Gene editing for surgery-related fibrosis treatment

Abstract

Provided herein are compositions and methods for treating musculoskeletal fibrosis and / or scarring by gene editing elimination of intracellular signaling through specific cell surface receptors. In some aspects, the compositions and methods relate to TGFB1 ligands. In other aspects, the compositions and methods relate to the TGFB1 receptor (TGFBR1 / TGFBR2). In some aspects, the compositions and methods are useful for treating or preventing post-traumatic fibrosis and / or scarring. In some aspects, the compositions and methods are used to treat or prevent postoperative fibrosis and / or scarring. In some aspects, the compositions and methods are used to treat or prevent local nociceptive feelings, inflammation, degeneration, or morphological changes associated with fibrosis and / or scarring. In some aspects, the compositions and methods are useful for treating fibrosis.

Description

[0001] Cross-referencing related applications

[0002] This application claims priority to International Application No. PCT / US23 / 61392, filed January 26, 2023, and U.S. Provisional Patent Application No. 63 / 480,642, filed January 19, 2023, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0003] References to sequence lists submitted electronically

[0004] This application contains a sequence list that has been submitted electronically in XML file format and is hereby incorporated in its entirety by reference. The XML file is named 123994-5004-WO50 Sequence Listing.xml, was created on January 17, 2024, and has a size of 3,360 kilobytes. Background Technology

[0005] Musculoskeletal tissue is particularly prone to fibrosis and / or scarring after trauma. In many cases, this trauma may be caused by surgical procedures. Fibrosis results from the accumulation of extracellular matrix (ECM) components at specific sites and leads to the formation of scar tissue. See Sheets, K. et al. (2022). Journal of Cellular Biochemistry. This musculoskeletal scar tissue can cause a variety of problems, such as limited range of motion or pain, and may even lead to other diseases, complicating the medical or surgical management of musculoskeletal tissue and joints.

[0006] Transforming growth factor-β (TGFB) transduces signals via typical receptor-ligand interactions, responsible for extracellular signal transduction across the plasma membrane of receptor-expressing cells. In many cases, circulating ligands bind to specific receptors anchored on the cell membrane, and these circulating ligands, through the activity of the receptor's cytoplasmic domains, transduce the signaling pathway from the cell surface to the cell interior. TGFB signaling events can subsequently affect various cellular activities, leading to changes at the tissue and organismal levels.

[0007] One such change is fibrosis in musculoskeletal tissue. Indeed, increased expression of factors such as TGFB1 has been observed in fascial fibroblasts at sites of musculoskeletal injury. See Ihn, H. (2019). Allergology International, 68(4), 437-439. This expression pattern is associated with higher expression of type I collagen and fibronectin (ECM components), the aggregation of which may lead to fibrosis. TGFB is a known driver of pro-inflammatory signaling, providing a link between fibrosis and / or scar formation and chronic inflammation.

[0008] Current treatments for this type of musculoskeletal condition, 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 lead to further trauma. Therefore, there is an urgent need for new approaches and compositions that address the root causes of this condition. Summary of the Invention

[0009] This article provides compositions and methods for silencing the signaling function of TGFB1 or its cellular receptor in animals in need of treating diseases, ailments, or symptoms caused by abnormal or excessive signaling of said receptor.

[0010] 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, the CRISPR editing results in the ablation of the transmembrane domain (i.e., the production of a soluble receptor). In some embodiments, the CRISPR editing results in the ablation of the cytoplasmic domain.

[0011] The ubiquity of the receptor-ligand paradigm in cell biology means that many diseases, ailments, and symptoms are caused, in whole or in part, by abnormal or excessive signaling from various cellular receptors, and various approaches have been employed to address this issue. Small and large molecules can be used to disrupt receptor-ligand interactions, but off-target effects and potential immunogenicity remain concerns.

[0012] Recently, genetic approaches have been investigated to temporarily reduce (i.e., knock down) the expression of a given ligand or receptor (e.g., in the case of siRNA) or permanently eliminate (i.e., gene knockout) the expression of a given ligand or receptor. However, more advanced genetic tools are needed to address the vast number of diseases, ailments, and symptoms caused by this aberrant signaling.

[0013] It is worth noting that the compositions and methods disclosed herein also cover various routes of administration of CRISPR components, with a particular focus on post-traumatic and postoperative interventions to treat and prevent fibrotic tissues such as musculoskeletal tissue, cardiac tissue, lung tissue, kidney tissue, and so on. Attached Figure Description

[0014] The embodiments disclosed herein will be further explained with reference to the accompanying drawings. The drawings are not necessarily drawn to scale, but rather focus on illustrating the principles of the disclosed embodiments.

[0015] Figure 1A , 1B 1C, 1D, 1E, 1F, 1G, and 1H collectively represent SEQ ID NO: 1-198, (AD) a spacer sequence of CRISPR RNA (crRNA) generated by the bioinformatics methods described herein, which enables CRISPR / Cas9 to target human TGFB1 to produce gene knockout, and (EH) additional information regarding the bound DNA, the targeted DNA strand, the genomic coordinates of the targeted exon, and several quality control parameters.

[0016] Figure 2A , 2B Images 2C, 2D, 2E, and 2F together illustrate SEQ ID NO: 199-320, (AC) a spacer sequence of crRNA generated by the bioinformatics methods described herein, which enables CRISPR / Cas9 to target human TGFBR1 to produce a gene knockout, soluble, or membrane-bound decoy receptor, and (DF) additional information regarding the bound DNA, the targeted DNA strand, the genomic coordinates of the targeted exon, and several quality control parameters.

[0017] Figure 3A , 3B 3C, 3D, 3E, 3F, 3G, and 3H collectively represent SEQ ID NO: 321-519, (AD) a spacer sequence of crRNA generated by the bioinformatics methods described herein, which enables CRISPR / Cas9 to target human TGFBR2 to produce a gene knockout, soluble, or membrane-bound decoy receptor, and (EH) additional information regarding the bound DNA, the targeted DNA strand, the genomic coordinates of the targeted exon, and several quality control parameters.

[0018] Figure 4A and 4BThe diagram shows (A) a schematic representation of the targeted domains of TGFBR1 and the orientation of various spacer sequences that are predicted to produce knockout Cas9-recruiting single-guide RNAs (sgRNAs), and (B) a set of parameters considered when designing sgRNAs targeting TGFBR1, as well as example protospacer DNA sequences that target sgRNAs.

[0019] Figure 5A and 5B The diagram shows (A) a schematic representation of the targeted domains of TGFBR1 and the orientation of various protospacer sequences targeted by CRISPR / Cas9, which are predicted to produce 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 when designing sgRNAs targeting TGFBR1, and example protospacer DNA sequences targeting sgRNAs.

[0020] Figure 6A and 6B The diagram shows (A) a schematic representation of the targeted domains of TGFBR2 and the orientation of various protospacer sequences targeted by CRISPR / Cas9, which are predicted to produce 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 when designing sgRNAs targeting TGFBR2, and example protospacer DNA sequences targeting sgRNAs.

[0021] Figure 7A and 7B A summary of the efficiency of editing the human TGFBR1 gene in THP-1 cells using identified sgRNAs and (A) wild-type Cas9 (WTCas9) or (B) high-fidelity (R691A) Cas9 (ARCas9) proteins is presented. Bar charts show the top-ranking and all other frameshift edits contributing to the knockout (KO) score, inferred from Sanger trace deconvolution.

[0022] Figure 8A and 8B This section summarizes the efficiency of top-level editing of the human TGFBR1 gene in THP-1 cells using identified sgRNAs and (A) wild-type Cas9 (WTCas9) or (B) high-fidelity (R691A) Cas9 (ARCas9) proteins. Bar charts show the top-level and all other frameshift edits contributing to the knockout (KO) score, inferred from Sanger trace deconvolution.

[0023] Figure 9A and 9B This paper summarizes the efficiency of editing the human TGFBR2 gene in THP-1 cells using identified sgRNAs and (A) wild-type Cas9 (WTCas9) or (B) high-fidelity (R691A) Cas9 (ARCas9) proteins. Bar charts show the top-ranking and all other frameshift edits contributing to the knockout (KO) score, inferred from Sanger trace deconvolution.

[0024] Figure 10A , 10B Figures 10C and 10D show the relative levels of TGFB1 and TIMP1 gene expression in unedited (WT) or edited cells (edited to knock out TGFB1 (OHTG), TGFBR1 (OHTIR), or TGFBR2 (OHTIIR) cells after 6 hours of stimulation with lipopolysaccharide (LPS) (Figures A and B) or TGF-β (Figures C and D).

[0025] Figure 11A , 11B 11C and 11D show the relative levels of expression of (A) SERPINE1, (B) Col1A2, (C) FN1 and (D) CTGF genes in unedited (WT) or edited TGF-β cells to knock out TGFB1 (OHTG), TGFBR1 (OHTIR) or TGFBR2 (OHTIIR) after 6 hours of TGF-β stimulation.

[0026] Figure 12A , 12B Together with 12C, it presents spacer sequences for sgRNA targeting human (A)TGFB1 (SEQ ID NO:520-527), (B)TGFBR1 (SEQ ID NO:528-552), and (C)TGFBR2 (SEQ ID NO:553-604) to validate in vitro edited crRNAs with different delivery modes. Detailed Implementation

[0027] I. Introduction

[0028] This article provides compositions and methods for silencing the signaling function of one or more cellular receptors in animals in need of treating diseases, ailments, or symptoms caused by abnormal or excessive signaling of said receptors.

[0029] In some embodiments, receptor signaling is reduced or eliminated by editing the TGFB1, TGFBR1, and / or TGFBR2 genes using the compositions and methods described herein. In some embodiments, the TGFB1, TGFBR1, and / or TGFBR2 genes are edited to knock out (gene editing) so that they no longer encode functional proteins. In some embodiments, the editing results in the ablation of transmembrane domains of proteins encoded by the TGFBR1 or TGFBR2 genes, for example, generating soluble receptor decoys. In some embodiments, the editing results in the ablation of cytoplasmic domains of proteins encoded by the TGFBR1 or TGFBR2 genes, for example, generating membrane-bound receptor decoys. In some embodiments, TGFB1, TGFBR1, and / or TGFBR2 are regulated in expression by altering coding or non-coding gene regulatory sequences or their epigenetic landscape. In some embodiments, the receptor is TGFBR1. In some embodiments, the receptor is TGFBR2. In some embodiments, receptor signaling is reduced or eliminated by editing the TGFB1 ligand using the compositions and methods described herein.

[0030] II. Definition

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All patents and publications mentioned herein are incorporated herein by reference in their entirety.

[0032] The term “transforming growth factor β1” or “TGFB1” refers to the gene (NCBI Gene ID:7040 [human], NCBI Gene ID:403998 [dog], NCBI Gene ID:100033900 [horse], NCBI Gene ID:768263 [cat]) or its encoded gene product (e.g., UniProt:P01137; AAH00125..1 [human], XP_038512824.1 [dog], XP_014716505.1 [horse], XP_006941294.1 [cat]), as well as sequence variants, proteins with conserved amino acid substitutions, and their glycoforms. Typically, the cytokine proteins encoded by the genes listed above can bind to the TGFBR complex and mediate intracellular signaling pathways regulating multiple physiological and pathological processes, including inflammatory processes, through the release of SMAD2. SMAD2 can then translocate to the nucleus or activate other cytoplasmic signaling mediators. In some cases, and solely for the purpose of disambiguation, prefixes are added when referring to proteins or genes of specific species (h, c, e, and f refer to humans, dogs, horses, and cats, respectively).

