic methods in mammals requiring treatment. In some embodiments, the methods treat fibrosis and / or scarring. In some embodiments, the methods treat one or more musculoskeletal disorders, conditions, and diseases, including but not limited to Loeys-Dietz syndrome, osteoarthritis, Marfan syndrome, aortic aneurysms (e.g., familial thoracic aortic aneurysms), craniofacial malformations, and combinations thereof. In other embodiments, the methods treat neoplastic disorders, conditions, and diseases, including pancreatic cancer, multiple spontaneously resolving 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.

[0251] 1. Treatment of musculoskeletal disorders by targeting the transforming growth factor beta receptor 2 gene. In one embodiment, a method and pharmaceutical composition for treating musculoskeletal disorders using a CRISPR pharmaceutical are provided. In one embodiment, a method and pharmaceutical composition for treating musculoskeletal disorders using a CRISPR pharmaceutical composition that targets a transforming growth factor beta receptor gene are provided. In some embodiments, the CRISPR pharmaceutical composition comprises (i) an RNA guide nuclease or a nucleic acid encoding an RNA guide 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, wherein the gRNA specifically binds to a target sequence adjacent to the protospacer-adjacent motif (PAM) sequence of the RNA guide nuclease. In some embodiments, the CRISPR pharmaceutical composition comprises (i) mRNA encoding an RNA guide nuclease, and (ii) at least one gRNA that targets the transforming growth factor beta receptor gene. In some embodiments, the RNA guide nuclease and the mRNA encoding at least one gRNA are packaged in lipid nanoparticles (LNPs). In some embodiments, the musculoskeletal disorder is Loeys-Dietz syndrome, osteoarthritis, Marfan syndrome, aortic aneurysm (familial thoracic aneurysm 3), or craniofacial malformation, and is treated with a CRISPR pharmaceutical composition that targets the TGFBR1 or TGFBR2 gene.

[0252] 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 includes a spacer sequence selected from SEQ ID NOs. 321-519. In some embodiments, editing of the TGFBR2 gene results in the knockout of the TGFBR2 gene. In some such embodiments, the TGFBR2 gene is the human TGFBR2 gene, and the guide RNA includes a spacer 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 includes a spacer 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 the human TGFBR2 gene, and the guide RNA includes a spacer sequence selected from sequence numbers 499-508.

[0253] 2. Treatment of fibrosis and / or scarring by targeting the transforming growth factor beta receptor 2 gene In one embodiment, a method and pharmaceutical composition for treating fibrosis and / or scarring using a CRISPR pharmaceutical is provided. In one embodiment, a method and pharmaceutical composition for treating fibrosis and / or scarring using a CRISPR pharmaceutical composition that targets the transforming growth factor beta receptor gene is provided. In some embodiments, the CRISPR pharmaceutical composition comprises (i) an RNA guide nuclease or a nucleic acid encoding an RNA guide 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, wherein the gRNA specifically binds to a target sequence adjacent to the protospacer-adjacent motif (PAM) sequence of the RNA guide nuclease. In some embodiments, the CRISPR pharmaceutical composition comprises (i) an mRNA encoding an RNA guide nuclease, and (ii) at least one gRNA that targets the transforming growth factor beta receptor gene. In some embodiments, fibrosis and / or scarring is caused by 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 compositions described herein are postoperative treatments for preventing or reducing fibrosis and / or scarring. For example, in some embodiments, the surgical procedure is selected from ligament reconstruction, anterior cruciate ligament (ACL) reconstruction, autologous ACL reconstruction, allogeneic ACL reconstruction, tendon repair, fracture repair, total knee arthroplasty (TKA), or microscopic discectomy, and subsequently, fibrosis and / or scarring is treated with a CRISPR pharmaceutical composition targeting the TGFBR1 or TGFBR2 gene. In some embodiments, fibrosis and / or scarring is a consequence of a condition that may be induced or aggravated by surgical procedures.

[0254] 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 includes a spacer sequence selected from SEQ ID NOs. 321-519. In some embodiments, editing of the TGFBR2 gene results in the knockout of the TGFBR2 gene. In some such embodiments, the TGFBR2 gene is the human TGFBR2 gene, and the guide RNA includes a spacer 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 includes a spacer 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 the human TGFBR2 gene, and the guide RNA includes a spacer sequence selected from sequence numbers 499-508.

[0255] VII. Route of administration The methods and compositions described herein include the use of pharmaceutical compositions comprising a CRISPR gene editing system as an active ingredient.

[0256] Depending on the method / route of administration, pharmaceutical dosage forms are available 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, as well as 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. Liquid pharmaceutical dosage forms are liquid forms of doses of compounds used as drugs or pharmaceuticals intended for administration or consumption.

[0257] As described below, the compositions of the present disclosure may be delivered to a target subcutaneously (e.g., by intra-articular or intra-discal injection), through the skin (e.g., percutaneously 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 dissolved tablets (ODTs), gel capsules, thin films, adhesive topical patches, lollipops, lozenges, chewing gum, dry powder inhalers (DPIs), vaporizers, nebulizers, medium-dose inhalers (MDIs), ointments, percutaneous patches, and intradermal implants.

[0258] As used herein, “cutaneous delivery” or “cutaneous administration” may refer to a route of administration in which a pharmaceutical dosage form is delivered to or through the dermis (i.e., the layers of skin between the epidermis (which make up the skin) and the subcutaneous tissue). “Subcutaneous delivery” may refer to a route of administration in which a pharmaceutical dosage form is delivered to or beneath the subcutaneous tissue layer.

[0259] 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, a solution or suspension used for parenteral, intradermal, or subcutaneous application may contain the following components: sterile diluents such as water for injection, physiological saline, fixative oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; and agents for adjusting toxicity such as sodium chloride or glucose. The pH may be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be sealed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0260] Pharmaceutical compositions suitable for injectable use may include sterile aqueous solutions (water-soluble) or dispersions and sterile powders for the immediate 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 fluid enough to allow for injection. It must be stable under manufacturing and storage conditions and stored in a manner resistant to contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it is preferable to include isotonic agents in the composition, such as sugars, mannitol, sorbitol, and polyalcohols such as sodium chloride. Long-term absorption of the injectable composition can be achieved by including absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition.

[0261] Pharmaceutical compositions suitable for injectable use may also contain cryopreservatives, which improve the stability of the formulation, particularly when frozen. For further consideration of conditions useful for storing LNPs, see Kim B, et al., "Optimization of storage conditions for lipid nanoparticle-formulated self-replicating RNA vaccine," J. Control Release, 353:241-53 (2023), the disclosure thereof, which is incorporated herein by reference in its entirety for all purposes.

[0262] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in a suitable solvent using one or a combination of the components listed above, and subsequently sterilizing by filtration as necessary. Generally, dispersions are prepared by incorporating the active compound, which contains a basic dispersion medium and other necessary components from those listed above, into a sterile vehicle. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying, which are obtained by adding any additional desired components from the previously sterile filtered solution of the active ingredient to the powder.

[0263] Nucleic acids, or therapeutic compounds containing nucleic acids, may be administered by any method suitable for administering nucleic acid agents, such as DNA vaccines. These methods include gene guns, bioinjectors, and skin patches, as well as needle-free methods such as mammalian percutaneous needle-free vaccination with particulate DNA vaccine technology disclosed in U.S. Patent No. 6,194,389 and powder-form vaccines disclosed in U.S. Patent 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., described in U.S. Patent No. 6,472,375) and microencapsulation may also be used. Biodegradable targetable particulate delivery systems may also be used (e.g., described in U.S. Patent No. 6,471,996).

[0264] Therapeutic compounds may be prepared using carriers that protect the therapeutic compounds from rapid elimination from the body, such as sustained-release formulations, including implants and microencapsulated delivery systems. Biodegradable and biocompatible polymers 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 may be used. Such formulations may be prepared using standard techniques or may be commercially obtained, for example, from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (containing liposomes targeted to selected cells having monoclonal antibodies against cellular antigens) may also be used as pharmaceutically acceptable carriers. These can 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, gelled, softgel, or other formulations (including sustained-release) may be used, for example, when administration to a surgical site is desired. Methods for preparing such formulations are known in the art and may involve the use of biodegradable, biocompatible polymers. See, for example, Sawyer et al., Yale J Biol Med. 2006 December, 79(3-4): 141-152.

[0265] The pharmaceutical compositions described herein may be included in a container, kit, pack, or dispenser, along with instructions for administration.

[0266] A. Systemic administration In some embodiments, the pharmaceutical composition containing the CRISPR gene editing system is administered systemically to a mammal requiring it. 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.

[0267] B. Local administration In some embodiments, a pharmaceutical composition containing a CRISPR gene editing system is administered topically to a mammal requiring it. In some embodiments, the topical administration is 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 administration to the facet joints of the spine.

[0268] In some embodiments, a pharmaceutical composition containing a CRISPR gene editing system is administered topically to a mammal requiring it during surgery. In some embodiments, a pharmaceutical composition containing a CRISPR gene editing system is administered topically to a mammal requiring it 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or 30 days after surgery.

[0269] 1. Administration after musculoskeletal trauma In some embodiments, the compositions and methods disclosed herein are administered to a subject after musculoskeletal trauma. In some embodiments, the musculoskeletal trauma is the result of surgery (i.e., surgical incision). In some embodiments, the musculoskeletal trauma is a wound.

[0270] In some embodiments, the composition containing RNA guide nuclease and 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.

[0271] Additional exemplary compositions to which RNA guide nucleases and sgRNAs are applied to wounds or incisions include (each of which is incorporated herein by reference in whole for all purposes): U.S. Patent Nos. 8,574,627, 9,204,953, 9,272,073, 9592,324, 9597,426, 9993,298, 1,0471,181, 1,0765,423, 1,0835,235, 1,133,8062, 1,1426,156, and U.S. Patent Applications Nos. 2008 / 0039877A1, 2020 / 0345366A1, and 2020 / 0069478A1.

[0272] In some embodiments, the Disclosure provides pharmaceutical compositions for treating or preventing disorders having symptoms at least partially caused by intercellular signaling mediated through the transforming growth factor beta (TGFβ) signaling pathway, the compositions comprising (i) an RNA guide nuclease or a nucleic acid encoding an RNA guide nuclease, and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA that targets the TGFB1 gene, the TGFBR1 gene, the TGFBR2 gene, or a combination thereof.

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

[0274] In some embodiments, the fibrosis is postoperative fibrosis.

[0275] In some embodiments, the fibrosis is post-ligament reconstruction fibrosis.

[0276] In some embodiments, fibrosis is fibrosis after anterior cruciate ligament (ACL) reconstruction, fibrosis after autologous ACL reconstruction, fibrosis after allogeneic ACL reconstruction, or fibrosis after tendon repair.

[0277] In some embodiments, the fibrosis is knee joint fibrosis.

[0278] In some embodiments, fibrosis is caused by intra-articular fibrous nodules.

[0279] In some embodiments, fibrosis occurs after artificial joint replacement surgery.