[0033] In some 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 intercalated intron regions, intron / exon junctions, the 3' UTR, or polyadenylation signals) can be targeted by an RNA-guided nuclease to alter the gene. In some embodiments, the TGFB1 gene targeted by the RNA-guided nuclease is derived from mammals. In some embodiments, the TGFB1 gene targeted by the RNA-guided nuclease is derived from humans (hTGFB1). In some embodiments, the TGFB1 gene targeted by the RNA-guided nuclease is derived from dogs (cTGFB1). In some embodiments, the TGFB1 gene targeted by the RNA-guided nuclease is derived from horses (eTGFB1). In some embodiments, the TGFB1 gene targeted by the RNA-guided nuclease is derived from cats (fTGFB1).

[0034] The term “transforming growth factor β receptor 1” or “TGFBR1” refers to the gene (NCBI Gene ID:7046 [human], NCBI Gene ID:481628 [dog], NCBI Gene ID:100034117 [horse], NCBI Gene ID:101094057 [cat]) or its encoded gene product (e.g., UniProt:P36897; NP_004603.1 [human], XP_038538191.1 [dog], XP_023485510.1 [horse], XP_023098269.1 [cat]), as well as sequence variants, proteins with conserved amino acid substitutions, and their glycoforms. Typically, the proteins encoded by the genes listed above are transmembrane serine / threonine kinases that, together with TGFBR2, form the natural receptors for the TGF-β cytokines TGFB1, TGFB2, and TGFB3. When TGFBR1 binds to its ligand, it is phosphorylated by TGFBR2, which activates intracellular signaling via the release of SMAD2. This intracellular signaling regulates multiple physiological and pathological processes. SMAD2 can then translocate to the nucleus or activate other cytoplasmic signaling mediators. In some cases, and solely for the purpose of disambiguation, prefixes are added when referring to proteins or genes of specific species (h, c, e, and f refer to humans, dogs, horses, and cats, respectively).

[0035] In some 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 intercalated intron regions, intron / exon junctions, the 3' UTR, or polyadenylation signals) can be targeted by an RNA-guided nuclease to alter the gene. In some embodiments, the TGFBR1 gene targeted by the RNA-guided nuclease is derived from mammals. In some embodiments, the TGFBR1 gene targeted by the RNA-guided nuclease is derived from humans (hTGFBR1). In some embodiments, the TGFBR1 gene targeted by the RNA-guided nuclease is derived from dogs (cTGFBR1). In some embodiments, the TGFBR1 gene targeted by the RNA-guided nuclease is derived from horses (eTGFBR1). In some embodiments, the TGFBR1 gene targeted by the RNA-guided nuclease is derived from cats (fTGFBR1).

[0036] The term “transforming growth factor β 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 its encoded gene product (e.g., UniProt:P37173; NP_003233.4 [human], XP_038288013.1 [dog], XP_023475502.1 [horse], XP_023116415.1 [cat]), as well as sequence variants, proteins with conserved amino acid substitutions, and their glycoforms. Typically, the proteins encoded by the genes listed above are transmembrane serine / threonine kinases that, together with TGFBR2, form the natural receptors for the TGF-β cytokines TGFB1, TGFB2, and TGFB3. When TGFBR1 binds to its ligand, it is phosphorylated by TGFBR2, activating intracellular signaling through the release of SMAD2. This intracellular signaling regulates multiple physiological and pathological processes. SMAD2 can then translocate to the nucleus or activate other cytoplasmic signaling mediators. In some cases, and solely for the purpose of disambiguation, prefixes are added when referring to proteins or genes of specific species (h, c, e, and f refer to humans, dogs, horses, and cats, respectively).

[0037] In some 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 intercalated intron regions, intron / exon junctions, the 3' UTR, or polyadenylation signals) can be targeted by an RNA-guided nuclease to alter the gene. In some embodiments, the TGFBR2 gene targeted by the RNA-guided nuclease is derived from mammals. In some embodiments, the TGFBR2 gene targeted by the RNA-guided nuclease is derived from humans (hTGFBR2). In some embodiments, the TGFBR2 gene targeted by the RNA-guided nuclease is derived from dogs (cTGFBR2). In some embodiments, the TGFBR2 gene targeted by the RNA-guided nuclease is derived from horses (eTGFBR2). In some embodiments, the TGFBR2 gene targeted by the RNA-guided nuclease is derived from cats (fTGFBR2).

[0038] The term "treatment" refers to achieving a desired pharmacological and / or physiological effect. The effect may be preventative in relation to the complete or partial prevention of a disease or its symptoms, and / or therapeutic in relation to the partial or complete cure of a disease and / or adverse effects attributable to said disease. For example, the compositions, methods, or systems of this disclosure may be administered as preventative treatment to subjects predisposed to a given symptom (e.g., arthritis). As used herein, "treatment" encompasses any treatment of a disease in mammals (particularly in humans, canines, felines, or equines) and includes: (a) preventing the development of said disease in subjects who may be susceptible but have not yet been diagnosed with said disease; (b) inhibiting the disease, i.e., preventing the development or progression of the disease; and (c) alleviating the disease, i.e., causing the remission of the disease and / or alleviating one or more symptoms of the disease.

[0039] "Therapeutics" also means the delivery of a pharmaceutical agent to provide a pharmacological effect even in the absence of disease or symptoms. For example, "therapeutics" encompasses the delivery of a composition that can elicit an immune response or confer immunity in the absence of a disease condition, such as in the case of a vaccine. It should be understood that the compositions and methods disclosed herein are suitable for treating all mammals, including but not limited to human, canine, feline, equine, and bovine subjects.

[0040] The term "therapeuticly effective" refers to an amount sufficient to achieve the intended application, including but not limited to the treatment of a disease, using a composition or combination of compositions as described herein. Therapeuticly effective amounts may vary depending on the intended application (in vitro or in vivo), the subject being treated and the disease condition (e.g., the subject's weight, age, and sex), the severity of the disease condition, or the method 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). Specific doses will vary depending on the particular composition selected, the dosing regimen followed, whether the composition is administered in combination with other compositions or compounds, the timing of administration, the tissue to be administered, and the physical delivery system in which the composition is carried.

[0041] The terms “fibrosis” and “fibrotic scarring” are used interchangeably and refer to the formation of an abnormal amount of fibrous tissue in an organ or tissue, usually resulting from inflammation, irritation, or healing of the tissue. Fibrosis encompasses clinical phenomena commonly referred to as scarring, such as tissue scarring or organ scarring, as well as adhesions, such as fibrosis forming between organs or different tissues. Fibrosis can occur in any tissue, including but not limited to: joint tissues (e.g., knee joints, shoulder joints—e.g., adhesive capsulitis—elbow joints, etc.), other musculoskeletal tissues (e.g., tendon scarring), kidney tissues (e.g., in chronic kidney disease, etc.), skin tissues (e.g., related to wound healing—e.g., post-traumatic or post-operative conditions in humans—keloid scarring, renal systemic fibrosis or scleroderma / systemic sclerosis, equine edema, etc.), and lung tissues (e.g., fibrothorax, pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, radiation-induced lung injury, progressive massive fibrosis, scleroderma / systemic sclerosis, etc.). This includes tissues such as liver (e.g., cirrhosis, bridging fibrosis), heart (e.g., interstitial fibrosis—often associated with congestive heart failure, hypertension, and normal aging—alternative fibrosis—often associated with a history of myocardial infarction—etc.), brain (e.g., glial scars), intestinal tissue (e.g., Crohn's disease), hands and / or fingers (e.g., Dupuytren's contracture), lymphoid tissue (e.g., mediastinal fibrosis), bone marrow (e.g., myelofibrosis), penis (e.g., Peyronie's disease), retroperitoneal soft tissue (e.g., retroperitoneal fibrosis), and musculoskeletal tissue.

[0042] In some embodiments, fibrosis is caused by tissue trauma. 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, autologous ACL reconstruction, allogeneic ACL reconstruction, fracture repair, total knee arthroplasty (TKA), and microsurgical discectomy. Additionally, fibrosis can be caused by any of the following conditions that may be induced or exacerbated by surgical procedures: knee fibrosis, intra-articular fibrotic nodules, or epidural fibrosis.

[0043] As used herein, the term "musculoskeletal trauma" refers to any injury affecting bones, muscles, ligaments, nerves, or tendons. In some embodiments, the trauma is the result of a surgical procedure.

[0044] 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 spliced ​​or unspliced ​​mRNA, rRNA, tRNA, lncRNA, RNAantagomirs, and repressive DNA or RNA (RNAi, e.g., small or short hairpin (sh) RNA, microRNA (miRNA), aptamers, small or short interfering (si) RNA, trans-spliced ​​RNA, or antisense RNA). Polynucleotides also include non-coding RNAs, including but not limited to RNAi, miRNA, lncRNA, RNA antagomirs, aptamers, and any other non-coding RNA known to those skilled in the art. Polynucleotides include naturally occurring, synthetic, and intentionally altered or modified polynucleotides, as well as analogs and derivatives. The term “polynucleotide” also refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides or their analogs, and is synonymous with a nucleic acid sequence. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. Modifications to the nucleotide structure may be imparted, if present, before or after polymer assembly. As used herein, the term polynucleotide is interchangeable with both double-stranded and single-stranded molecules. Unless otherwise stated or required, any embodiment of polynucleotides as described herein encompasses both double-stranded forms and each of two complementary single-stranded forms known or predicted to constitute a double-stranded form. Polynucleotides may be single-stranded, double-stranded, or triple-stranded, linear or cyclic, and can be of any length. When discussing polynucleotides, the sequence or structure of a particular polynucleotide may be described herein according to the convention of providing sequences in the 5' to 3' directions.

[0045] The term "gene" or "nucleotide sequence encoding a polypeptide" refers to a segment of DNA involved in the production of a polypeptide chain. DNA segments can include regions before and after the coding regions (precursors and trailers) involved in the transcription / translation of the gene product and the regulation of transcription / translation, as well as insertion sequences (introns) between the individual coding segments (exons). For example, a gene includes a polynucleotide containing at least one open reading frame capable of encoding a specific protein or polypeptide after transcription and translation.

[0046] The term "extracellular domain" refers to a transmembrane cell receptor, defined as a portion of a protein that is exposed to the extracellular environment and is capable of binding and / or interacting with ligands.

[0047] The terms "cytoplasmic domain" and "intrinsic domain" are used interchangeably; when referring to transmembrane receptors, it defines a portion of a protein exposed in the cytoplasm. In many cases, these portions of a protein contain signaling domains that recruit and associate various intracellular factors. After binding to ligands via extracellular domains, interactions change, and these changes can result in new associations, dissociations, or recruitments of various cytoplasmic factors that contribute to signal transduction.

[0048] The term "transmembrane domain" refers to a transmembrane receptor, defined as a portion of a protein embedded within the plasma membrane (i.e., not exposed to the extracellular environment or cytosol). Transmembrane domains typically possess stronger hydrophobic properties than their extracellular or cytoplasmic portions and often employ a higher-order helical structure. While the primary role of ligand-induced conformational changes in a particular receptor is anchoring, these changes have been shown to affect the transmembrane domain, enabling it to become part of subsequent intracellular signaling.

[0049] The term "receptor" refers to a protein that can bind to another homologous protein (i.e., its ligand) with high affinity. This receptor-ligand interaction can be 1:1 or induce polymerization, in which multiple protein aggregates bind one or more ligands. Receptors are typically located on the cell surface so that they can most effectively encounter ligands and initiate intracellular signaling.

[0050] The term "intracellular signaling" refers to cellular changes resulting from events occurring at the cell surface. Typically, soluble ligands bind to receptors on the cell surface, inducing changes in these receptors, which in turn affect related intracellular factors. These factors can then influence other factors within the cell, and this cascade reaction continues. In many cases, a specific factor can respond to surface stimuli by altering gene expression in the cell nucleus.

[0051] The term "gene regulatory sequence" refers to a double-stranded DNA sequence in the genome that controls the level of gene transcription.

[0052] The term "RNA-guided nuclease" refers to an enzyme capable of finding and breaking the backbone of, for example, a DNA molecule. The activity of an RNA-guided nuclease is directed by a nucleic acid molecule (i.e., guide RNA). Once the enzyme is correctly oriented to form a functional ribonucleoprotein complex, it localizes to a specific location within a target nucleic acid (e.g., a gene or locus) through sequence complementarity with a portion of the gRNA. Incomplete examples of RNA-guided nucleases include Cas9, Cpf1, and Cas12.

[0053] The term "Cas9" refers to an RNA-guided double-stranded DNA-binding nuclease protein, a cleavage enzyme protein, or a protein without enzymatic activity, or a variant thereof, and can be used to refer to naturally occurring or recombinant Cas9 nuclease variants (e.g., ES-Cas9, HF-Cas9, PE-Cas9, and AR-Cas9). Cas9 can be further tethered to other functional protein domains, such as those of nucleobase deaminases, reverse transcriptases, epigenetic modifiers, or transcriptional effectors. Wild-type Cas9 nucleases have two functional domains, such as RuvC and HNH, which simultaneously cleave different strands of double-stranded DNA, resulting in double-strand breaks. The Cas9 enzymes described herein can contain an HNH or HNH-like nuclease domain and / or a RuvC or RuvC-like nuclease domain without affecting the ability to induce double-strand breaks in genomic DNA (e.g., at target loci) when both functional domains are active. Cas9 enzymes may contain one or more catalytic domains of a Cas9 protein derived from bacteria belonging to the group consisting of: *Corynebacterium*, *Sutterella*, *Legionella*, *Treponema*, *Filifactor*, *Eubacterium*, *Streptococcus*, *Lactobacillus*, *Mycoplasma*, and *Bacteroides*. s), Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, and Campylobacter. In some embodiments, the two catalytic domains originate from different bacterial species.

[0054] As used herein, “PAM” refers to the protospacer adjacent motif, which is essential for RNA-guided nuclease binding to target nucleic acids. In many cases, the PAM is directly adjacent to the complementary sequence in the target. For example, naturally occurring Cas9 molecules can recognize specific PAM sequences (see, for example, Table 1). In some embodiments, Cas9 molecules have the same PAM specificity as naturally occurring Cas9 molecules. In other embodiments, Cas9 molecules have PAM specificity unrelated to naturally occurring Cas9 molecules. In other embodiments, the PAM specificity of a Cas9 molecule is independent of its closest sequence homology with that of a naturally occurring Cas9 molecule. For example, naturally occurring Cas9 molecules can be modified such that PAM sequence recognition is altered to reduce off-target sites, improve specificity, or eliminate the need for PAM recognition. In one embodiment, the Cas9 molecule can be modified (e.g., to lengthen the PAM recognition sequence, improve Cas9 specificity to a high level of identity, reduce off-target sites, and / or increase specificity). In one embodiment, the length of the PAM recognition sequence is at least 4, 5, 6, 7, 8, 9, 10, or 15 amino acids. In some embodiments, the Cas9 molecule can be modified to ablate PAM recognition.

[0055] The terms "guide RNA," "gRNA," or "sgRNA" are used interchangeably and refer to an RNA molecule consisting of a target spacer sequence and a scaffold sequence, preferably a single guide RNA molecule synthesized. Once these molecules are added to a functional RNA-guided nuclease, they can direct the sequence-specific cleavage of the target nucleic acid.

[0056] sgRNA can be administered or formulated, for example, as synthetic RNA, or as a nucleic acid containing a sequence encoding gRNA, and then the nucleic acid is expressed in target cells. It will be apparent to those skilled in the art that various tools can be used to design and / or optimize sgRNA to, for example, increase the specificity and / or precision of genome editing at specific sites.

[0057] Generally, candidate sgRNAs can be designed and identified through the following steps: First, a suitable PAM is located within the genomic sequence. Then, additional computations can be used to predict on-target and off-target efficiencies. Available web-based tools that aid in the initial design and modeling of candidate sgRNAs include, but are not limited to, CALITAS, SWOffinder, CRISPRseek, CRISPR Design Tool, Cas-OFFinder, E-CRISP, ChopChop, CasOT, CRISPR direct, CRISPOR, BREAKING-CAS, CrispRGold, and CCTop. See, for example, Safari et al., Current Pharma Biotechol. (2017) 18(13), which is incorporated herein by reference in its entirety for all purposes. Such tools are also described, for example, in PCT Publication No. WO2014093701A1 and Liu et al., “Computational approached for effective CRISPR guide RNA design and evaluation,” *Computational StructBiotechnol J.*, 2020; 18:35-44, each of which is incorporated herein by reference in its entirety for all purposes. Candidate sgRNAs can be further evaluated through experimental screening or other methods.

[0058] The term "spacer" refers to a portion of an sgRNA molecule that is complementary to the target nucleic acid. Typically, a spacer is a portion of a CRISPR RNA (crRNA) that hybridizes to the target nucleic acid, for example, via a spacer sequence, and to a trans-activating crrRNA (tracrRNA), for example, via a non-target sequence. However, when crRNA is used herein in conjunction with the sequence of a target gene (e.g., the TNFB or TNFBR gene), the sequence referred to is the spacer sequence, not the spacer and non-target sequences typically present in a crRNA molecule. In sgRNA, crRNA and tracrRNA are combined into a single molecule.

[0059] The phrase “pharmaceutically acceptable” means those compounds, materials, compositions, and / or dosage forms that, to a reasonable extent of medical judgment, are suitable for use in contact with tissues of humans and animals without excessive toxicity, irritation, allergic reactions, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0060] The terms "pharmaceutically acceptable carrier" or "pharmaceuticalally acceptable excipient" are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delay agents, as well as inert components. The use of such pharmaceutically acceptable carriers or excipients for the active pharmaceutical ingredient is well known in the art. Their use in the therapeutic compositions of this disclosure is contemplated, except that any conventional pharmaceutically acceptable carrier or excipient is incompatible with the active pharmaceutical ingredient. Additional active pharmaceutical ingredients, such as other pharmaceutical agents, may also be incorporated into the described compositions and methods.

[0061] The term "pharmaceutically acceptable excipient" is intended to include media and carriers that can be co-administered with a compound to promote its intended function. The use of such media for pharmaceutically active substances is well known in the art. Examples of such media and carriers include solutions, solvents, dispersion media, delay agents, emulsions, etc. Any other conventional carriers suitable for use with multi-binding compounds also fall within the scope of this disclosure.

[0062] As used herein, the terms “a,” “an,” or “the” are generally interpreted to encompass both the singular and plural forms.

[0063] The terms “about” and “approximately” mean a range that is statistically significant for a value. This range may be within the 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 permissible variation covered by the terms “about” or “approximately” depends on the specific system under study and will be readily understood by those skilled in the art. Furthermore, as used herein, the terms “about” and “approximately” mean that compositions, amounts, formulations, parameters, shapes, and other quantities and characteristics are not precise and do not need to be precise, but may be approximate and / or larger or smaller as required, reflecting tolerances, conversion factors, rounding, measurement errors, and other factors known to those skilled in the art. Generally, dimensions, sizes, formulations, parameters, shapes, or other quantities or characteristics are “about” or “approximate”, whether or not they are explicitly stated. It should be noted that embodiments of very different sizes, shapes, and dimensions may be adopted in the described arrangements.

[0064] As used herein, the term “substantially” can mean most or most, 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.

[0065] When used in the appended claims, both in their original and modified forms, the transitional terms “comprising,” “substantially consisting of,” and “consisting of” define the scope of the claims with respect to which unrecited elements or steps of the appended claims (if any) are excluded from the scope of the claims. The term “comprising” is intended to be inclusive or open-ended and does not exclude any additional, unrecited elements, methods, steps, or materials. The term “consisting of” excludes any elements, steps, or materials other than those specified in the claims, and in the latter case, excludes impurities typically associated with the specified materials. The term “substantially consisting of” limits the scope of the claims to the specified elements, steps, or materials, and those elements, steps, or materials that do not substantially affect the essential and novel features of the claimed method and composition. In alternative embodiments, all compositions, methods, and kits embodying this disclosure described herein may be more specifically defined by any of the transitional terms “comprising,” “substantially consisting of,” and “consisting of.”

[0066] III. Methods

[0067] A.CRISPR

[0068] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While methods and materials similar to or equivalent to those described herein may be used to practice or test this disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. Furthermore, materials, methods, and examples are illustrative only and are not intended to be limiting.

[0069] In one aspect, this disclosure covers compositions involving clustered regularly spaced short palindromic repeats (CRISPR) and CRISPR-associated RNA-guided nucleases, as well as related methods, components, and compositions (hereinafter referred to as CRISPR / Cas systems). Such systems require at least one isolated or non-naturally occurring RNA-guided nuclease (e.g., Cas9 protein) and at least one isolated or non-naturally occurring guide RNA (e.g., sgRNA) to achieve enhancement of a nucleic acid sequence (e.g., genomic DNA) or its epigenetic characteristics (e.g., DNA methylation).

[0070] In some embodiments, the CRISPR / Cas system achieves alterations to a targeted gene or locus in eukaryotic cells by changing the sequence at the target location, for example, by producing an insertion or deletion (collectively, insertion / deletion) that results in loss of function (i.e., knockout) of the affected gene or allele; for example, by truncating, nonsense, or other types of loss-of-function nucleotide substitutions that result in truncation, nonsense mutation, or other types of loss of function of the coding products (i.e., mRNA or protein) of one or more TGFB1, TGFBR1, or TGFBR2 genes; by deleting one or more nucleotides that result in truncation, nonsense mutation, or other types of loss of function of the coding products of one or more TGFB1, TGFBR1, or TGFBR2 genes; for example, by loss of function of the coding mRNA or protein resulting from a single nucleotide, dinucleotide, or other frameshift deletion, or by a deletion that produces a premature stop codon; or by inserting a gene product (e.g., the coding product of one or more TGFB1, TGFBR1, or TGFBR2 genes) (i.e., mRNA or protein) that results in truncation, nonsense mutation, or other types of loss of function; for example, by a single nucleotide, dinucleotide, or other frameshift insertion, or by an insertion that produces a premature stop codon. In some embodiments, the CRISPR / Cas system of this disclosure provides alterations to genes and / or gene-encoded products, such alterations resulting in a loss of function and becoming a dominant-negative product or a decoy (e.g., a transmembrane receptor or soluble receptor that cannot elicit intracellular signaling). In some embodiments, the CRISPR / Cas system of this disclosure alters the transcriptional level of one or more TGFB1, TGFBR1, or TGFBR2 genes.