[0280] In some embodiments, fibrosis occurs after total knee arthroplasty (TKA).

[0281] In some embodiments, fibrosis is associated with tendon injury or surgery.

[0282] In some embodiments, the fibrosis is post-fracture repair fibrosis.

[0283] In some embodiments, the fibrosis is fibrosis following microscopic discectomy.

[0284] In some embodiments, the fibrosis is epidural fibrosis following microscopic discectomy.

[0285] In some embodiments, the fibrosis is post-lumbar laminectomy fibrosis.

[0286] In some embodiments, the fibrosis is epidural fibrosis following lumbar laminectomy.

[0287] In some embodiments, the fibrosis is post-anterior acromionoplasty fibrosis.

[0288] In some embodiments, the fibrosis is post-subacromial decompression fibrosis.

[0289] In some embodiments, the fibrosis is soft tissue fibrosis following anterior acromionoplasty.

[0290] In some embodiments, fibrosis is joint contracture after anterior acromionoplasty.

[0291] In some embodiments, the fibrosis is post-arthroscopic fibrosis.

[0292] In some embodiments, the fibrosis is arthroscopic fibrosis.

[0293] In some embodiments, the fibrosis is fibrosis of the trabecular network.

[0294] In some embodiments, fibrosis of the trabecular network is fibrosis following glaucoma surgery.

[0295] In some embodiments, the fibrosis is epidural fibrosis.

[0296] In some embodiments, fibrosis is atrial fibrosis, cardiac fibrosis, myocardial fibrosis, and / or post-thoracotomy fibrosis.

[0297] In some embodiments, the fibrosis is post-liposuction fibrosis.

[0298] In some embodiments, the fibrosis is idiopathic pulmonary fibrosis (IPF).

[0299] In some embodiments, the fibrosis is fibrosis of the knee, shoulder, or elbow joint.

[0300] In some embodiments, the disorder includes fibrosis in the kidney tissue.

[0301] In some embodiments, the disorder is chronic kidney disease.

[0302] In some embodiments, the disorder includes fibrosis in the skin tissue.

[0303] In some embodiments, the disorder is post-wound scarring, keloid disorder, nephrogenic systemic fibrosis, or scleroderma / systemic sclerosis.

[0304] In some embodiments, the disorder includes fibrosis in the lung tissue.

[0305] In some embodiments, the disorder is fibrothorax, pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, radiation-induced lung injury, progressive widespread fibrosis, or scleroderma / systemic sclerosis.

[0306] In some embodiments, the disorder includes fibrosis in liver tissue.

[0307] In some embodiments, the disorder is cirrhosis or bridging fibrosis.

[0308] In some embodiments, the disorder includes fibrosis in cardiac tissue.

[0309] In some embodiments, the fibrosis is interstitial fibrosis.

[0310] In some embodiments, the disorder is congestive heart failure or hypertension.

[0311] In some embodiments, the fibrosis is replacement fibrosis.

[0312] In some embodiments, the disorder is a myocardial infarction.

[0313] In some embodiments, the disorder includes fibrosis in brain tissue.

[0314] In some embodiments, fibrosis is glial scarring.

[0315] In some embodiments, the disorder includes fibrosis in the intestinal tissue.

[0316] In some embodiments, the disorder is Crohn's disease.

[0317] In some embodiments, the disorder includes fibrosis of the hand or fingers.

[0318] In some embodiments, the disorder is Dupuytren's contracture.

[0319] In some embodiments, the disorder includes fibrosis in the lymphatic tissue.

[0320] In some embodiments, the fibrosis is mediastinal fibrosis.

[0321] In some embodiments, the disorder includes fibrosis in the bone marrow tissue.

[0322] In some embodiments, fibrosis is myelofibrosis.

[0323] In some embodiments, the disorder includes fibrosis in the penile tissue.

[0324] In some embodiments, the disorder is Peyronie's disease.

[0325] In some embodiments, the disorder includes fibrosis in the soft tissues of the retroperitoneum.

[0326] In some embodiments, the fibrosis is retroperitoneal fibrosis.

[0327] In some embodiments, the disorder includes fibrosis in the musculoskeletal tissue.

[0328] In some embodiments, the disorder is a musculoskeletal disorder.

[0329] In some embodiments, the musculoskeletal disorder is a hereditary musculoskeletal disorder.

[0330] In some embodiments, the musculoskeletal disorder is muscular dystrophy.

[0331] 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).

[0332] In some embodiments, the musculoskeletal disorder is a type II collagen disorder.

[0333] In some embodiments, the musculoskeletal disorder is selected from achondroplasia type II, chondrodysplasia, Stickler syndrome, and Czech dysplasia.

[0334] In some embodiments, the musculoskeletal disorder is osteogenesis imperfecta (OI).

[0335] In some embodiments, the musculoskeletal disorder is an autoimmune musculoskeletal disease.

[0336] In some embodiments, musculoskeletal disorders are autoimmune musculoskeletal diseases associated with polygenic susceptibility traits.

[0337] In some embodiments, the musculoskeletal disorder is spondyloarthropathy (SA).

[0338] In some embodiments, the musculoskeletal disorder is ankylosing spondylitis.

[0339] In some embodiments, the musculoskeletal disorder is rheumatoid arthritis.

[0340] In some embodiments, the musculoskeletal disorder is autoimmune osteoarthritis.

[0341] In some embodiments, the musculoskeletal disorder is osteoarthritis (OA).

[0342] In some embodiments, the musculoskeletal disorder is Kamrachi-Engelmann disease.

[0343] In some embodiments, the musculoskeletal disorder is Sjögren's syndrome.

[0344] In some embodiments, the musculoskeletal disorder is a mechanical musculoskeletal injury.

[0345] In some embodiments, the musculoskeletal disorder is selected from chronic muscle injury, tendon rupture, post-traumatic osteoarthritis, and post-traumatic arthropathy.

[0346] In some embodiments, the disorder is cancer. In some embodiments, the cancer is musculoskeletal cancer. In some embodiments, the cancer is cancer involving TGF-beta or TGFBR.

[0347] In some embodiments, at least one guide RNA or a nucleic acid encoding at least one guide RNA targets the mammalian TGFB1 gene.

[0348] In some embodiments, the mammalian TGFB1 gene is the TGFB1 gene of a dog, horse, or cat.

[0349] In some embodiments, the mammalian TGFB1 gene is the human TGFB1 gene.

[0350] In some embodiments, at least one guide RNA includes a spacer sequence selected from the group consisting of sequences (sequence numbers 1 to 198) shown in Figures 1A to 1D.

[0351] In some embodiments, at least one guide RNA includes a spacer sequence selected from the group consisting of sequences shown in Figure 12A (sequences 520-527).

[0352] In some embodiments, at least one guide RNA includes a spacer sequence of OHTG03 (sequence number 522) or OHTG04 (sequence number 523).

[0353] In some embodiments, at least one guide RNA or a nucleic acid encoding at least one guide RNA targets the mammalian TGFBR1 gene.

[0354] In some embodiments, the mammalian TGFBR1 gene is the TGFBR1 gene of a dog, horse, or cat.

[0355] In some embodiments, the mammalian TGFBR1 gene is the human TGFBR1 gene.

[0356] In some embodiments, at least one guide RNA includes a spacer sequence selected from the group consisting of sequences shown in Figures 2A-2C (sequence numbers 199-320).

[0357] In some embodiments, at least one guide RNA includes a spacer sequence selected from the group consisting of sequences shown in Figure 12B (sequences 528-552).

[0358] In some embodiments, the guide RNA includes at least one spacer sequence selected from the group of sequences shown in Figure 5B.

[0359] In some embodiments, at least one guide RNA includes the spacer sequence OHTIR04 (sequence number 532) or OHTIR08 (sequence number 539).

[0360] In some embodiments, at least one guide RNA or a nucleic acid encoding at least one guide RNA targets the mammalian TGFBR2 gene.

[0361] In some embodiments, the mammalian TGFBR2 gene is the TGFBR2 gene of a dog, horse, or cat.

[0362] In some embodiments, the mammalian TGFBR2 gene is the human TGFBR2 gene.

[0363] In some embodiments, at least one guide RNA includes a spacer sequence selected from the group consisting of sequences (sequence numbers 321-519) shown in Figures 3A-3D.

[0364] In some embodiments, at least one guide RNA includes a spacer sequence selected from the group consisting of sequences shown in Figure 12C (SEQ ID NOs. 553-604).

[0365] In some embodiments, the guide RNA includes at least one spacer sequence selected from the group of sequences shown in Figure 6B.

[0366] In some embodiments, at least one guide RNA includes the spacer sequence OHTIIR04 (sequence number 563).

[0367] In some embodiments, the RNA guide nuclease or the nucleic acid encoding the RNA guide nuclease is the RNA guide nuclease.

[0368] In some embodiments, the RNA guide nuclease or the nucleic acid encoding the RNA guide nuclease is the DNA encoding the RNA guide nuclease.

[0369] In some embodiments, the RNA guide nuclease or the nucleic acid encoding the RNA guide nuclease is the mRNA encoding the RNA guide nuclease.

[0370] In some embodiments, the RNA guide nuclease is a Cas protein.

[0371] In some embodiments, the Cas protein is the Cas9 protein.

[0372] In some embodiments, the Cas9 protein is the S. pyogenes Cas9 polypeptide.

[0373] In some embodiments, the Cas9 protein is selected from the group consisting of ESCas9, HFCas9, PECas9, and ARCas9.

[0374] In some embodiments, at least one guide RNA or a nucleotide encoding at least one guide RNA is at least one guide RNA.

[0375] In some embodiments, the at least one guide RNA or the nucleic acid encoding the at least one guide RNA is the DNA encoding the at least one guide RNA.

[0376] In some embodiments, the pharmaceutical composition comprises nucleic acids encoding both an RNA guide nuclease and at least one guide RNA.

[0377] In some embodiments, at least one guide RNA is a single guide RNA (sgRNA).

[0378] In some embodiments, the composition comprises one or more viral vectors, each comprising collectively (i) an RNA guide nuclease or a nucleic acid encoding an RNA guide 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.

[0379] In some embodiments, one of the more viral vectors includes a recombinant virus selected from retroviruses, adenoviruses, adeno-associated viruses, lentiviruses, and herpes simplex virus-1.

[0380] In some embodiments, one of the multiple viral vectors includes recombinant adeno-associated virus (AAV).

[0381] In some embodiments, recombinant AAV is of serotype 5 (AAV5).

[0382] In some embodiments, recombinant AAV is of serotype 6 (AAV6).

[0383] In some embodiments, the composition comprises one or more lipid nanoparticles (LNPs) collectively comprising (i) an RNA guide nuclease or a nucleic acid encoding an RNA guide 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.

[0384] In some embodiments, one or more LNPs include a first group of LNPs encapsulating an RNA guide nuclease or a nucleic acid encoding an RNA guide nuclease, and a second group of LNPs encapsulating at least one guide RNA or a nucleic acid encoding at least one guide RNA.