[0071] In one respect, the CRISPR / Cas system achieves changes in nucleic acid sequences through nuclease activity. For example, with respect to genomic DNA, RNA-guided nucleases locate target sites within genes or loci through sequence complementarity with target genomic sequences (e.g., spacer sequences of CRISPR RNA (crRNA) or synthetic guide RNA (sgRNA))—cutting genomic DNA upon recognition of a specific nuclease-specific motif called a protospacer neighbor motif (PAM). See Collias, D., & Beisel, CL (2021). Nature Communications, 12(1), 1-12.

[0072] In the case of genomic DNA, nuclease activity (i.e., cleavage) induces double-strand breaks (DSBs). Due to interference from the components of the CRISPR / Cas system, the endogenous cellular mechanisms of DSB repair—namely, non-homologous end joining (NHEJ), microhomologous-mediated end joining (MMEJ), and homologous recombination—lead to misrepair at a calculable frequency at a given target site, thereby introducing substitutions or insertions / deletions into the genomic DNA. See generally Scully, R. et al. (2019). Nature Reviews Molecular Cell Biology, 20(11), 698-714. At some frequency, these insertions / deletions and / or substitutions can lead to frameshifts, nonsense mutations (i.e., early stop codons), or truncations that affect the availability of gene products such as mRNA and / or proteins. In some embodiments, the CRISPR / Cas system can induce homologous directed repair (HDR) mechanisms by using a template (e.g., an HDR template) provided to the cell along with the nuclease and gRNA as part of the system, resulting in the insertion of non-random sequences at the target site. See Bloh, K., & Rivera-Torres, N. (2021). International Journal of Molecular Sciences, 22(8), 3834.

[0073] Generally, the minimum requirements for a CRISPR / Cas system will depend on the nuclease (i.e., Cas protein) provided with it. In this regard, these bacterial-derived nucleases have been functionally classified into types I, III, and V (all belonging to class 1) and types II, IV, and VI (which are grouped into class 2).

[0074] Class 1 CRISPR / Cas systems:

[0075] The exact components, compositions, and methods for achieving targeted changes in nucleic acid sequences using a Class 1 CRISPR / Cas system will vary, but should at a minimum include: a nuclease (e.g., selected from at least type I and type III), and at least one guide RNA, such as selected from 1) sgRNA or 2) a combination of crRNA and tracrRNA. These CRISPR / Cas systems are grouped together as Class 1 CRISPR / Cas systems due to their similarities in requirements and modes of action within eukaryotic cells. Therefore, the compositions, components, and methods in Class 1 components can be considered functionally interchangeable, and the following details, provided for illustrative purposes only, do not constitute an exhaustive list of class members:

[0076] As part of a larger complex capable of genome editing (a cascade of Cse1 and Cse2), Cas3 (see Table 1) is a prototype type I DNA nuclease acting as an effector protein. See He, L. et al. (2020). Genes, 11(2), 208. Unlike other CRISPR / Cas systems, the type I system targets DNA without the Cas3 nuclease via the cascade complex, which then recruits Cas3 to cleave the DNA upon binding and localization to 3'PAM. The cascade complex also processes crRNAs, making them available for guiding to the target site. Due to this function, the cascade has the ability to process multiple arrays of crRNAs from a single molecule. See Luo, M. (2015). Nucleic Acids Research, 43(1), 674-681. Thus, the type I system can be used to edit multiple targeted genes or loci from a single molecule.

[0077] Because the natural substrate of Cas3 is ssDNA, its function in genome editing is assumed to be that of a nicking enzyme; however, when targeted in tandem, the resulting editing is a blunt-end cleavage of the opposing strand to approximate the blunt cleavage of endonucleases such as Cas9. See Pickar-Oliver, A., & Gersbach, CA (2019). Nature Reviews Molecular Cell Biology, 20(8), 490-507.

[0078] Like type I nucleases, type III systems rely on protein complexes to achieve nucleic acid cleavage. Specifically, Cas10 possesses nuclease activity for cleaving ssDNA in prokaryotes. See Tamulaitis, G. Trends in Microbiology, 25(1), 49-61. Interestingly, the CRISPR / Cas system (native to archaea) exhibits dual specificity, targeting both ssDNA and ssRNA. Aside from this variation, the system functions very similarly to type I, as the crRNA targets the effector complex in a sequence-dependent manner (similar to a cascade). Similarly, the effector complex processes the crRNA prior to association. This dual nature of the nuclease could make its application in genome editing potentially more powerful, as both genomic DNA and, in some cases, mRNA with the same sequence can be targeted to silence specific targeted genes.

[0079] Two types of CRISPR / Cas systems:

[0080] The exact components, compositions, and methods for achieving targeted changes in nucleic acid sequences using Class 2 CRISPR / Cas systems will vary, but should at a minimum include: a nuclease (selected from at least type II and type V), and at least one guide RNA selected from 1) sgRNA or 2) a combination of crRNA and tracrRNA. These CRISPR / Cas systems are grouped together as Class 2 CRISPR / Cas systems due to their similarities in requirements and modes of action within eukaryotic cells. Therefore, the compositions, components, and methods in Class 2 components can be considered functionally interchangeable, and the following details, provided for illustrative purposes only, do not constitute an exhaustive list of class members:

[0081] Type II nucleases are the best-characterized CRISPR / Cas systems, particularly the typical genome-editing nuclease Cas9 (see Table 1). Multiple Cas9 proteins derived from various bacterial species have been isolated. The main difference between these nucleases is the PAM, a desired recognition site within the targeted dsDNA. After association with a gRNA molecule, the spacer sequence orients the nuclease to the appropriate location, but protein recognition of the PAM is what induces a cleavage event near the site, resulting in a blunted DSB.

[0082] In addition to the naturally occurring Cas9 protein, several engineered variants have been similarly reported. These range from Cas9 with enhanced specificity (i.e., lower off-target activity), such as espCas9. Others have been catalytically modified via point mutations in the RuvC (e.g., D10A) and HNH (e.g., H840A) domains, causing them to induce only single-strand breaks (i.e., Cas9 nickases). See Frock, R. et al. (2015). Nature Biotechnology, 33(2), 179-186. These variants have also been shown to be less prone to errors during editing. Such mitigation of off-target effects becomes crucial when the desired insertion (i.e., knock-in mutation, where the desired nucleotide sequence is introduced into the target nucleic acid molecule) is chosen rather than a deletion. In fact, fewer off-target effects can contribute to the preferred DNA repair mechanism (HDR, which is used in most cases for knock-in mutations). See Naeem, M. et al. (2020). Cells, 9(7), 1608.

[0083] In some embodiments, the CRISPR-related endonuclease is a Cas9 endonuclease. In some embodiments, the Cas9 endonuclease contains amino acid substitutions of K848A, K1003A, and R1060A. In some aspects, the Cas9 endonuclease contains an amino acid substitution of R691A. In some embodiments, the Cas9 endonuclease contains amino acid substitutions of N497A, R661A, Q695A, and Q926A. In some embodiments, the Cas9 endonuclease contains amino acid substitutions of N692A, M694A, Q695A, and H698A. In some aspects, the Cas9 endonuclease contains amino acid substitutions of R63A and Q768A. In some aspects, the Cas9 endonuclease contains amino acid substitutions of M495V, Y515N, K526E, and R661Q. In some aspects, the Cas9 endonuclease contains amino acid substitutions of K526E or K526N. In some embodiments, the Cas9 endonuclease contains amino acid substitutions of F539S, M763I, and K890N. In some embodiments, the Cas9 endonuclease contains amino acid substitutions of E1007L. In some embodiments, the Cas9 endonuclease contains amino acid substitutions of N690C, T769I, G915M, and N980K. In some embodiments, the Cas9 endonuclease contains amino acid substitutions of E1007L.

[0084] Other exemplary engineered variants of the typical Cas protein (e.g., mutants, chimeras) include the following patents (each of which is incorporated herein by reference in its entirety for all purposes): WO2015035162A2, WO2019126716A1, WO2019126774A1, WO2014093694A1, WO2014150624A1, US20190225955A1, U.S. Patent 11427818, U.S. Patent 11242542, U.S. Patent 11098297, U.S. Patent 10876100, U.S. Patent 10767193, U.S. Patent 10494621, and U.S. Patent 10100291.

[0085] To avoid ambiguity, the Cas9 (SpCas9) of Streptococcus pyogenes is collectively referred to as any one of the following groups: espCas9 (also referred to as ESCas9 or esCas9 in this article), HFCas9, PECas9, and ARCas9.

[0086] Like the typical Cas9 system, type V nucleases require only synthetic sgRNAs with target domains complementary to the genomic sequence for genome editing. These nucleases contain the RuvC domain but lack the HNH domain of type II nucleases. Furthermore, for example, Cas12 leaves staggered cuts in the dsDNA substrate distal to PAM, compared to the blunt cut of Cas9 adjacent to PAM. Both Cas12a (also known as Cpf1) and Cas12b (also known as C2c1) (see Table 1) are part of a larger complex of two gRNA-associated nucleases that act on dsDNA in a quaternary structure, cleaving each strand simultaneously. See Zetsche B, et al. Cell. 2015; 163(3):759-771; see also Liu L, Chen P, Wang M, et al. Molecular Cell. 2017; 65(2):310-322. In addition, Cas12b (C2c1) is a highly accurate nuclease with little tolerance to mismatches. See Yang H, et al. Cell. 2016; 167(7):1814-1828.e12.

[0087] Table 1. Exemplary list of Cas nucleases and their requirements

[0088]

[0089] See generally Wang, J., Zhang, C., & Feng, B. (2020). Journal of Cellular and Molecular Medicine, 24(6), 3256-3270, where N = any nucleotide; R = any purine (A or G); Y = any pyrimidine (C or T); W = A or T; V = A, C or G.

[0090] In one aspect, the CRISPR / Cas system of this disclosure comprises at least one RNA-guided nuclease (e.g., a Cas protein) derived from one or more of the following selected bacterial genera: Corynebacterium, Sartorius, Legionella, Spirochetes, Filamentosa, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flavobacterium, Spirochetes, Azotobacter, Gluconobacterium, Neisseria, Rosaceae, Corynebacterium, Nitrifying Bacteria, Campylobacter, Pseudomonas, Streptococcus, Staphylococcus, Francisella, Acidobacterium, Trichophyton, Trichophyton, and Prevotella. In some embodiments, the Cas protein is derived from a species of bacteria in the class Delta-Proteobacteria or the phylum Planctomycetes.

[0091] Some aspects of this disclosure provide strategies, methods, compositions, and therapeutic approaches for causing loss of function of a targeted gene product by altering a targeted sequence within a gene locus (e.g., altering the sequence of wild-type and / or mutant sequences within cells or mammals) through the insertion or deletion of one or more nucleotides mediated by RNA-guided nucleases and one or more guide RNAs (gRNAs). In some embodiments, loss of function “knocks out” the target gene by ablating gene expression (i.e., producing a “knockout”). In some embodiments, loss of function produces a non-functional gene product (i.e., a gene product that does not have all the functions of the wild-type gene product). In some embodiments, loss of function induces the expression of a gene product with different characteristics (e.g., different binding affinity or different cellular localization).

[0092] In some embodiments, the targeted gene is selected from TGFB1, TGFBR1, TGFBR2, and combinations thereof. In some embodiments, any region of the targeted gene (e.g., promoter region, 5' untranslated region, 3' untranslated region, exon, intron, or exon / intron boundary) may be targeted by an RNA-guided nuclease to alter the gene, its expression, or both. In some embodiments, non-coding regions of the targeted gene (e.g., enhancer region, promoter region, intron, 5'UTR, 3'UTR, polyadenylation signal) are targeted to alter the gene, its expression, or both.

[0093] CRISPR guide RNA:

[0094] In one aspect, the CRISPR / Cas system of this disclosure also provides a gRNA molecule (e.g., isolated or non-naturally occurring RNA molecule) that interacts with an RNA-guided nuclease. In some 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 crRNA and the 5' end of tracrRNA) that interacts with an RNA-guided nuclease, thereby locating the spacer sequence to scan for complementarity in the target nucleic acid. In some embodiments, the system is further optionally composed of oligonucleotides, which are HDR templates homologous to either side of the target site. See Bloh, K., & Rivera-Torres, N, at 3836.

[0095] In one embodiment, the RNA-guided nuclease and sgRNA are configured as directionally correlated nucleases such that cleavage events (e.g., double-strand breaks or single-strand breaks) occur sufficiently close to a complementary sequence in the targeted nucleic acid, thereby facilitating changes in 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.

[0096] In some embodiments, the spacer sequence-directed RNA-guided nuclease causes a cleavage event to occur 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. Double-strand DNA breaks or single-strand DNA breaks can be located upstream or downstream of the complementary sequence in the targeted 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.

[0097] In some embodiments, a second gRNA molecule containing a second spacer sequence directs the second RNA-guided nuclease such that the cleavage event occurs sufficiently close to a complementary sequence in the targeted nucleic acid, thereby promoting a change in the nucleic acid sequence. In some embodiments, the first gRNA and the second gRNA promote cleavage events within a single targeted gene. In some embodiments, the first gRNA and the second gRNA promote cleavage events within different targeted genes. In some embodiments, the length of the second spacer sequence is 20 nucleotides. In some embodiments, the length of the second spacer sequence is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

[0098] In some embodiments, the second spacer sequence-directed RNA-guided nuclease causes a cleavage event to occur 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. Double-strand DNA breaks or single-strand DNA breaks can be located upstream or downstream of the complementary sequence in the targeted 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.

[0099] In some embodiments, the targeting domains of the first and second gRNAs are configured such that the cleavage event independently localizes to 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 other cleavage events for each gRNA molecule. In some embodiments, the first and second gRNA molecules simultaneously alter the targeted nucleic acid sequence. In some embodiments, the first and second gRNA molecules sequentially alter the targeted nucleic acid sequence.

[0100] In some embodiments, a single-strand break is accompanied by a second single-strand break, the breaks being located by spacer sequences of the first and second gRNAs, respectively. In some embodiments, the spacer sequences can direct the associated RNA-guided nuclease such that the cleavage events (e.g., two single-strand breaks) are located within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, or 200 nucleotides from each other. In some embodiments, the first and second spacer sequences are configured as direction-associated RNA-guided nucleases such that, for example, two single-strand breaks occur at the same position on opposite strands of the genomic DNA, or differ from each other by 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides, thereby substantially approximating a double-strand break.

[0101] In some embodiments, the nucleic acid encoding one or more spacer sequences is selected from any sequences disclosed in Table 2. In some embodiments, the nucleic acid encoding one or more spacer sequences is selected from any sequences disclosed in Table 3. In some embodiments, the nucleic acid encoding one or more spacer sequences is selected from any sequences disclosed in Table 4. In some embodiments, the nucleic acid encodes a second sgRNA molecule. In some embodiments, the nucleic acid encodes a third sgRNA molecule. In some embodiments, the nucleic acid encodes a fourth sgRNA molecule. In some embodiments, one or more sgRNAs are encoded by nucleic acids selected from any of SEQ ID NO:1-519.

[0102] In some embodiments, the nucleic acid may comprise (a) a sequence encoding a first sgRNA containing a spacer sequence complementary to a sequence in the targeted gene, (b) a sequence encoding a second sgRNA containing a spacer sequence complementary to a sequence in the second targeted gene, and (c) a sequence encoding an RNA-guided nuclease (e.g., Cas9). Optionally, (d) and (e) are sequences encoding a third sgRNA and a fourth sgRNA, respectively. In some embodiments, the second targeted gene is the same as the first targeted gene. In other embodiments, the second targeted gene is different from the first targeted 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 a single nucleic acid molecule. When more than two sgRNAs are used, any combination of (a), (b), (c), (d), and (e) can be encoded within a single or separate nucleic acid molecule.

[0103] In one aspect, nucleic acid molecules (i.e., those encoding (a), (b), (c), (d), or (e)) are delivered to target cells (i.e., any combination of an RNA-guided nuclease encoding (c) and at least one gRNA molecule encoding (a), (b), (d), or (e) contacts the target cell). In some embodiments, the nucleic acid molecules are delivered to target cells in vivo. In other embodiments, the nucleic acid molecules are delivered to target cells ex vivo. In some embodiments, the nucleic acid molecules are delivered to target cells in vitro. In some embodiments, the nucleic acid molecules are delivered to target cells as DNA. In other embodiments, the nucleic acid molecules are delivered to target cells as RNA (e.g., mRNA). In some embodiments, the product of the nucleic acid molecules is delivered as an assembled ribonucleoprotein (RNP).

[0104] In some embodiments, the contact target cell comprises delivery of a nuclease guided by the RNA of (c) as a protein having at least one of the nucleic acid molecules selected from (a), (b), (d), and (e). In some embodiments, the contact target cell comprises delivery of a nuclease guided by the RNA encoded by (c) as DNA having at least one of the nucleic acid molecules selected from (a), (b), (d), and (e). In some embodiments, the contact target cell comprises delivery of a nuclease guided by the RNA encoded by (c) as mRNA having at least one of the nucleic acid molecules selected from (a), (b), (d), and (e).

[0105] In some 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, organometallic nanoparticles, gold nanoparticles, and magnetic nanoparticles. See generally Xu, CF, et al. (2021). Advanced Drug Delivery Reviews, 168, 3-29.

[0106] B.TALEN

[0107] In one aspect, this disclosure envisions the use of methods, components, and compositions involving transcription activator-like effector nucleases (TALENs) to enhance nucleic acid sequences (e.g., targeted genes).

[0108] TALE stands for “transcription activator-like effector” protein, which includes TALEN (“transcription activator-like effector nuclease”). The gene editing method using the TALE system may also be referred to herein as the TALE approach. TALE is a naturally occurring protein from the plant pathogenic bacterium genus Xanthomonas and contains a DNA-binding domain consisting of a series of 33-35 amino acid repeating domains, each recognizing a single base pair. TALE specificity is determined by two highly variable amino acids called repetitive variable double residues (RVDs). Modular TALE repeat sequences are linked together to recognize adjacent DNA sequences. Specific RVDs in the DNA-binding domain recognize bases at target loci, providing structural features for assembling predictable DNA-binding domains. The DNA-binding domain of TALE is fused with the catalytic domain of an IIS-type FokI endonuclease to form a targetable TALE nuclease. To induce site-specific mutations, two separate TALEN arms, separated by 14-20 base-pair spacers, bring the FokI monomers very close together to dimerize and generate targeted double-strand breaks.

[0109] Several large-scale systematic studies utilizing various assembly methods have demonstrated that TALE repeat sequences can be combined to identify virtually any user-defined sequence. Custom-designed TALE arrays are also commercially available from Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuticals (Lexington, KY, USA), and Life Technologies (Grand Island, NY, USA). The TALE and TALEN methods applicable to this disclosure are described in U.S. Patent Application Publications US 2011 / 0201118A1; US2013 / 0117869 A1; US2013 / 0315884 A1; US2015 / 0203871A1 and US2016 / 0120906 A1, the disclosures of which are incorporated herein by reference.

[0110] Non-limiting examples of genes that can be silenced or suppressed by permanent gene editing using the TALE method include TGFB1, TGFBR1, TGFBR2, and combinations thereof. Non-limiting examples of genes that can be enhanced (so that their products can act as decoys or dominant-negative products) by permanent gene editing using the TALE method include TGFB1, TGFBR1, TGFBR2, and combinations thereof. Non-limiting examples of genes that can be enhanced by permanent gene editing using the TALE method include TGFB1, TGFBR1, TGFBR2, and combinations thereof. In one aspect, this disclosure provides compositions for upregulating protein receptors (including wild-type or gene-edited ones), including compositions that bind to anti-inflammatory cytokines via the TALE method.

[0111] Examples of systems, methods, and compositions for altering the expression of target gene sequences via the TALE method and for use in accordance with embodiments of this disclosure are described in U.S. Patent No. 8,586,526, which is incorporated herein by reference.

[0112] C. Zinc finger nucleases (ZFN)

[0113] In one aspect, this disclosure envisions the use of methods, components, and compositions involving zinc finger nucleases (ZFNs) to enhance nucleic acid sequences (e.g., targeted genes).

[0114] A single zinc finger contains approximately 30 amino acids in its conserved ββα conformation. Several amino acids on the surface of the α-helix typically contact 3 bp in the major groove of DNA, exhibiting varying levels of selectivity. The zinc finger possesses two protein domains. The first domain is a DNA-binding domain, which includes eukaryotic transcription factors and contains the zinc finger. The second domain is a nuclease domain, which includes the FokI restriction enzyme and is responsible for the catalytic cleavage of DNA.

[0115] Individual zinc finger (ZFN) DNA-binding domains typically contain three to six separate zinc finger repeat sequences, each recognizing 9 to 18 base pairs. Theoretically, even a pair of 3-finger ZFNs recognizing a total of 18 base pairs could target a single locus in the mammalian genome if the zinc finger domains are specific to their intended target sites. One approach to generating novel zinc finger arrays is to combine smaller zinc finger “modules” with known specificity. The most common modular assembly process involves combining three separate zinc fingers, each recognizing 3 base pairs of DNA sequences, to generate a 3-finger array that can recognize 9 base pairs of target sites. Alternatively, selection-based methods such as oligomer pooling (OPEN) can be used to select new zinc finger arrays from a random library that takes into account context-dependent interactions between adjacent fingers. Engineered zinc fingers are commercially available; Sangamo Biosciences (Richmond, CA, USA) has partnered with Sigma-Aldrich (St. Louis, MO, USA) to develop a proprietary platform for zinc finger construction.