[0385] In some embodiments, one or more LNPs comprise multiple LNPs that encapsulate both (i) an RNA guide nuclease or a nucleic acid encoding an RNA guide 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.

[0386] In some embodiments, one or more LNPs are 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethaneamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethaneamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), and 1,2-dilinoleyloxy-N,N-dimethylaminopropyl 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-dimethylaminopropyl Pan(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 The material contains components selected from the group consisting of -diene-1-yloxy]propan-1-amine(octyl-CLinDMA(2R)), (2S)-2-({8-[(3.beta.)-cholest-5-ene-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z-,12Z)-octadeca-9,12-diene-1-yloxy]propan-1-amine(octyl-CLinDMA(2S)), lipids containing cyclic amine groups, and mixtures thereof.

[0387] In some embodiments, LNP is 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-diu Ndecanoyl-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-difitanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 The material contains components selected from the group consisting of 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.

[0388] In some embodiments, the LNP comprises components 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-DLPE, PEG-DMPE, PEG-DPPC, PEG-DMA, PEG-DSPE lipids, and mixtures thereof.

[0389] In some embodiments, LNP comprises components selected from the group consisting of cholesterol, fecosterol, stigmasterol, stigmanol, sitosterol, β-sitosterol, lupeol, betulin, ursolic acid, oleanolic acid, campesterol, fucosterol, brassicasterol, ergosterol, 9,11-dehydroergosterol, tomatidine, tomatine, α-tocopherol, and mixtures thereof.

[0390] In some embodiments, the composition comprises one or more liposomes, each collectively comprising (i) an RNA guide nuclease or a nucleic acid encoding an RNA guide 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.

[0391] In some embodiments, one or more liposomes include a first group of liposomes encapsulating an RNA guide nuclease or a nucleic acid encoding an RNA guide nuclease, and a second group of liposomes encapsulating at least one guide RNA or a nucleic acid encoding at least one guide RNA.

[0392] In some embodiments, one or more liposomes comprise a plurality of liposomes each containing both (i) an RNA guide nuclease or a nucleic acid encoding an RNA guide 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.

[0393] In some embodiments, the composition comprises one or more virus-like particles, each collectively comprising (i) an RNA guide nuclease or a nucleic acid encoding an RNA guide 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.

[0394] In some embodiments, one or more virus-like particles include: In some embodiments, a first plurality of virus-like particles encapsulating an RNA guide nuclease or a nucleic acid encoding an RNA guide 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.

[0395] In some embodiments, one or more virus-like particles comprise a plurality of virus-like particles that encapsulate both (i) an RNA guide nuclease or a nucleic acid encoding an RNA guide 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.

[0396] In some embodiments, the composition is formulated for parenteral administration.

[0397] In some embodiments, the composition is formulated for intra-articular injection into the target joint.

[0398] In some embodiments, the composition is formulated for intradiscal injection.

[0399] In some embodiments, the composition is formulated for peridiscal injection.

[0400] In some embodiments, the composition is formulated for intraspinal injection.

[0401] In some embodiments, the Disclosure provides a method for treating or preventing free oxygen radicals in a subject requiring treatment or prevention by administering a therapeutically effective amount of a composition comprising (i) an RNA guide nuclease or a nucleic acid encoding an RNA guide nuclease, and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA that targets the TGFB1 gene, the TGFBR1 gene, the TGFBR2 gene, or a combination thereof.

[0402] In some embodiments, the Disclosure provides a method for treating or preventing a disorder having symptoms at least partially caused by intercellular signaling mediated through the transforming growth factor beta (TGFβ) signaling pathway, the method comprising administering a therapeutically effective amount of a composition, the composition comprising (i) an RNA guide nuclease or a nucleic acid encoding an RNA guide nuclease, and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA that targets the TGFB1 gene, the TGFBR1 gene, the TGFBR2 gene, or a combination thereof.

[0403] In some embodiments, the disorder is fibrosis.

[0404] In some embodiments, the fibrosis is postoperative fibrosis.

[0405] In some embodiments, the fibrosis is post-ligament reconstruction fibrosis.

[0406] In some embodiments, fibrosis is fibrosis after anterior cruciate ligament (ACL) reconstruction, fibrosis after autologous ACL reconstruction, or fibrosis after allogeneic ACL reconstruction.

[0407] In some embodiments, the fibrosis is knee joint fibrosis.

[0408] In some embodiments, fibrosis is caused by intra-articular fibrous nodules.

[0409] In some embodiments, fibrosis occurs after artificial joint replacement surgery.

[0410] In some embodiments, fibrosis occurs after total knee arthroplasty (TKA).

[0411] In some embodiments, fibrosis is associated with tendon injury or surgery.

[0412] In some embodiments, the fibrosis is post-fracture repair fibrosis.

[0413] In some embodiments, the fibrosis is fibrosis following microscopic discectomy.

[0414] In some embodiments, the fibrosis is epidural fibrosis following microscopic discectomy.

[0415] In some embodiments, the fibrosis is post-lumbar laminectomy fibrosis.

[0416] In some embodiments, the fibrosis is epidural fibrosis following lumbar laminectomy.

[0417] In some embodiments, the fibrosis is post-anterior acromionoplasty fibrosis.

[0418] In some embodiments, the fibrosis is post-subacromial decompression fibrosis.

[0419] In some embodiments, the fibrosis is soft tissue fibrosis following anterior acromionoplasty.

[0420] In some embodiments, fibrosis is joint contracture after anterior acromionoplasty.

[0421] In some embodiments, the fibrosis is post-arthroscopic fibrosis. [Examples]

[0422] VIII. Examples

[0423] Embodiments included herein will be described here with reference to the following examples. These examples are provided for illustrative purposes only, and the disclosure included herein should not be construed as being limited in any way to these examples, but rather as being construed as encompassing all possible modifications that may become apparent as a result of the teachings provided herein.

[0424] Example 1: Bioinformatics design of a guide for disrupting TGF-beta signaling We developed in silico and in vitro screening pipelines to identify candidate CRISPR-Cas guides for gene editing. The pipelines were applied to identify guides for effectively editing the human TGFB1 (hTGFB1), TGFBR1 (hTGFRB1), and TGFBR2 (hTGFRB2) genes, thereby disrupting TGF-beta signaling in vivo to treat musculoskeletal fibrosis and / or scarring. The first step in the pipeline was to identify all possible crRNA or sgRNA spacer sequences for specific CRISPR-Cas proteins within the coding portions of hTGFB1, hTGFRB1, and hTGFRB2. Many algorithms for identifying such sequences are known in the art. Generally, these algorithms work by identifying protospacer-adjacent motif (PAM) sequences for specific CRISPR-Cas proteins, and then, according to the requirements of the specific Cas protein, spaced sequences typically directly at the 5' of the PAM site. For example, the S. pyogenes Cas9 (SpCas9) protein used in this embodiment recognizes the 5'-NGG-3'PAM sequence. Therefore, all sequences directly at the 5' of the NGG trinucleotide are possible spacer sequences for crRNA or sgRNA. The spacer sequences were identified along with biometric information related to the spacer sequences in the manner shown in Figure 27.

[0425] Next, each identified spacer sequence (see Figures 1–3) was evaluated using multiple algorithms for each metric across three different metrics: potential off-target editing at genomic locations other than the target gene, on-target editing efficiency, and the potential for editing that causes frameshift mutations, as shown in Figures 4B, 5B, and 6B. The basis of the combined approach used is the assumption that all models have blind spots that can distort 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.

[0426] Off-target editing effects were predicted by averaging 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). This model is based on a positional penalty matrix (1×20) generated from a library of 15 EMX1 sgRNAs with mismatches to the target at each position. The CFD algorithm (cleavage frequency determination) is based on a threat matrix (12×20) that considers both position and mismatch type, as well as PAM integrity (27,897 'CD33' sgRNA + 10,618 negative control sgRNAs). Doench, JG et 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 on 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)). Columns labeled "Off" show the average score provided by two or three models, respectively. Columns labeled "Number of Off-Targets" show 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).

[0427] On-target editing efficiency was predicted by averaging the scores generated by the Azimuth model, the DeepSpCas9 model, and the CrispRcan model. 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, JG 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 edited 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). Columns labeled "ON" show the average score provided by the three models.

[0428] The likelihood of the estimated guide generating frameshift mutations was predicted by averaging the scores generated by the Lindel and InDelphi models. Lindel is a machine learning model trained on profiling data of 1.16 million independent mutation events induced by CRISPR / Cas9-mediated and non-homologous end-join-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, M. 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 "Precision" shows the frequency distribution of indels estimated by inDelphi. Precision is close to 100 and represents sgRNAs characterized by one or a very small number of repair results.

[0429] Next, candidate spacer sequences were evaluated for the presence of Graf motifs, TT, or GCC in the four PAM proximal bases of the spacer 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 characteristic of inefficient sgRNAs. Spacer sequences containing Graf motifs, especially GCC motifs, were avoided where possible. In contrast to TT motifs, GCC motifs remain important when sgRNAs are synthesized de novo rather than by transcription.

[0430] Next, three consensus scores were calculated for each spacer sequence, as shown in Figures 1-3, for example. 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, the Bioscore of VBCs was used to predict whether potential in-frame mutations could interfere with protein function. This is more likely to occur in conserved genomic DNA sequences encoding important protein domains. Therefore, the Bioscore is based on protein domain annotation, systematic 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 the overall score (most often above 70), a relatively low count of potential off-targets (most often below 200), the absence of Graf motifs (if possible), and the genomic cleavage site within the coding sequence for knockout, or, in the case of hTGFBR1 and hTGFBR2, for producing a cleaved protein with reduced functionality (e.g., a decoy receptor).

[0431] Finally, the CRISPR targeting domain was determined based on its nucleotide position within the gene (Figures 4A, 5A, 6A). The domain position was used to predict whether the editing would result in a wild-type-like protein (editing the most C-terminal sequence of the receptor, which is less likely to disrupt receptor-mediated signaling), complete knockout of any functional protein (editing the sequence encoding the N-terminal and / or essential functional domain), and, where applicable, a soluble decoy receptor (editing the sequence encoding the transmembrane domain of the receptor) or a membrane-bound decoy receptor (editing the sequence encoding the intracellular signaling domain of the receptor).

[0432] In fact, this in silico analysis demonstrated that several candidate sgRNAs were predicted to generate dominant-negative (DN) TGFB receptors, thereby resulting in genome editing that affects pro-inflammatory intracellular signaling. These predictive studies provide a high-throughput method for validating candidates without requiring time- and resource-intensive brute-force methods and demonstrate the feasibility of predicting gene knockout (or dominant-negative TGFB receptor) induction as a unique approach for treating musculoskeletal fibrosis and / or scarring.

[0433] Example 2: Verification of TGFB1 editing in human cells After editing TGFB1 to design and analyze numerous sgRNAs predicted to result in gene knockout by the bioinformatics method described in Example 1 (see Figure 1), the next step was to evaluate their ability to act in vitro. Of particular interest was whether one preferred edit would be observed.