[0116] Non-limiting examples of genes that can be silenced or suppressed by permanent gene editing using a zinc finger method include TGFB1, TGFBR1, TGFBR2, and combinations thereof. Non-limiting examples of genes that can be enhanced (so that their products can act as decoys or dominant-negative products) by permanent gene editing using a zinc finger method include TGFB1, TGFBR1, TGFBR2, and combinations thereof. Non-limiting examples of genes that can be enhanced by permanent gene editing using a zinc finger method include TGFB1, TGFBR1, TGFBR2, and combinations thereof. In one aspect, this disclosure provides compositions for upregulating protein receptors (including wild-type or gene-edited ones), including compositions that bind to anti-inflammatory cytokines via a zinc finger method.

[0117] Examples of systems, methods, and compositions for altering the expression of target gene sequences via a zinc finger method, which can be used according to embodiments of this disclosure, are described in U.S. Patent Nos. 6,534,261, 6,607,882, 6,746,838, 6,794,136, 6,824,978, 6,866,997, 6,933,113, 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.

[0118] Other examples of systems, methods, and compositions for altering the expression of target gene sequences using the zinc finger method, which can be used according to embodiments of this disclosure, are described in Beane et al., Molecular Therapy, 2015, 23 1380-1390, the disclosure of which is incorporated herein by reference.

[0119] IV. Fibrosis / Scar Formation

[0120] A. Introduction

[0121] Musculoskeletal tissue is particularly prone to fibrosis and / or scarring after trauma. In many cases, this trauma may be caused by surgical procedures. Indeed, musculoskeletal scar tissue formation can cause a variety of problems, including limited range of motion, pain, and complicate subsequent surgical access to the injury site. Without being bound by any particular theory, increased expression of factors such as TGFB1 has been observed in fascial fibroblasts at the site of musculoskeletal trauma. See Ihn, H. (2019). *International Journal of Allergy*, 68(4), 437-439. This expression pattern is associated with high expression of type I collagen and fibronectin (which contribute to the fibrotic process).

[0122] In one aspect, the compositions and methods described herein relate to the treatment of excessive fibrosis and / or scarring. In some embodiments, this scarring is caused by disease (e.g., liver fibrosis) or trauma (e.g., tendon injury). In some embodiments, the fibrosis and / or scarring is the result of a surgical procedure. In some embodiments, the surgical procedure is ligament reconstruction. In some embodiments, the surgical procedure is anterior cruciate ligament (ACL) reconstruction. In some embodiments, the surgical procedure is autologous ACL reconstruction. In some embodiments, the surgical procedure is allogeneic ACL reconstruction. In some embodiments, the surgical procedure is fracture repair. In some embodiments, the surgical procedure is total knee arthroplasty (TKA). In some embodiments, the surgical procedure is microsurgical discectomy.

[0123] In some embodiments, fibrosis and / or scarring are a result of a condition that may be induced or exacerbated by surgical procedures. In some embodiments, the condition leading to musculoskeletal fibrosis and / or scarring is knee fibrosis. In some embodiments, the condition leading to musculoskeletal fibrosis and / or scarring is intra-articular fibrous nodules. In some embodiments, the condition leading to musculoskeletal fibrosis and / or scarring is epidural fibrosis.

[0124] B. Fibrosis

[0125] In one aspect, this disclosure covers the treatment of fibrosis. In some embodiments, a CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting TGFB1. In some embodiments, a CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting hTGFB1. In some embodiments, a CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting cTGFB1. In some embodiments, a CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting eTGFB1. In some embodiments, a CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting fTGFB1.

[0126] In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 1 of hTGFB1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 2 of hTGFB1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 3 of hTGFB1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 4 of hTGFB1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 5 of hTGFB1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 6 of hTGFB1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 7 of hTGFB1.

[0127] In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 1 of cTGFB1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 2 of cTGFB1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 3 of cTGFB1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 4 of cTGFB1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 5 of cTGFB1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 6 of cTGFB1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 7 of cTGFB1.

[0128] In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 1 of eTGFB1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 2 of eTGFB1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 3 of eTGFB1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 4 of eTGFB1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 5 of eTGFB1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 6 of eTGFB1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 7 of eTGFB1.

[0129] In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 1 of fTGFB1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 2 of fTGFB1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 3 of fTGFB1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 4 of fTGFB1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 5 of fTGFB1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 6 of fTGFB1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 7 of fTGFB1.

[0130] In some embodiments, a CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting TGFBR1. In some embodiments, a CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting hTGFBR1. In some embodiments, a CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting cTGFBR1. In some embodiments, a CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting eTGFBR1. In some embodiments, a CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting fTGFBR1.

[0131] In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 1 of hTGFBR1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 2 of hTGFBR1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 3 of hTGFBR1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 4 of hTGFBR1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 5 of hTGFBR1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 6 of hTGFBR1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 7 of hTGFBR1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 8 of hTGFBR1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 9 of hTGFBR1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 10 of hTGFBR1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 11 of hTGFBR1.

[0132] In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 1 of cTGFBR1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 2 of cTGFBR1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 3 of cTGFBR1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 4 of cTGFBR1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 5 of cTGFBR1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 6 of cTGFBR1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 7 of cTGFBR1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 8 of cTGFBR1. In some embodiments, a CRISPR gene editing system for treating fibrosis includes one or more sgRNAs that target exon 9 of cTGFBR1.

[0133] In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 1 of eTGFBR1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 2 of eTGFBR1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 3 of eTGFBR1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 4 of eTGFBR1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 5 of eTGFBR1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 6 of eTGFBR1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 7 of eTGFBR1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 8 of eTGFBR1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 9 of eTGFBR1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 10 of eTGFBR1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 11 of eTGFBR1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 12 of eTGFBR1.

[0134] In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 1 of fTGFBR1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 2 of fTGFBR1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 3 of fTGFBR1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 4 of fTGFBR1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 5 of fTGFBR1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 6 of fTGFBR1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 7 of fTGFBR1. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 8 of fTGFBR1. In some embodiments, a CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 9 of fTGFBR1. In some embodiments, a CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 10 of fTGFBR1. In some embodiments, a CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 11 of fTGFBR1.

[0135] In some embodiments, a CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting TGFBR2. In some embodiments, a CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting hTGFBR2. In some embodiments, a CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting cTGFBR2. In some embodiments, a CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting eTGFBR2. In some embodiments, a CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting fTGFBR2.

[0136] In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 1 of hTGFBR2. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 2 of hTGFBR2. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 3 of hTGFBR2. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 4 of hTGFBR2. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 5 of hTGFBR2. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 6 of hTGFBR2. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 7 of hTGFBR2. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 8 of hTGFBR2. In some embodiments, a CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 9 of hTGFBR2.

[0137] In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 1 of cTGFBR2. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 2 of cTGFBR2. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 3 of cTGFBR2. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 4 of cTGFBR2. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 5 of cTGFBR2. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 6 of cTGFBR2. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 7 of cTGFBR2. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 8 of cTGFBR2.

[0138] In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 1 of eTGFBR2. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 2 of eTGFBR2. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 3 of eTGFBR2. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 4 of eTGFBR2. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 5 of eTGFBR2. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 6 of eTGFBR2. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 7 of eTGFBR2. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 8 of eTGFBR2. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 9 of eTGFBR2. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 10 of eTGFBR2.

[0139] In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 1 of fTGFBR2. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 2 of fTGFBR2. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 3 of fTGFBR2. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 4 of fTGFBR2. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 5 of fTGFBR2. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 6 of fTGFBR2. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 7 of fTGFBR2. In some embodiments, the CRISPR gene editing system for treating fibrosis includes one or more sgRNAs targeting exon 8 of fTGFBR2.

[0140] C. Musculoskeletal scarring

[0141] In one aspect, this disclosure covers the treatment of musculoskeletal scarring. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting TGFB1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting hTGFB1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting cTGFB1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting eTGFB1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting fTGFB1.

[0142] In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 1 of hTGFB1. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 2 of hTGFB1. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 3 of hTGFB1. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 4 of hTGFB1. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 5 of hTGFB1. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 6 of hTGFB1. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 7 of hTGFB1.

[0143] In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 1 of cTGFB1. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 2 of cTGFB1. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 3 of cTGFB1. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 4 of cTGFB1. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 5 of cTGFB1. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 6 of cTGFB1. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 7 of cTGFB1.

[0144] In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 1 of eTGFB1. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 2 of eTGFB1. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 3 of eTGFB1. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 4 of eTGFB1. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 5 of eTGFB1. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 6 of eTGFB1. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 7 of eTGFB1.

[0145] In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 1 of fTGFB1. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 2 of fTGFB1. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 3 of fTGFB1. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 4 of fTGFB1. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 5 of fTGFB1. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 6 of fTGFB1. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 7 of fTGFB1.

[0146] In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting TGFBR1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting hTGFBR1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting cTGFBR1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting eTGFBR1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting fTGFBR1.

[0147] In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 1 of hTGFBR1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 2 of hTGFBR1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 3 of hTGFBR1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 4 of hTGFBR1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 5 of hTGFBR1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 6 of hTGFBR1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 7 of hTGFBR1. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 8 of hTGFBR1. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 9 of hTGFBR1. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 10 of hTGFBR1. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 11 of hTGFBR1.

[0148] In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 1 of cTGFBR1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 2 of cTGFBR1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 3 of cTGFBR1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 4 of cTGFBR1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 5 of cTGFBR1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 6 of cTGFBR1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 7 of cTGFBR1. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 8 of cTGFBR1. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 9 of cTGFBR1.

[0149] In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 1 of eTGFBR1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 2 of eTGFBR1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 3 of eTGFBR1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 4 of eTGFBR1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 5 of eTGFBR1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 6 of eTGFBR1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 7 of eTGFBR1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 8 of eTGFBR1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 9 of eTGFBR1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 10 of eTGFBR1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 11 of eTGFBR1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 12 of eTGFBR1.

[0150] In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 1 of fTGFBR1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 2 of fTGFBR1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 3 of fTGFBR1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 4 of fTGFBR1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 5 of fTGFBR1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 6 of fTGFBR1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 7 of fTGFBR1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 8 of fTGFBR1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 9 of fTGFBR1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 10 of fTGFBR1. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 11 of fTGFBR1.

[0151] In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting TGFBR2. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting hTGFBR2. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting cTGFBR2. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting eTGFBR2. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting fTGFBR2.

[0152] In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 1 of hTGFBR2. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 2 of hTGFBR2. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 3 of hTGFBR2. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 4 of hTGFBR2. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 5 of hTGFBR2. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 6 of hTGFBR2. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 7 of hTGFBR2. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 8 of hTGFBR2. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 9 of hTGFBR2.

[0153] In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 1 of cTGFBR2. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 2 of cTGFBR2. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 3 of cTGFBR2. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 4 of cTGFBR2. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 5 of cTGFBR2. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 6 of cTGFBR2. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 7 of cTGFBR2. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 8 of cTGFBR2.

[0154] In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 1 of eTGFBR2. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 2 of eTGFBR2. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 3 of eTGFBR2. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 4 of eTGFBR2. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 5 of eTGFBR2. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 6 of eTGFBR2. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 7 of eTGFBR2. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 8 of eTGFBR2. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 9 of eTGFBR2. In some embodiments, the CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 10 of eTGFBR2.