[0434] To test this, an 80pmole sgRNA candidate was introduced into THP-1 human monocytes via electroporation as part of a ribonucleoprotein (i.e., pre-assembled with a 25pmole Cas9 protein for at least 5 minutes at room temperature). Briefly, approximately 400,000 cells resuspended in 20 μl of SG buffer (Lonza) were added to the pre-assembled Cas9 ribonucleoprotein complex, transferred to a 16-well Nucleocuvette, and electroporated using a 4D Nucleofector (Lonza) with pulse code FF-100. After holding the electroporated cells in SG buffer for approximately 10 minutes, 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 the editing effectiveness 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.

[0435] Several sgRNA candidates were able to induce mutations in the hTGFB1 gene with high efficiency, some showing frequencies of over 50%, and only one candidate, OHTG03 (SEQ ID NO: 682), showed the ability to introduce the same editing in a high percentage of cells when paired with either wild-type (WT) Cas9 or high-fidelity AR-Cas9 (IDT, catalog number IDT, catalog number 1081060, Vaclskas, 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.

[0436] Example 3: Verification of TGFBR1 editing in human cells After editing the TGFBR1 receptor and designing and analyzing a number of sgRNAs predicted to produce either knockout or (soluble or membrane-bound) dominant-negative decoys by the bioinformatics method described in Example 1 (see Figure 2), the next step was to evaluate their ability to act in vitro. Of particular interest was whether a preferred on-target edit was observed in any of the candidates.

[0437] To test this, candidate sgRNAs were complexed with Cas9 nuclease to form ribonucleoprotein (RNP) complexes, and then human THP-1 monocytes were electroporated to introduce RNP into the cells as described above. Editing efficacy was then measured via Sanger sequencing. Several candidates were able to induce mutations in the hTGFBR1 gene with high efficiency, some showing a frequency of over 50% with WT-Cas9 (Figure 8A), and this efficacy was universally reduced with the use of AR-Cas9 (Figure 8B). These results generally confirm the in-silico analysis and suggest that several candidate sgRNAs, including OHTIR04, were able to efficiently edit TGFBR1 with some frequency in human cells.

[0438] Example 4: Verification of TGFBR2 editing in human cells After editing the TGFBR2 receptor and designing and analyzing a number of sgRNAs predicted to produce either knockout or (soluble or membrane-bound) dominant-negative decoys by the bioinformatics method described in Example 1 (see Figure 3), the next step was to evaluate their ability to act in vitro. Of particular interest was whether one preferred edit was observed in any of the candidates.

[0439] To test this, candidate sgRNAs were complexed with Cas9 nuclease to form ribonucleoprotein (RNP) complexes, and then human THP-1 monocytes were electroporated to introduce RNP into the cells as described above. Editing efficiency was then measured via Sanger sequencing. Several candidates were able to induce mutations in the hTGFBR2 gene with high efficacy, some showing frequencies of nearly 50% or more with WT-Cas9 (Figure 9A), and this efficacy was universally reduced with the use of AR-Cas9 (Figure 9B). In particular, pairing AR-Cas9 with OHTIIR11 (SEQ ID NO: 1065) resulted in increased editing superiority (i.e., the percentage of edits composed by the desired on-target edit), although with lower editing efficacy than WT-Cas9. These results generally confirm the in-silico analysis and suggest that several candidate sgRNAs, including OHTIIR11, were able to efficiently edit TGFBR2 with some frequency in human cells.

[0440] Example 5: Editing TGFBR1 or TGFBR2 makes human monocytes unresponsive to TGF-beta stimulation. After designing and validating numerous guides targeting the TGF-beta pathway, the next step involved functional testing of edited cells. As part of this testing, approximately 400,000 edited human THP-1 monocytes were treated for 6 hours with either 1 μg / ml LPS or 20 ng / μl recombinant TGF-beta. 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 TGFB1 transcription as previously reported. For general information, see Hall, MC, et al. (2003). Journal of Biological Chemistry, 278(12), 10304-10313, and Xiang Yin, et al. 2017, J Atheroscler Thromb, 2017, 24:55-67. doi:10.5551 / jat.35204. LPS treatment induced a mild increase in TGFB1 transcription in control cells (WT-Cas9 injection only) (Figure 10A). However, TGFB1 transcription levels were reduced by approximately 5-fold in TGFBR1-edited cells compared to unedited controls, while TGFBR2-edited cells showed a 2-fold reduction.

[0441] As an additional test, the same cellular RNA was assayed for TIMP1 induction, which is normatively upregulated by SMAD protein-mediated TGF-beta signaling (see Hall, MC, et al. (2003). Journal of Biological Chemistry, 278(12), 10304-10313). In LPS-treated cells, robust induction of TIMP1 was observed compared to unedited controls compared to untreated cells (Figure 10B). TGFBR1-edited monocytes demonstrated a 1:1 reduction in TIMP1 induction compared to unedited controls. No change was observed in TGFBR2-edited cells.

[0442] The results were even more pronounced after treatment with natural ligands. In response to TGF-beta treatment, TGFB1 induction was more robust in cells injected with unedited WTCas9 alone, and editing of either TGFBR1 or TGFBR2 reduced this response threefold (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 TGFB1 levels in the edited cells remained far below those of the control. Taken together, these results demonstrate that both positive feedback (TGFB1 induction) and downstream signaling (TIMP1 induction) of TGF-beta were impaired in cells with edited TGFBR1 and TGFBR2.

[0443] Example 6: Verification of TGFB1 editing in human cells After editing the human TGFB1 gene and designing and analyzing numerous sgRNA candidates predicted to produce either gene knockout (see Figure 11A), the next step is to evaluate their ability to act in vitro. Of particular interest is whether the preferred (i.e., on-target) editing is observed in the edited cells.

[0444] To test this, candidate sgRNAs (SEQ ID NOs: 1-198) were introduced into human THP-1 monocytes via electroporation as part of ribonucleoproteins, as described above in Example 2.

[0445] The results of these studies are expected to further confirm the robustness of in silico analysis and identify preferred candidates that can indeed 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.

[0446] Example 7: In vitro delivery of TGFB1 editing guide RNA to human cells via lipid nanoparticles Additional experiments evaluate the suitability of lipid nanoparticles (LNPs) for delivering 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 as described in Example 5 are performed.

[0447] Example 8: In vitro delivery of TGFB1 editing guide RNA to human cells via adeno-associated virus Since all validation experiments are performed via electroporation of cell cultures to introduce RNA-dependent nucleases and sgRNA, additional experiments will be conducted to evaluate the suitability of using adeno-associated virus (AAV) vectors for delivering sgRNA to human cells. [Table 4]

[0448] Exemplary AAV vectors are shown in Table 2. After preparing the viral vector according to the manufacturer's specifications so that one or more sgRNAs containing SEQ ID NOs. 1-198 are incorporated, the vector is incubated with approximately 500,000 Cas9-transfected U2OS cells (MOI = 0.1-10) (see, for example, Rojas-Fernandez, A., et al. (2015). Scientific Reports, 5(1), 1-6). Two to seven days after infection, the editing is verified using Sanger sequencing as described in Example 2. After confirmation of editing, various functional assays as described in Example 5 are performed.

[0449] Example 9: Verification of TGFBR1 editing in human cells After designing and analyzing numerous sgRNA candidates predicted to edit the human transforming growth factor beta receptor 1 protein to produce either a knockout or dominant-negative receptor (see Figure 11B), the next step is to evaluate their ability to act in vitro. Of particular interest is whether the preferred (i.e., on-target) editing is observed in the edited cells.

[0450] To test this, candidate sgRNAs (SEQ ID NOs. 199-320) were introduced into human THP-1 monocytes via electroporation as part of ribonucleoproteins, as described above in Example 2.

[0451] The results of these studies are expected to further confirm the robustness of in silico analysis and identify several preferred candidates that can indeed induce mutations in the TGFBR1 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.

[0452] Example 10: In vitro delivery of TGFBR1 editing 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 sgRNAs, additional experiments are conducted to evaluate the suitability of using lipid nanoparticles (LNPs) for delivering mRNA to human cells. LNPs containing either sgRNA (SEQ ID NOs. 199-320) or Cas9 mRNA were incubated with THP-1 monocytes, and then the editing was validated as described in Example 7. After editing confirmation, various functional assays described in Example 5 were performed.

[0453] Example 11: In vitro delivery of TGFBR1 editing guide RNA to human cells via adeno-associated virus Since all validation experiments are performed via electroporation of cell cultures to introduce RNA-dependent nucleases and sgRNAs, additional experiments are conducted to evaluate the suitability of using adeno-associated virus (AAV) vectors for delivering sgRNAs to human cells. Exemplary AAV vectors are shown in Table 2. After preparation according to the manufacturer's specifications so that one or more sgRNAs containing SEQ ID NOs. 199-320 are integrated into the viral vector, the vector is introduced into human U2OS cells and the editing is validated as described in Example 8. After editing confirmation, various functional assays described in Example 5 are performed.

[0454] Example 12: Verification of TGFBR2 editing in human cells After designing and analyzing numerous sgRNA candidates predicted to edit the human transforming growth factor beta receptor 2 protein to produce either a knockout or dominant-negative receptor (see Figure 11C), the next step is to evaluate their ability to act in vitro. Of particular interest is whether the preferred (i.e., on-target) editing is observed in the edited cells.

[0455] To test this, candidate sgRNAs (SEQ ID NOs. 321-519) were introduced into human THP-1 monocytes via electroporation as part of ribonucleoproteins, as described above in Example 2.

[0456] The results of these studies are expected to further confirm the robustness of in silico analysis and identify several preferred candidates that can indeed induce mutations in the TGFBR2 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.

[0457] Example 13: In vitro delivery of TGFBR2 editing 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 sgRNAs, additional experiments are conducted to evaluate the suitability of using lipid nanoparticles (LNPs) for delivering mRNA to human cells. LNPs containing either sgRNA (SEQ ID NOs. 321-519) or Cas9 mRNA were incubated with THP-1 monocytes, and then the editing was validated as described in Example 7. After editing confirmation, various functional assays described in Example 5 were performed.

[0458] Example 14: In vitro delivery of TGFBR2 editing guide RNA to human cells via adeno-associated virus Since all validation experiments are performed via electroporation of cell cultures to introduce RNA-dependent nucleases and sgRNAs, additional experiments are conducted to evaluate the suitability of using adeno-associated virus (AAV) vectors for delivering sgRNAs to human cells. Exemplary AAV vectors are shown in Table 2. After preparation according to the manufacturer's specifications so that one or more sgRNAs containing SEQ ID NOs. 321-519 are integrated into the viral vector, the vector is introduced into human U2OS cells and the editing is validated as described in Example 8. After editing confirmation, various functional assays described in Example 5 are performed.

[0459] Example 15: Postoperative treatment of arthral fibrosis Additional experiments will be conducted to demonstrate the efficacy of in vivo treatment of arthritis fibrosis in a postoperative setting. Briefly, a pre-assembled RNP complex composition containing sgRNAs induced by the rabbit TGFB1, TGFBR1, and TGFBR2 genes, along with the Cas9 enzyme, will be prepared as described in Example 2. These RNPs will be injected intra-articularly into rabbits exhibiting arthritis fibrosis.

[0460] In short, two cohorts of rabbits undergo contracture reduction surgery with knee fixation, followed by remobilization surgery at 8 weeks. At remobilization, one cohort undergoes capsular release while the other does not. Each cohort is then divided into subcohorts. Within each cohort, one subcohort receives injections of the RNP prepared above, while the other subcohort does not. Clinical assessments for arthral fibrosis are then performed at several time points after injection, e.g., at 10, 16, and 24 weeks, and the amputated limbs are assessed for fibrosis at the end of the study. An example of a study using this rabbit model of arthral fibrosis is described in Trousdale et al. Bone Joint Res., 2022, 11(1):32-39, doi: 10.1302 / 2046-3758.111.BJR-2021-0546.R1.

[0461] Example 16: Editing TGFB1, TGFBR1, or TGFBR2 renders human monocytes unresponsive to the expression of multiple TGF-beta-responsive gene products. Additional functional studies were conducted on edited THP-1 monocytes and control THP-1 monocytes, with TGF-beta added for 6 hours, followed by collection of total cellular RNA for RT-qPCR analysis as described 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, particularly TGFBR1 and TGFBR2 edited monocytes, experienced strongly reduced induction. Similarly, Col1A2 was induced by TGF-beta treatment, and all edited monocytes showed approximately a twofold decrease in induction (Figure 11B). More robust induction was observed, particularly for FN1 (Figure 11C) and CTGF (Figure 11D). Therefore, it should be noted that relative induction is not very clear in edited cells, but TGFBR1 and TGFBR2 edited monocytes showed FN1 and CTGF levels equivalent to those of the unedited vehicle control. These results suggested that these cells were unable to induce RNA beyond background levels, even after 6 hours of TGF-beta treatment. Collectively, these results indicate that, after exposure to TGF-beta, several additional TGF-beta-responsive gene products showed little to no expression above background levels in TGFBR1 and TGFBR2 edited cells, while TGFB1 edited cells also showed a potent reduction in the induction of these TGF-beta-responsive gene products.

[0462] The above embodiments are provided to give a complete disclosure and explanation to those skilled in the art how to prepare and use embodiments of the compositions, systems, and methods of the Disclosure, and are not intended to limit the scope of what the inventors consider to be their invention. Modifications of the above methods for carrying out embodiments of the Disclosure that would be obvious to those skilled in the art are intended to be within the scope of the following claims. All patents and publications described herein represent the skill level of those skilled in the art to which the Disclosure relates.

[0463] All headings and section names are used for clarity and reference purposes only and are not intended to be considered restrictive in any way. For example, a person skilled in the art will recognize the usefulness of combining various forms from different headings and sections as appropriate, in accordance with the intent and scope of the disclosure described herein.

[0464] It will be understood that the methods described herein are not limited to, and are therefore subject to modification, the specific methodologies, protocols, subjects, and sequencing techniques described herein. Furthermore, the terms used herein are solely for the purpose of describing specific embodiments and are not intended to limit the scope of the methods and compositions described herein, which are limited only by the appended claims. While several embodiments of the Disclosure are shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided merely as examples. Numerous modifications, changes, and substitutions will be made by those skilled in the art without departing from the Disclosure. It should be understood that various alternatives to the embodiments of the Disclosure described herein may be used in the practice of the Disclosure. The following claims are intended to encompass the scope of the Disclosure, the methods and structures within these claims, and their equivalents.

[0465] The above embodiments are provided to give a complete disclosure and explanation to those skilled in the art how to prepare and use embodiments of the compositions, systems, and methods of the Disclosure, and are not intended to limit the scope of what the inventors consider to be their invention. Modifications of the above methods for carrying out embodiments of the Disclosure that would be obvious to those skilled in the art are intended to be within the scope of the following claims. All patents and publications described herein represent the skill level of those skilled in the art to which the Disclosure relates.

[0466] All headings and section names are used for clarity and reference purposes only and are not intended to be considered restrictive in any way. For example, a person skilled in the art will recognize the usefulness of combining various forms from different headings and sections as appropriate, in accordance with the intent and scope of the disclosure described herein.

[0467] It will be understood that the methods described herein are not limited to, and are therefore subject to modification, the specific methodologies, protocols, subjects, and sequencing techniques described herein. Furthermore, the terms used herein are solely for the purpose of describing specific embodiments and are not intended to limit the scope of the methods and compositions described herein, which are limited only by the appended claims. While several embodiments of the Disclosure are shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided merely as examples. Numerous modifications, changes, and substitutions will be made by those skilled in the art without departing from the Disclosure. It should be understood that various alternatives to the embodiments of the Disclosure described herein may be used in the practice of the Disclosure. The following claims are intended to encompass the scope of the Disclosure, the methods and structures within these claims, and their equivalents.

[0468] Several embodiments are described with reference to illustrative uses. Unless otherwise suggested, any embodiment may be combined with any other embodiment. It should be understood that numerous specific details, relationships, and methods are specified in order to provide a complete understanding of the features described herein. However, those skilled in the art will readily recognize that the features described herein may be implemented without one or more of the specific details, or in other ways. The features described herein are not limited by the illustrated order of actions or events, as some actions may occur in different orders and / or simultaneously with other actions or events. Furthermore, not all illustrated actions or events are required to implement the methodology according to the features described herein.

[0469] While several embodiments are shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided merely as examples. This disclosure is not intended to be limited by any particular embodiment provided herein. Although this disclosure has been described with reference to the above-mentioned specification, the descriptions and examples of embodiments herein are not intended to be construed as limiting. Thereafter, those skilled in the art will likely make numerous modifications, changes, and substitutions without departing from this disclosure.

[0470] Furthermore, it should be understood that all aspects of this disclosure are not limited to any specific descriptions, configurations, or relative proportions described herein, which depend on various conditions and variables. It should be understood that various alternatives to the embodiments of this disclosure described herein may be used in the implementation of this disclosure. Therefore, this disclosure is intended to encompass any such alternatives, modifications, variations, or equivalents. The following claims are intended to encompass the scope of this disclosure, the methods and structures within these claims, and their equivalents.

[0471] All publications, patents, and patent applications herein are incorporated by reference to the same extent as any individual publication, patent, or patent application would be specifically and individually indicated as being incorporated by reference. In the event of any conflict between the terminology herein and the terminology of any incorporated reference, the terminology herein shall prevail.

Claims

1. A pharmaceutical composition for treating or preventing disorders having symptoms at least partially caused by intercellular signaling mediated through the transforming growth factor beta (TGFβ) signaling pathway, (i) RNA guide nuclease or nucleic acid encoding RNA guide nuclease, (ii) A pharmaceutical composition comprising at least one guide RNA or nucleic acid encoding at least one guide RNA that targets the TGFB1 gene, the TGFBR1 gene, the TGFBR2 gene, or a combination thereof.

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

3. The pharmaceutical composition according to 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. The pharmaceutical composition according to claim 4, wherein the fibrosis is fibrosis after anterior cruciate ligament (ACL) reconstruction, fibrosis after autologous ACL reconstruction, or fibrosis after allogeneic ACL reconstruction.

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

7. The pharmaceutical composition according to claim 2, wherein the fibrosis is caused by intra-articular fibrous nodules.

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

9. The pharmaceutical composition according to claim 2, wherein the fibrosis is related to tendon injury or surgery.

10. The pharmaceutical composition according to claim 2, wherein the fibrosis is fibrosis after fracture repair.

11. The pharmaceutical composition according to claim 2, wherein the fibrosis is fibrosis following microscopic discectomy.

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

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

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

15. The pharmaceutical composition according to claim 2, wherein the fibrosis is fibrosis following anterior acromiontoplasty.

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

17. The pharmaceutical composition according to claim 2, wherein the fibrosis is soft tissue fibrosis following anterior acromiontoplasty.

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

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

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

21. The pharmaceutical composition according to claim 2, wherein the fibrosis is fibrosis of the trabecular network.

22. The pharmaceutical composition according to claim 21, wherein the fibrosis of the trabecular network is fibrosis following glaucoma surgery.

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

24. The pharmaceutical composition according to claim 2, wherein the fibrosis is atrial fibrosis, cardiac fibrosis, myocardial fibrosis, and / or fibrosis after thoracotomy.

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

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

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

28. The pharmaceutical composition according to claim 1, wherein the disorder includes fibrosis in renal tissue.

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

30. The pharmaceutical composition according to claim 1, wherein the disorder includes fibrosis in skin tissue.

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

32. The pharmaceutical composition according to claim 1, wherein the disorder includes fibrosis in lung tissue.

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

34. The pharmaceutical composition according to claim 1, wherein the disorder includes fibrosis in liver tissue.

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

36. The pharmaceutical composition according to claim 1, wherein the disorder includes fibrosis in cardiac tissue.

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

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

39. The pharmaceutical composition according to claim 36, wherein the fibrosis is replacement fibrosis.

40. The pharmaceutical composition according to claim 36, wherein the disorder is myocardial infarction.

41. The pharmaceutical composition according to claim 1, wherein the disorder includes fibrosis in brain tissue.

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

43. The pharmaceutical composition according to claim 1, wherein the disorder includes fibrosis in intestinal tissue.

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

45. The pharmaceutical composition according to claim 1, wherein the disorder includes fibrosis of the hand or fingers.

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

47. The pharmaceutical composition according to claim 1, wherein the disorder includes fibrosis in lymphoid tissue.

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

49. The pharmaceutical composition according to claim 1, wherein the disorder includes fibrosis in bone marrow tissue.

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

51. The pharmaceutical composition according to claim 1, wherein the disorder includes fibrosis in penile tissue.

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

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

54. The pharmaceutical composition according to claim 53, wherein the fibrosis is retroperitoneal fibrosis.

55. The pharmaceutical composition according to claim 1, wherein the disorder includes fibrosis in musculoskeletal tissue.

56. The pharmaceutical composition according to claim 1, wherein the disorder is cancer.

57. The pharmaceutical composition according to claim 1, wherein the cancer is a musculoskeletal cancer.

58. The pharmaceutical composition according to claim 1, wherein the disorder is a musculoskeletal disorder.

59. The pharmaceutical composition according to claim 58, wherein the musculoskeletal disorder is a hereditary musculoskeletal disorder.

60. The pharmaceutical composition according to claim 58, wherein the musculoskeletal disorder is muscular dystrophy.

61. The pharmaceutical composition according to claim 58, wherein the musculoskeletal disorder is selected from Duchenne muscular dystrophy (DMD), myotonic dystrophy (DM), facioscapulohumeral muscular dystrophy (FSHD), and limb-girdle muscular dystrophy (LGMD).

62. The pharmaceutical composition according to claim 58, wherein the musculoskeletal disorder is a type II collagen disorder.