[0155] In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 1 of fTGFBR2. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 2 of fTGFBR2. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 3 of fTGFBR2. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 4 of fTGFBR2. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 5 of fTGFBR2. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 6 of fTGFBR2. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 7 of fTGFBR2. In some embodiments, a CRISPR gene editing system for treating musculoskeletal scarring includes one or more sgRNAs targeting exon 8 of fTGFBR2.

[0156] V. Delivery

[0157] A. Viral vector

[0158] In one aspect, this disclosure covers methods for delivering a CRISPR gene editing system targeting genes selected from TGFB1, TGFBR1, TGFBR2, and combinations thereof using one or more recombinant viral particles. In some embodiments, the one or more viral vectors comprise a recombinant virus selected from retroviruses, adenoviruses, adeno-associated viruses, lentiviruses, and herpes simplex virus-1. In some embodiments, the one or more viral vectors comprise recombinant adeno-associated virus (AAV). In some embodiments, the recombinant AAV belongs to serotype 5 (AAV5). In some embodiments, the recombinant AAV belongs to serotype 6 (AAV6). In some embodiments, the one or more viral vectors comprise: a first viral vector comprising a first nucleic acid encoding a Cas protein from one or more nucleic acids; and a second viral vector comprising a second nucleic acid encoding at least one guide RNA from one or more nucleic acids. In some embodiments, the one or more viral vectors comprise a viral vector comprising a single nucleic acid, wherein the single nucleic acid encodes a Cas9 protein and at least one guide RNA.

[0159] 1. Adeno-associated virus (AAV)

[0160] The viral vector system used to deliver nucleic acids is adeno-associated virus (AAV). AAV is a naturally occurring defective virus that requires another virus (such as 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). It is also one of the few viruses capable of integrating its DNA into non-dividing cells and exhibits a high frequency of stable integration (see, for example, Flotte et al., *Am. J. Respir. Cell. Mol. Biol.*, 7:349-356 (1992); Samulski et al., *Journal of Virology*, 63:3822-38). 28 (1989); and McLaughlin et al., Journal of Virology, 62:1963-1973 (1989)). AAV vectors containing as few as 300 base pairs can be packaged and integrated. The space for exogenous DNA is confined to about 4.5 kb. AAV vectors, such as those described in Molecular Cell Biology (Mol. Cell. Biol.) by Tratschin et al., 5:3251-3260 (1985), can be used to package DNA. Introduced into cells. AAV vectors have been used to introduce various nucleic acids into different cell types (see, for example, Hermonat et al., Proceedings of the National Academy of Sciences, 81:6466-6470 (1984); Tratschin et al., Molecular Cell Biology, 4:2072-2081 (1985); Wondisford et al., Molecular Endocrinology, 2:32-39 (1988); Tratschin et al. Human, *Journal of Virology*, 51:611-619 (1984); and Flotte et al., *Journal of Biochemistry*, 268:3781-3790 (1993). The identification of Staphylococcus aureus (SaCas9) and other smaller Cas9 enzymes has paved new avenues for therapeutic gene editing, which can be packaged into highly stable and efficient adeno-associated virus (AAV) vectors that are easy to produce, FDA-approved, and tested in multiple clinical trials.

[0161] According to specific embodiments, the CRISPR gene editing system targeting genes 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 nucleic acids, wherein the nucleic acids are recombinant AAV genomes. In some embodiments, the AAV vector is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10 vectors.

[0162] In some respects, AAV vectors contain serotypes 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 respects, the AAV vector contains serotypes selected from the group consisting of: AAV1, AAV5, AAV6, AAV6(Y705F / Y731F / T492V), AAV8, AAV9, and AAV9(Y731F).

[0163] In one aspect, the use of a CRISPR gene-editing system further comprising one or more AAV vectors targeting TGFB1, TGFBR1, and / or TGFBR2 is therapeutic. In some embodiments, the use of said system can treat fibrosis and / or scarring. In some embodiments, the use of said system can treat one or more musculoskeletal diseases, conditions, and patients, including but not limited to Loeys-Dietz syndrome, osteoarthritis, Marfan syndrome, aortic aneurysm (e.g., familial T3 aortic aneurysm), craniofacial abnormalities, and combinations thereof. In other embodiments, the use of said system can treat vegetation diseases, conditions, and patients, including but not limited to pancreatic cancer, multiple self-healing squamous cell carcinoma (Ferguson-Smith disease), gastrointestinal stromal tumor (GIST), hereditary nonpolyposis colorectal cancer (Lynch syndrome), metastatic colorectal cancer, bone vegetations, anaplastic carcinoma, spindle cell carcinoma, lung vegetations, brain vegetations, and combinations thereof.

[0164] 2. Lentiviral virus

[0165] In some respects, the viral vector is a lentivirus. Lentivirals are selected from the group consisting of: human immunodeficiency virus-1 (HIV-1), human immunodeficiency virus-2 (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Jembrana disease virus (JDV), equine infectious anemia virus (EIAV), and caprine arthritis encephalitis virus (CAEV).

[0166] Lentiviral transduction systems are known in the art and described, for example, in Levine et al., Proceedings of the National Academy of Sciences, 2006, 103, 17372-77; Zufferey et al., Nature Biotechnology, 1997, 15, 871-75; Dull et al., Journal of Virology, 1998, 72, 8463-71 and U.S. Patent No. 6,627,442, the disclosure of each of which is incorporated herein by reference.

[0167] In one aspect, the use of a CRISPR gene-editing system comprising one or more lentiviral vectors targeting TGFB1, TGFBR1, and / or TGFBR2 is therapeutic. In some embodiments, the use of the system can treat fibrosis and / or scarring. In some embodiments, the use of the system can treat one or more musculoskeletal diseases, conditions, and patients, including but not limited to Loeys-Dietz syndrome, osteoarthritis, Marfan syndrome, aortic aneurysm (e.g., familial T3 aortic aneurysm), craniofacial abnormalities, and combinations thereof. In other embodiments, the use of the system can treat vegetation diseases, conditions, and patients, including but not limited to pancreatic cancer, multiple self-healing squamous cell carcinoma (Ferguson-Smith disease), gastrointestinal stromal tumor (GIST), hereditary nonpolyposis colorectal cancer (Lynch syndrome), metastatic colorectal cancer, bone vegetations, anaplastic carcinoma, spindle cell carcinoma, lung vegetations, brain vegetations, and combinations thereof.

[0168] B. Lipid nanoparticles (LNP)

[0169] In some embodiments, the CRISPR gene editing system is delivered by nanoparticles. Not wishing to be bound by any particular theory, in some embodiments, nucleic acids, when present in the nanoparticles, are resistant to degradation by nucleases in aqueous solution. In other embodiments, proteins are protected from degradation by proteases. In some embodiments, proteins and nucleic acids encapsulated in the nanoparticles are capable of penetrating the cell membrane.

[0170] Lipid nanoparticles containing nucleic acids and methods for preparing the same are disclosed in at least the following patents: WO2017 / 019935, WO2017 / 049074, WO2017 / 201346, WO2017 / 218704, WO2018 / 006052, WO2018 / 013525, WO2018 / 089540, WO2018 / 119115, WO2018 / 126084, WO2018 / 157009, WO2018 / 170336, WO2018 / 222890, WO2019 / 046809, WO2019 / 08 9828、WO2020 / 061284、WO2020 / 061317、WO2020 / 081938、WO2020 / 097511、WO2020 / 097520、WO2020 / 097540、WO2020 / 097548、WO2020 / 214946, WO2020 / 219941, WO2020 / 232276, WO2020 / 227615, WO2020 / 061295, WO2021 / 007278, WO2021 / 016430, WO2021 / 021988, EP patent EP 2 972 360, US20200155691, US20200237671, US Patents 8,058,069, 8,492,359, 8,822,668, 9,364,435, 9,404,127, 9,504,651, 9,593,077, 9,738,593, 9,868,691, 9,868,692, 9,950,068 Patents 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, are incorporated herein by reference in their entirety for all purposes.

[0171] Lipid nanoparticle composition

[0172] In some embodiments, the maximum size of the nanoparticle composition is 1 micrometer or shorter (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 shorter), for example when measured by dynamic light scattering (DLS), transmission electron microscopy, scanning electron microscopy or another method. The nanoparticle composition includes, for example, lipid nanoparticles (LNPs), liposomes, lipid vesicles, and lipid complexes. In some embodiments, the nanoparticle composition is a vesicle comprising one or more lipid bilayers. In some embodiments, the nanoparticle composition comprises two or more concentric bilayers separated by aqueous compartments. The lipid bilayers may be functionalized and / or cross-linked with each other. The lipid bilayer may include one or more ligands, proteins, or channels. In various embodiments, the lipid nanoparticles described herein have an average diameter of about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 nm to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and are substantially non-toxic.

[0173] In some embodiments, the lipid nanoparticles described herein comprise one or more components, including a lipid component and (optionally) a structural component. The lipid component comprises lipids selected from ionizable 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-conjugated lipids (e.g., PEGylated lipids). In some embodiments, the lipid component comprises a single ionizable lipid. In other embodiments, the lipid component comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 ionizable lipids. In some embodiments, the lipid component comprises a single neutral lipid. In other embodiments, the lipid component comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 neutral lipids. In some embodiments, the lipid component comprises a single polymer-conjugated lipid. In other embodiments, the lipid component comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 polymer-conjugated lipids. In some embodiments, the structural component comprises a single structural lipid. In other embodiments, the structural component comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 structural lipids. In some embodiments, the lipid component comprises at least one cationic lipid, at least one neutral lipid, and at least one polymer-conjugated lipid. This disclosure contemplates that the lipid component may comprise any combination of the foregoing components.

[0174] Ionizable lipids / cationic lipids

[0175] In some embodiments, the lipid component comprises ionizable lipids. In some embodiments, the ionizable lipids are anionic. In other embodiments, the ionizable lipids are cationic lipids. In some embodiments, the lipid component comprises cationic lipids, including but not limited to cationic lipids selected from the group consisting of: 3-(bisdodecylamino)-N1,N1,4-tris(dodecyl)-1-piperazine ethylamine (KL10), N1-[2-(bisdodecylamino)ethyl]-N1,N4,N4-tris(dodecyl)-1,4-piperazine diethylamine (KL22), 14,25-bis(tridecyl)-15,18,21,24-tetraaza-octacosan (KL25), 1,2-dilinyl Alkenyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinylenyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptadecyl-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA), 2,2-dilinylenyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethyl Aminopropane (DODMA), 2-({8-[(3.β.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3.β.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z-,12Z)-octadec-9,12 [-dien-1-yloxy]prop-1-amine (octyl-CLinDMA(2R)(Octyl-CLinDMA(2R))), (2S)-2-({8-[(3.β.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z-,12Z)-octadec-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA(2S)(Octyl-CLinDMA(2S))), lipids including cyclic amine groups, and mixtures thereof.