63. The pharmaceutical composition according to claim 58, wherein the musculoskeletal disorder is selected from achondroplasia type II, hypochondrodysplasia, Stickler syndrome, and Czech dysplasia.

64. The pharmaceutical composition according to claim 58, wherein the musculoskeletal disorder is osteogenesis imperfecta (OI).

65. The pharmaceutical composition according to claim 58, wherein the musculoskeletal disorder is an autoimmune musculoskeletal disease.

66. The pharmaceutical composition according to claim 58, wherein the musculoskeletal disorder is an autoimmune musculoskeletal disease accompanied by polygenic susceptibility traits.

67. The pharmaceutical composition according to claim 58, wherein the musculoskeletal disorder is spondyloarthropathy (SA).

68. The pharmaceutical composition according to claim 58, wherein the musculoskeletal disorder is ankylosing spondylitis.

69. The pharmaceutical composition according to claim 58, wherein the musculoskeletal disorder is rheumatoid arthritis.

70. The pharmaceutical composition according to claim 58, wherein the musculoskeletal disorder is autoimmune osteoarthritis.

71. The pharmaceutical composition according to claim 58, wherein the musculoskeletal disorder is osteoarthritis (OA).

72. The pharmaceutical composition according to claim 58, wherein the musculoskeletal disorder is Kamrachi-Engelmann disease.

73. The pharmaceutical composition according to claim 58, wherein the musculoskeletal disorder is Sjögren's syndrome.

74. The pharmaceutical composition according to claim 58, wherein the musculoskeletal disorder is a mechanical musculoskeletal injury.

75. The pharmaceutical composition according to claim 58, wherein the musculoskeletal disorder is selected from chronic muscle injury, tendon rupture, post-traumatic osteoarthritis, and post-traumatic arthropathy.

76. The pharmaceutical composition according to any one of claims 1 to 75, wherein the at least one guide RNA or nucleic acid encoding the at least one guide RNA targets the mammalian TGFB1 gene.

77. The pharmaceutical composition according to claim 76, wherein the mammalian TGFB1 gene is a dog, horse, or cat TGFB1 gene.

78. The pharmaceutical composition according to claim 76, wherein the mammalian TGFB1 gene is the human TGFB1 gene.

79. The pharmaceutical composition according to claim 78, wherein the at least one guide RNA includes a spacer sequence selected from the group consisting of sequences (sequences 1 to 198) shown in Figures 1A to 1D.

80. The pharmaceutical composition according to claim 78, wherein the at least one guide RNA includes a spacer sequence selected from the group consisting of sequences shown in Figure 12A (Sequence IDs 520 to 527).

81. The pharmaceutical composition according to claim 78, wherein the at least one guide RNA comprises the spacer sequence OHTG03 (SEQ ID NO: 522) or OHTG04 (SEQ ID NO: 523).

82. The pharmaceutical composition according to any one of claims 1 to 75, wherein the at least one guide RNA or nucleic acid encoding the at least one guide RNA targets the mammalian TGFBR1 gene.

83. The pharmaceutical composition according to claim 82, wherein the mammalian TGFBR1 gene is a dog, horse, or cat TGFBR1 gene.

84. The pharmaceutical composition according to claim 82, wherein the mammalian TGFBR1 gene is the human TGFBR1 gene.

85. The pharmaceutical composition according to claim 84, wherein the at least one guide RNA includes a spacer sequence selected from the group consisting of sequences shown in Figures 2A to 2C (Sequence IDs 199 to 320).

86. The pharmaceutical composition according to claim 84, wherein the at least one guide RNA includes a spacer sequence selected from the group consisting of sequences shown in Figure 12B (Sequence IDs 528 to 552).

87. The pharmaceutical composition according to claim 84, wherein the at least one guide RNA includes a spacer sequence selected from the group consisting of sequences shown in Figure 5B.

88. The pharmaceutical composition according to claim 84, wherein the at least one guide RNA comprises the spacer sequence OHTIR04 (SEQ ID NO: 532) or OHTIR08 (SEQ ID NO: 539).

89. The pharmaceutical composition according to any one of claims 1 to 75, wherein the at least one guide RNA or nucleic acid encoding the at least one guide RNA targets the mammalian TGFBR2 gene.

90. The pharmaceutical composition according to claim 89, wherein the mammalian TGFBR2 gene is a dog, horse, or cat TGFBR2 gene.

91. The pharmaceutical composition according to claim 89, wherein the mammalian TGFBR2 gene is the human TGFBR2 gene.

92. The pharmaceutical composition according to claim 91, wherein the at least one guide RNA includes a spacer sequence selected from the group consisting of sequences shown in Figures 3A to 3D (Sequence IDs 321 to 519).

93. The pharmaceutical composition according to claim 91, wherein the at least one guide RNA includes a spacer sequence selected from the group consisting of sequences shown in Figure 12C (Sequence IDs 553 to 604).

94. The pharmaceutical composition according to claim 91, wherein the at least one guide RNA includes a spacer sequence selected from the group consisting of sequences shown in Figure 6B.

95. The pharmaceutical composition according to claim 91, wherein the at least one guide RNA comprises the spacer sequence OHTIIR04 (SEQ ID NO: 563).

96. The pharmaceutical composition according to any one of claims 1 to 95, wherein the RNA guide nuclease or the nucleic acid encoding the RNA guide nuclease is the RNA guide nuclease.

97. The pharmaceutical composition according to any one of claims 1 to 95, wherein the RNA guide nuclease or the nucleic acid encoding the RNA guide nuclease is the DNA encoding the RNA guide nuclease.

98. The pharmaceutical composition according to any one of claims 1 to 95, wherein the RNA guide nuclease or the nucleic acid encoding the RNA guide nuclease is the mRNA encoding the RNA guide nuclease.

99. The pharmaceutical composition according to any one of claims 1 to 98, wherein the RNA guide nuclease is a Cas protein.

100. The pharmaceutical composition according to claim 99, wherein the Cas protein is a Cas9 protein.

101. The pharmaceutical composition according to claim 99, wherein the Cas9 protein is S. pyogenes Cas9 polypeptide.

102. The pharmaceutical composition according to claim 99, wherein the Cas9 protein is selected from the group consisting of ESCas9, HFCas9, PECas9, and ARCas9.

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

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

105. A pharmaceutical composition according to any one of claims 1 to 102, comprising a nucleic acid encoding both the RNA guide nuclease and the at least one guide RNA.

106. The pharmaceutical composition according to any one of claims 1 to 105, wherein the at least one guide RNA is a single guide RNA (sgRNA).

107. The pharmaceutical composition according to any one of claims 1 to 106, wherein the composition comprises one or more viral vectors, the composition comprising (i) an RNA guide nuclease or a nucleic acid encoding an RNA guide nuclease, and (ii) at least one guide RNA that targets a gene encoding the transmembrane receptor or a nucleic acid encoding at least one guide RNA.

108. The pharmaceutical composition according to claim 107, wherein one of the more viral vectors comprises a recombinant virus selected from retroviruses, adenoviruses, adeno-associated viruses, lentiviruses, and herpes simplex virus-1.

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

110. The pharmaceutical composition according to claim 109, wherein the recombinant AAV is of serotype 5 (AAV5).

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

112. The pharmaceutical composition according to any one of claims 1 to 106, wherein the composition comprises one or more lipid nanoparticles (LNPs) collectively comprising (i) an RNA guide nuclease or a nucleic acid encoding an RNA guide nuclease, and (ii) at least one guide RNA that targets a gene encoding the transmembrane receptor or a nucleic acid encoding at least one guide RNA.

113. The one or more LNPs mentioned above A first plurality of LNPs that encapsulate the RNA guide nuclease or nucleic acid encoding the RNA guide nuclease, The pharmaceutical composition according to claim 112, comprising: a second plurality of LNPs encapsulating at least one guide RNA or nucleic acid encoding at least one guide RNA.

114. The pharmaceutical composition according to claim 112, wherein one or more LNPs comprise a plurality of LNPs that encapsulate both (i) an RNA guide nuclease or a nucleic acid encoding an RNA guide nuclease and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA that targets a gene encoding the transmembrane receptor.

115. The one or more LNPs are 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazinediethanamine (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 A pharmaceutical composition according to any one of claims 112 to 114, comprising a component selected from the group consisting of n(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 cyclic amine groups, and mixtures thereof.

116. The LNPs are 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), and 1,2-diundecanoi 1,2-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 dietherPC), 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 A pharmaceutical composition according to any one of claims 112 to 115, comprising a component selected from the group consisting of 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.

117. The pharmaceutical composition according to any one of claims 112 to 116, 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 PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DMA, PEG-DSPE lipid, and mixtures thereof.

118. The composition according to any one of claims 112 to 117, wherein the LNP comprises 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.

119. The pharmaceutical composition according to any one of claims 112 to 114, wherein the one or more LNPs comprises an LP01 ionic lipid, a neutral / helper lipid component, a steroid system component, and a PEG-modified lipid component.

120. The pharmaceutical composition according to any one of claims 112 to 114, wherein the one or more LNPs comprises an SM-102 ionic lipid, a neutral / helper lipid component, a steroid system component, and a PEGylated lipid component.

121. The pharmaceutical composition according to any one of claims 112 to 114, wherein the one or more LNPs comprises an ALC0315 ionic lipid, a neutral / helper lipid component, a steroid system component, and a PEG-modified lipid component.

122. The pharmaceutical composition according to any one of claims 119 to 121, wherein the neutral / helper lipid component comprises DSPC.

123. The pharmaceutical composition according to claims 119 to 122, wherein the steroid system component comprises cholesterol.

124. The pharmaceutical composition according to claims 119 to 123, wherein the steroid system component comprises a glucocorticoid steroid.

125. The pharmaceutical composition according to claim 124, wherein the glucocorticoid steroid is dexamethasone.

126. The pharmaceutical composition according to claims 119 to 125, wherein the PEGylated lipid component comprises DMG-PEG.

127. The pharmaceutical composition according to claim 126, wherein the DMG-PEG is DMG-PEG2000.

128. The pharmaceutical composition according to claims 119 to 127, wherein one or more LNPs contain LP01, a neutral / helper lipid component, a steroid system component, and a PEG-modified lipid component in a molar ratio of (43-47):(7.5-10.5):(42-46):(1.5-2.5).

129. The pharmaceutical composition according to any one of claims 119 to 127, wherein the one or more LNPs contain SM-102, a neutral / helper lipid component, a steroid system component, and a PEGylated lipid component in a molar ratio of (48-52):(8.5-11.5):(36.5-40.5):(1-2).

130. The pharmaceutical composition according to any one of claims 119 to 127, wherein the one or more LNPs contain ALC0315, a neutral / helper lipid component, a steroid system component, and a PEG-modified lipid component in a molar ratio of (48-52):(8.5-11.5):(36.5-40.5):(1-2).

131. The pharmaceutical composition according to any one of claims 1 to 106, wherein the composition comprises one or more liposomes, each comprising (i) an RNA guide nuclease or a nucleic acid encoding an RNA guide nuclease, and (ii) at least one guide RNA that targets a gene encoding the transmembrane receptor or a nucleic acid encoding at least one guide RNA.

132. The one or more liposomes described above A first group of liposomes encapsulating the RNA guide nuclease or nucleic acid encoding the RNA guide nuclease, The pharmaceutical composition according to claim 131, comprising a second plurality of liposomes encapsulating at least one guide RNA or a nucleic acid encoding at least one guide RNA.

133. The pharmaceutical composition according to claim 131, wherein one or more liposomes comprise a plurality of liposomes each encapsulating (i) an RNA guide nuclease or a nucleic acid encoding an RNA guide nuclease, and (ii) at least one guide RNA that targets a gene encoding the transmembrane receptor or a nucleic acid encoding at least one guide RNA.

134. The pharmaceutical composition according to any one of claims 1 to 106, wherein the composition comprises one or more virus-like particles, each comprising (i) an RNA guide nuclease or a nucleic acid encoding an RNA guide nuclease, and (ii) at least one guide RNA that targets a gene encoding the transmembrane receptor or a nucleic acid encoding at least one guide RNA.

135. The one or more virus-like particles described above A first plurality of virus-like particles encapsulating the RNA guide nuclease or nucleic acid encoding the RNA guide nuclease, The pharmaceutical composition according to claim 134, comprising: a second plurality of virus-like particles encapsulating at least one guide RNA or a nucleic acid encoding at least one guide RNA.

136. The pharmaceutical composition according to claim 134, wherein one or more virus-like particles encapsulate both (i) an RNA guide nuclease or a nucleic acid encoding an RNA guide nuclease and (ii) at least one guide RNA that targets a gene encoding the transmembrane receptor or a nucleic acid encoding at least one guide RNA.

137. The pharmaceutical composition according to any one of claims 1 to 136, wherein the composition is formulated for parenteral administration.

138. The pharmaceutical composition according to any one of claims 1 to 136, wherein the composition is formulated for intra-articular injection into the joint of interest.

139. The pharmaceutical composition according to any one of claims 1 to 136, wherein the composition is formulated for intradiscal injection.

140. The pharmaceutical composition according to any one of claims 1 to 136, wherein the composition is formulated for peridiscal injection.

141. The pharmaceutical composition according to any one of claims 1 to 136, wherein the composition is formulated for intraspinal injection.

142. A method for treating or preventing a disorder in a subject requiring treatment or prevention of symptoms caused at least partially by intercellular signaling mediated through the transforming growth factor beta (TGFβ) signaling pathway, Administering a therapeutically effective amount of the composition, wherein the composition is (i) RNA guide nuclease or nucleic acid encoding RNA guide nuclease, (ii) A method comprising administering a nucleic acid that encodes at least one guide RNA or at least one guide RNA that targets the TGFB1 gene, the TGFBR1 gene, the TGFBR2 gene, and a combination thereof.

143. The method according to claim 142, wherein the disorder is fibrosis.

144. The method according to claim 143, wherein the fibrosis is postoperative fibrosis.

145. The method according to claim 143, wherein the fibrosis is post-ligament reconstruction fibrosis.

146. The method according to claim 145, wherein the fibrosis is fibrosis after anterior cruciate ligament (ACL) reconstruction, fibrosis after autologous ACL reconstruction, or fibrosis after allogeneic ACL reconstruction, or fibrosis after tendon injury or tendon repair.

147. The method according to claim 143, wherein the fibrosis is knee joint fibrosis.

148. The method according to claim 143, wherein the fibrosis is caused by intra-articular fibrous nodules.

149. The method according to claim 143, wherein the fibrosis is present after total knee arthroplasty (TKA).

150. The pharmaceutical composition according to claim 2, wherein the fibrosis is fibrosis after fracture repair.

151. The method according to claim 143, wherein the fibrosis is post-TKA knee joint fibrosis.

152. The method according to claim 143, wherein the fibrosis is fibrosis following microscopic discectomy.

153. The method according to claim 143, wherein the fibrosis is epidural fibrosis following microscopic discectomy.

154. The method according to claim 143, wherein the fibrosis is fibrosis following lumbar laminectomy.

155. The method according to claim 143, wherein the fibrosis is epidural fibrosis following lumbar laminectomy.

156. The method according to claim 143, wherein the fibrosis is postoperative fibrosis after anterior acromiontoplasty.

157. The method according to claim 143, wherein the fibrosis is fibrosis following subacromial decompression surgery.

158. The method according to claim 143, wherein the fibrosis is soft tissue fibrosis following anterior acromionoplasty.

159. The method according to claim 143, wherein the fibrosis is joint contracture after anterior acromionoplasty.

160. The method according to claim 143, wherein the fibrosis is post-arthroscopic fibrosis.

161. The method according to claim 143, wherein the fibrosis is articular fibrosis following arthroscopic surgery.

162. The method according to claim 143, wherein the fibrosis is fibrosis of the trabecular network.

163. The method according to claim 162, wherein the fibrosis of the trabecular network is fibrosis following glaucoma surgery.

164. The method according to claim 143, wherein the fibrosis is epidural fibrosis.

165. The method according to claim 143, wherein the fibrosis is atrial fibrosis, cardiac fibrosis, myocardial fibrosis, and / or fibrosis after thoracotomy.

166. The method according to claim 143, wherein the fibrosis is postoperative fibrosis.

167. The method according to claim 143, wherein the fibrosis is idiopathic pulmonary fibrosis (IPF).

168. The method according to claim 143, wherein the fibrosis is fibrosis of the knee joint, shoulder joint, or elbow joint.

169. The method according to claim 142, wherein the disorder includes fibrosis in renal tissue.

170. The method according to claim 142, wherein the disorder is chronic kidney disease.

171. The method according to claim 142, wherein the disorder includes fibrosis in skin tissue.

172. The method according to claim 142, wherein the disorder is scarring after wound healing, keloid disorder, nephrogenic systemic fibrosis, or scleroderma / systemic sclerosis.

173. The method according to claim 142, wherein the disorder includes fibrosis in lung tissue.

174. The method according to claim 142, wherein the disorder is fibrothorax, pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, radiation-induced lung injury, progressive widespread fibrosis, or scleroderma / systemic sclerosis.

175. The method according to claim 142, wherein the disorder includes fibrosis in liver tissue.

176. The method according to claim 142, wherein the disorder is cirrhosis of the liver or bridging fibrosis.

177. The method according to claim 142, wherein the disorder includes fibrosis in cardiac tissue.

178. The method according to claim 143, wherein the fibrosis is interstitial fibrosis.

179. The method according to claim 142, wherein the disorder is congestive heart failure or hypertension.

180. The method according to claim 143, wherein the fibrosis is replacement fibrosis.

181. The method according to claim 142, wherein the disorder is myocardial infarction.

182. The method according to claim 142, wherein the disorder includes fibrosis in brain tissue.

183. The method according to claim 143, wherein the fibrosis is a glial scar.

184. The method according to claim 142, wherein the disorder includes fibrosis in the intestinal tissue.

185. The method according to claim 142, wherein the disorder is Crohn's disease.

186. The method according to claim 142, wherein the disorder includes fibrosis of the hand or fingers.

187. The method according to claim 142, wherein the disorder is Dupuytren's contracture.

188. The method according to claim 142, wherein the disorder includes fibrosis in lymphoid tissue.

189. The method according to claim 143, wherein the fibrosis is mediastinal fibrosis.

190. The method according to claim 142, wherein the disorder includes fibrosis in bone marrow tissue.

191. The method according to claim 143, wherein the fibrosis is myelofibrosis.

192. The method according to claim 142, wherein the disorder includes fibrosis in penile tissue.

193. The method according to claim 142, wherein the disorder is Peyronie's disease.

194. The method according to claim 142, wherein the disorder includes fibrosis in the soft tissue of the retroperitoneum.

195. The method according to claim 143, wherein the fibrosis is retroperitoneal fibrosis.

196. The method according to claim 142, wherein the disorder is cancer.

197. The method according to claim 196, wherein the cancer is a musculoskeletal cancer.

198. The method according to claim 142, wherein the disorder includes fibrosis in musculoskeletal tissue.

199. The method according to claim 142, wherein the disorder is a musculoskeletal disorder.

200. The method according to claim 199, wherein the musculoskeletal disorder is a hereditary musculoskeletal disorder.

201. The method according to claim 199, wherein the musculoskeletal disorder is muscular dystrophy.

202. The method according to claim 199, wherein the musculoskeletal disorder is selected from Duchenne muscular dystrophy (DMD), myotonic dystrophy (DM), facioscapulohumeral muscular dystrophy (FSHD), and limb-girdle muscular dystrophy (LGMD).

203. The method according to claim 199, wherein the musculoskeletal disorder is a type II collagen disorder.

204. The method according to claim 199, wherein the musculoskeletal disorder is selected from achondroplasia type II, hypochondrodysplasia, Stickler syndrome, and Czech dysplasia.

205. The method according to claim 199, wherein the musculoskeletal disorder is osteogenesis imperfecta (OI).

206. The method according to claim 199, wherein the musculoskeletal disorder is an autoimmune musculoskeletal disease.

207. The method according to claim 199, wherein the musculoskeletal disorder is an autoimmune musculoskeletal disorder accompanied by polygenic susceptibility traits.

208. The method according to claim 199, wherein the musculoskeletal disorder is spondyloarthropathy (SA).

209. The method according to claim 199, wherein the musculoskeletal disorder is ankylosing spondylitis.

210. The method according to claim 199, wherein the musculoskeletal disorder is rheumatoid arthritis.

211. The method according to claim 199, wherein the musculoskeletal disorder is autoimmune osteoarthritis.

212. The method according to claim 199, wherein the musculoskeletal disorder is osteoarthritis (OA).

213. The method according to claim 199, wherein the musculoskeletal disorder is Kamrachi-Engelmann disease.

214. The method according to claim 199, wherein the musculoskeletal disorder is Sjögren's syndrome.

215. The method according to claim 199, wherein the musculoskeletal disorder is a mechanical musculoskeletal injury.

216. The method according to claim 199, wherein the musculoskeletal disorder is selected from chronic muscle injury, tendon rupture, post-traumatic osteoarthritis, and post-traumatic arthropathy.

217. The method according to any one of claims 142 to 216, wherein the at least one guide RNA or nucleic acid encoding the at least one guide RNA targets the mammalian TGFB1 gene.

218. The method according to claim 217, wherein the mammalian TGFB1 gene is a dog, horse, or cat TGFB1 gene.

219. The method according to claim 217, wherein the mammalian TGFB1 gene is the human TGFB1 gene.

220. The method according to claim 219, wherein the at least one guide RNA includes a spacer sequence selected from the group consisting of sequences (sequence numbers 1 to 198) shown in Figures 1A to 1D.

221. The method according to claim 219, wherein the at least one guide RNA includes a spacer sequence selected from the group consisting of sequences (sequence numbers 520 to 527) shown in Figure 12A.

222. The method according to claim 219, wherein the at least one guide RNA comprises a spacer sequence of OHTG03 or OHTG04.

223. The method according to any one of claims 142 to 216, wherein the at least one guide RNA or nucleic acid encoding the at least one guide RNA targets the mammalian TGFBR1 gene.

224. The method according to claim 223, wherein the mammalian TGFBR1 gene is a dog, horse, or cat TGFBR1 gene.

225. The method according to claim 223, wherein the mammalian TGFBR1 gene is the human TGFBR1 gene.

226. The method according to claim 225, wherein the at least one guide RNA includes a spacer sequence selected from the group consisting of sequences shown in Figures 2A to 2C (Sequence IDs 199 to 320).

227. The method according to claim 225, wherein the at least one guide RNA includes a spacer sequence selected from the group consisting of sequences shown in Figure 12B (Sequence IDs 528-552).

228. The method according to claim 225, wherein the at least one guide RNA includes a spacer sequence selected from the group consisting of sequences shown in Figure 5B.

229. The method according to claim 225, wherein the at least one guide RNA comprises a spacer sequence of OHTIR04 or OHTIR08.

230. The method according to any one of claims 142 to 216, wherein the at least one guide RNA or nucleic acid encoding the at least one guide RNA targets the mammalian TGFBR2 gene.

231. The method according to claim 230, wherein the mammalian TGFBR1 gene is a dog, horse, or cat TGFBR1 gene.

232. The method according to claim 230, wherein the mammalian TGFBR1 gene is the human TGFBR1 gene.

233. The method according to claim 232, wherein the at least one guide RNA includes a spacer sequence selected from the group consisting of sequences (sequence numbers 321 to 519) shown in Figures 3A to 3D.

234. The method according to claim 232, wherein the at least one guide RNA includes a spacer sequence selected from the group consisting of sequences (sequence numbers 553 to 604) shown in Figure 12C.

235. The method according to claim 232, wherein the at least one guide RNA includes a spacer sequence selected from the group consisting of sequences shown in Figure 6B.

236. The method according to claim 232, wherein the at least one guide RNA comprises the spacer sequence of OHTIIR04.

237. The method according to any one of claims 142 to 236, wherein the RNA guide nuclease or the nucleic acid encoding the RNA guide nuclease is the RNA guide nuclease.

238. The method according to any one of claims 142 to 236, wherein the RNA guide nuclease or the nucleic acid encoding the RNA guide nuclease is the DNA encoding the RNA guide nuclease.

239. The method according to any one of claims 142 to 236, wherein the RNA guide nuclease or the nucleic acid encoding the RNA guide nuclease is the mRNA encoding the RNA guide nuclease.

240. The method according to any one of claims 142 to 239, wherein the RNA guide nuclease is a Cas protein.

241. The method according to claim 240, wherein the Cas protein is a Cas9 protein.

242. The method according to claim 241, wherein the Cas9 protein is S. pyogenes Cas9 polypeptide.

243. The method according to claim 241, wherein the Cas9 protein is selected from the group consisting of ESCas9, HFCas9, PECas9, and ARCas9.

244. The method according to any one of claims 142 to 243, wherein the at least one guide RNA or the nucleic acid encoding the at least one guide RNA is the at least one guide RNA.

245. The method according to any one of claims 142 to 243, wherein the at least one guide RNA or the nucleic acid encoding the at least one guide RNA is the DNA encoding the at least one guide RNA.

246. The method according to any one of claims 142 to 243, comprising a nucleic acid encoding both the RNA guide nuclease and the at least one guide RNA.

247. The method according to any one of claims 142 to 246, wherein the at least one guide RNA is a single guide RNA (sgRNA).

248. The method according to any one of claims 142 to 247, wherein the composition comprises one or more viral vectors comprising (i) an RNA guide nuclease or a nucleic acid encoding an RNA guide nuclease, and (ii) at least one guide RNA that targets a gene encoding the transmembrane receptor or a nucleic acid encoding at least one guide RNA.

249. The method according to claim 248, wherein one of the more viral vectors comprises a recombinant virus selected from retroviruses, adenoviruses, adeno-associated viruses, lentiviruses, and herpes simplex virus-1.

250. The method according to claim 248, wherein one of the more viral vectors comprises recombinant adeno-associated virus (AAV).

251. The method according to claim 250, wherein the recombinant AAV is of serotype 5 (AAV5).

252. The method according to claim 250, wherein the recombinant AAV is of serotype 6 (AAV6).

253. The method according to any one of claims 142 to 247, wherein the composition comprises one or more lipid nanoparticles (LNPs) collectively comprising (i) an RNA guide nuclease or a nucleic acid encoding an RNA guide nuclease, and (ii) at least one guide RNA that targets a gene encoding the transmembrane receptor or a nucleic acid encoding at least one guide RNA.

254. The one or more LNPs mentioned above A first plurality of LNPs that encapsulate the RNA guide nuclease or nucleic acid encoding the RNA guide nuclease, The method according to claim 253, comprising: a second plurality of LNPs encapsulating at least one guide RNA or nucleic acids encoding at least one guide RNA.

255. The method according to claim 253, wherein the one or more LNPs include a plurality of LNPs that encapsulate both (i) an RNA guide nuclease or a nucleic acid encoding an RNA guide nuclease and (ii) at least one guide RNA that targets a gene encoding the transmembrane receptor or a nucleic acid encoding at least one guide RNA.

256. The one or more LNPs are 3-(didodecylamino)-N1,N1,4-tridedecyl-1-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridedecyl-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatricontane (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]octateca-9,12-dien-1-yloxy]propane-1-amine(octateca-CLinDMA),(2R)-2-({8-[(3.beta.)-cholest-5-en-3-yloxy]octateca-9,12-dien-1-yloxy]propane-1- The method according to any one of claims 253 to 255, comprising a component selected from the group consisting of amines (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 cyclic amine groups, and mixtures thereof.

257. The LNPs are 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), and 1,2-diundecanoi 1,2-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 dietherPC), 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 The method according to any one of claims 253 to 256, comprising a component selected from the group consisting of 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.

258. The method according to any one of claims 253 to 257, 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 PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DMA, PEG-DSPE lipids, and mixtures thereof.

259. The method according to any one of claims 253 to 258, wherein the LNP comprises 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.

260. The method according to any one of claims 253 to 255, wherein the one or more LNPs include an LP01 ionic lipid, a neutral / helper lipid component, a steroid system component, and a PEG-modified lipid component.

261. The method according to any one of claims 253 to 255, wherein the one or more LNPs include an SM-102 ionic lipid, a neutral / helper lipid component, a steroid system component, and a PEG-modified lipid component.

262. The method according to any one of claims 253 to 255, wherein the one or more LNPs include an ALC0315 ionic lipid, a neutral / helper lipid component, a steroid system component, and a PEG-modified lipid component.

263. The method according to any one of claims 260 to 262, wherein the neutral / helper lipid component comprises DSPC.

264. The method according to claims 260 to 263, wherein the steroid system component comprises cholesterol.

265. The method according to claims 260 to 264, wherein the steroid system component comprises a glucocorticoid steroid.

266. The method according to claim 265, wherein the glucocorticoid steroid is dexamethasone.

267. The method according to claims 260 to 266, wherein the PEGylated lipid component comprises DMG-PEG.

268. The method according to claim 267, wherein the DMG-PEG is DMG-PEG2000.

269. The method according to any one of claims 260 to 268, wherein the one or more LNPs contain LP01, a neutral / helper lipid component, a steroid system component, and a PEG-modified lipid component in a molar ratio of (43-47):(7.5-10.5):(42-46):(1.5-2.5).

270. The method according to any one of claims 260 to 268, wherein the one or more LNPs contain SM-102, a neutral / helper lipid component, a steroid system component, and a PEG-modified lipid component in a molar ratio of (48-52):(8.5-11.5):(36.5-40.5):(1-2).

271. The method according to any one of claims 260 to 268, wherein the one or more LNPs contain ALC0315, a neutral / helper lipid component, a steroid system component, and a PEG-modified lipid component in a molar ratio of (48-52):(8.5-11.5):(36.5-40.5):(1-2).

272. The method according to any one of claims 142 to 247, wherein the composition comprises one or more liposomes, each comprising (i) an RNA guide nuclease or a nucleic acid encoding an RNA guide nuclease, and (ii) at least one guide RNA that targets a gene encoding the transmembrane receptor or a nucleic acid encoding at least one guide RNA.

273. The one or more liposomes described above A first group of liposomes encapsulating the RNA guide nuclease or nucleic acid encoding the RNA guide nuclease, The method according to claim 272, comprising: a second plurality of liposomes encapsulating at least one guide RNA or a nucleic acid encoding at least one guide RNA.

274. The method according to claim 272, wherein one or more liposomes comprise a plurality of liposomes each encapsulating (i) an RNA guide nuclease or a nucleic acid encoding an RNA guide nuclease, and (ii) at least one guide RNA that targets a gene encoding the transmembrane receptor or a nucleic acid encoding at least one guide RNA.

275. The method according to any one of claims 142 to 247, wherein the composition comprises one or more virus-like particles, each comprising (i) an RNA guide nuclease or a nucleic acid encoding an RNA guide nuclease, and (ii) at least one guide RNA that targets a gene encoding the transmembrane receptor or a nucleic acid encoding at least one guide RNA.

276. The one or more virus-like particles described above A first plurality of virus-like particles encapsulating the RNA guide nuclease or nucleic acid encoding the RNA guide nuclease, The method according to claim 275, comprising: a second plurality of virus-like particles encapsulating at least one guide RNA or a nucleic acid encoding at least one guide RNA.

277. The method according to claim 275, wherein the one or more virus-like particles comprise a plurality of virus-like particles each containing (i) an RNA guide nuclease or a nucleic acid encoding an RNA guide nuclease, and (ii) at least one guide RNA that targets a gene encoding the transmembrane receptor or a nucleic acid encoding at least one guide RNA.

278. The method according to any one of claims 142 to 277, wherein the administration includes parenteral administration.

279. The method according to any one of claims 142 to 277, wherein the administration includes intra-articular injection into the joint of the subject.

280. The method according to any one of claims 142 to 277, wherein the administration includes intradiscal injection.

281. The method according to any one of claims 142 to 277, wherein the administration includes a peridiscal injection.

282. The method according to any one of claims 142 to 277, wherein the administration includes intraspinal injection.

283. The method according to any one of claims 142 to 277, wherein the administration includes administration to target cells or tissues.

284. The method according to any one of claims 142 to 277, wherein the administration is performed during surgery.

285. The method according to claim 284, wherein the composition is applied to the surgical site.

286. The method according to claim 284, wherein the composition is sprayed onto the surgical site.

287. The method according to claim 284, wherein the composition is coated onto a bandage applied to a surgical site.

288. The method according to claim 284, wherein the composition is coated onto a stent that is implanted during surgery.

289. The method according to claim 284, wherein the composition is immersed and coated onto an implant. ・

Images