[0176] Non-limiting examples of cationic lipids include:

[0177]

[0178]

[0179]

[0180]

[0181]

[0182] neutral lipids / phospholipids

[0183] In some embodiments, the lipid component further comprises neutral lipids, including but not limited to phospholipids selected from the group consisting of: 1,2-dilinoleoyl-sn-glycerol-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), 1,2-distearateoyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-di(undecanoyl)-sn-glycerol-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphocholine (18:0 Diether) 1,2-Oleoyl-2-cholesterol hemisuccino-sn-glycerol-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphocholine (C16LysoPC), 1,2-Dilinolenoyl-sn-glycerol-3-phosphocholine, 1,2-disarachidanoyl-sn-glycerol-3-phosphocholine, 1,2-bis(docohexanoyl)-sn-glycerol-3-phosphocholine, 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-diphydanyl-sn-glycerol-3-phosphoethanolamine (ME) 16.0PE), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docosahexaenoyl)-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate sodium salt (DOPG), sphingomyelin (SM) and mixtures thereof.

[0184] Polymer-conjugated lipids

[0185] In some embodiments, the lipid component further comprises polymer-conjugated lipids, including but not limited to 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, PEG lipids may be PEG-c-DOMG, PEG-DMG, PEG... 2000 -c-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DMA, or PEG-DSPE lipids. Non-exhaustive and non-limiting examples of PEG lipids include:

[0186]

[0187]

[0188] Structural lipids / sterols

[0189] In some embodiments, the LNP further comprises a structural component. See generally Patel, S. et al. (2020). Nature Communications, 11(1), 1-13. In some embodiments, the structural component comprises sterols, including but not limited to sterols selected from the group consisting of: cholesterol, fecosterol, stigmasterol, stigmasterol, sitosterol, β-sitosterol, lupeol, betulinol, ursolic acid, oleanolic acid, campesterol, fucosterol, brassosterol, ergosterol, 9,11-dehydroergosterol, tomatine, lycopene, α-tocopherol, and mixtures thereof. In other embodiments, the structural lipid comprises cholesterol and corticosteroids (e.g., prednisolone, dexamethasone, prednisone, and hydrocortisone) or combinations thereof.

[0190] Examples of the non-exhaustive and non-limiting nature of structural lipids include:

[0191]

[0192] formula

[0193] Nanoparticle compositions may include lipid components and one or more additional components, such as therapeutic and / or preventative agents. Nanoparticle compositions may be engineered for one or more specific applications or targets. The elements of a nanoparticle composition may be selected based on a specific application or target and / or based 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 specific applications or targets based on, for example, the efficacy and toxicity of a specific combination of elements.

[0194] The lipid component of the nanoparticle composition may include, for example, cationic lipids, phospholipids (e.g., unsaturated lipids, such as DOPE or DSPC), PEG lipids, and structural lipids. The elements of the lipid component may be provided in specific fractions.

[0195] In some embodiments, the lipid component of the nanoparticle composition includes ionizable lipids, phospholipids, PEG lipids, and structural lipids. In some embodiments, the lipid component of the nanoparticle composition includes about 30 mol% to about 60 mol% of ionizable 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 component of the nanoparticle composition includes about 35 mol% to about 55 mol% of an ionizable lipid compound, 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 one particular embodiment, the lipid component includes about 50 mol% of the compound, 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 component 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.

[0196] In some embodiments, ionizable lipids constitute about 20 mol% to about 60 mol% of the lipid component. In other embodiments, ionizable lipids constitute about 35 mol% to about 55 mol% of the lipid component. In various embodiments, ionizable lipids constitute about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, or 60 mol% of the lipid component.

[0197] In some embodiments, neutral lipids constitute about 0 to about 30 mol% of the lipid component. In other embodiments, neutral lipids constitute about 5 to about 25 mol% of the lipid component. In various embodiments, neutral lipids constitute about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 mol% of the lipid component.

[0198] In some embodiments, the polymer-conjugated lipids constitute about 0 to about 15 mol% of the lipid component. In other embodiments, the polymer-conjugated lipids constitute about 0.5 mol% to about 10 mol% of the lipid component. In various embodiments, the polymer-conjugated lipids constitute about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, or 15 mol% of the lipid component.

[0199] In some embodiments, the structural component constitutes about 17.5 mol% to about 50 mol% of the lipid component. In other embodiments, the structural component constitutes about 30 mol% to about 40 mol% of the lipid component. In various embodiments, the structural component constitutes about 17.5, 20, 22.5, 25, 27.5, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mol% of the lipid component.

[0200] The structural component can alternatively be expressed as a ratio relative to the lipid component. In some embodiments, the ratio of structural component to lipid component (sterol:lipid) is about 1:1. In other embodiments, the ratio of structural component to lipid component (sterol:lipid) is about 1:5. In various embodiments, the ratio of structural component to lipid component (sterol:lipid) is about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, or 1:25.

[0201] Nanoparticle compositions can be designed for one or more specific applications or targets. For example, nanoparticle compositions can be designed to deliver therapeutics and / or preventative agents, such as RNA, to specific cells, tissues, organs, or systems or groups thereof in a mammal. The physicochemical properties of the nanoparticle composition can be modified to increase selectivity for specific bodily targets. For example, particle size can be adjusted based on the fenestration size of different organs. The therapeutics and / or preventative agents included in the nanoparticle composition can also be selected based on one or more desired delivery targets. For example, therapeutics and / or preventative agents can be selected for specific indications, symptoms, diseases, or conditions and / or for delivery to specific cells, tissues, organs, or systems or groups thereof (e.g., local or specific delivery). In some embodiments, the nanoparticle composition may include mRNA encoding a polypeptide of interest, which is capable of being translated intracellularly to produce the polypeptide of interest. Such compositions can be designed for specific delivery to specific organs. In some embodiments, the composition can be designed for specific delivery to mammalian joints. In some embodiments, the nanoparticles are attached to or compounded with a targeting portion to improve delivery to specific target cells and / or tissues. For example, in some embodiments, the nanoparticles are attached to or compounded with an agent that binds to cell surface markers of target cells and / or tissues.

[0202] The amount of therapeutic and / or preventative agents in a nanoparticle composition can depend on the size, composition, desired target and / or application, or other properties of the nanoparticle composition, as well as the nature of the therapeutic and / or preventative agents. For example, the amount of RNA that can be used in a nanoparticle composition can depend on the size, sequence, and other characteristics of the RNA. The relative amounts of therapeutic and / or preventative agents and other elements (e.g., lipids) in the nanoparticle composition can also vary. In some embodiments, the wt / wt ratio of the lipid component to the therapeutic and / or preventative agent in the nanoparticle composition can be from 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 wt / wt ratio of the lipid component to the therapeutic and / or preventative agent can be from about 10:1 to about 40:1. In some embodiments, the wt / wt ratio is about 20:1. The amount of the therapeutic and / or preventative agent in the nanoparticle composition can be measured, for example, using absorption spectroscopy (e.g., UV-Vis spectroscopy).

[0203] In some embodiments, the therapeutic and / or preventative agents comprise nucleic acid components. In some embodiments, the nucleic acid components comprise RNA, including but not limited to RNA selected from the group consisting of: messenger RNA (mRNA), CRISPR RNA (crRNA), tracrRNA, single-guide RNA (sgRNA), short interfering RNA (siRNA), antisense oligonucleotides (ASO), and mixtures thereof. In other embodiments, the nucleic acid components comprise DNA, including but not limited to DNA selected from the group consisting of: linear DNA, plasmid DNA, antisense oligonucleotides, and mixtures thereof.

[0204] In some embodiments, the nanoparticle composition comprises one or more RNAs, and the amount of one or more RNAs, lipids, and their components can be selected to provide a specific N:P ratio. The N:P ratio of the composition refers to the molar ratio of the number of nitrogen atoms in one or more lipids to the number of phosphate groups in the RNA. Generally, a lower N:P ratio is preferred. The amount of one or more RNAs, lipids, and their components can be selected to provide an N:P ratio of about 2:1 to about 30:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1. In some embodiments, the N:P ratio can be about 2:1 to about 8:1. In other embodiments, the N:P ratio is about 5:1 to about 8:1. For example, the N:P ratio can be approximately 5.0:1, approximately 5.5:1, approximately 5.67:1, approximately 6.0:1, approximately 6.5:1, or approximately 7.0:1. For example, the N:P ratio can be approximately 5.67:1.

[0205] In some embodiments, the nucleic acid component is composed of modified nucleic acids. For example, RNA can be modified RNA. That is, RNA may include one or more non-naturally occurring nucleobases, nucleosides, nucleotides, or linkers. A “modified” species may also be referred to herein as an “altered” species. A species may be chemically, structurally, or functionally modified or altered. For example, a modified nucleobase species may include one or more non-naturally occurring substitutions.

[0206] In some embodiments, this disclosure includes methods for treating back or spinal symptoms or conditions. In other embodiments, this disclosure includes methods for treating discogenic conditions. In some embodiments, this disclosure includes methods for treating localized nociceptive sensations, inflammation, or morphological changes associated with back or spinal symptoms or conditions in a subject of need, the methods comprising administering a therapeutically effective amount of a CRISPR-Cas composition encapsulated within lipid nanoparticles (LNPs) or associated with LNPs, wherein the composition comprises one or more non-naturally occurring polynucleotides encoding the Cas9 protein and at least one sgRNA. In some embodiments, the LNP is administered intradiscally. In other embodiments, the LNP is administered epidurally. In some embodiments, the LNP is administered peridotidally. In some embodiments, the LNP is administered peridiscally.

[0207] Physical properties

[0208] The properties of nanoparticle compositions can depend on their components. For example, a nanoparticle composition comprising cholesterol as a structural lipid may have different properties than a nanoparticle composition comprising different structural lipids. Similarly, the properties of a nanoparticle composition may depend on the absolute or relative amounts of its components. For example, a nanoparticle composition comprising a higher molar fraction of phospholipids may have different properties than a nanoparticle composition comprising a lower molar fraction of phospholipids. Properties can also vary depending on the preparation method and conditions of the nanoparticle composition.

[0209] Nanoparticle compositions can be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of the nanoparticle composition. Dynamic light scattering or potentiometric measurements (e.g., potentiometric titration) can be used to measure the zeta potential. Dynamic light scattering can also be used to determine particle size. Instruments such as the Zetasizer Nano ZS (Malvern Instruments, Malvern, Worcestershire, UK) can also be used to measure various properties of nanoparticle compositions, such as particle size, polydispersity index, and zeta potential.

[0210] The average size of the nanoparticle composition can be between 10 nm and 1 micrometer, for example, as measured by dynamic light scattering (DLS). For example, the average size can be from about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the average size of the nanoparticle composition can be about 50 nm to about 100 nm, about 50 nm to about 90 nm, about 50 nm to about 80 nm, about 50 nm to about 70 nm, about 50 nm to about 60 nm, about 60 nm to about 100 nm, about 60 nm to about 90 nm, about 60 nm to about 80 nm, about 60 nm to about 70 nm, about 70 nm to about 100 nm, about 70 nm to about 90 nm, about 70 nm to about 80 nm, about 80 nm to about 100 nm, about 80 nm to about 90 nm, or about 90 nm to about 100 nm. In some embodiments, the average size of the nanoparticle composition can be about 70 nm to about 100 nm. In a particular embodiment, the average size can be about 80 nm. In other embodiments, the average size can be about 100 nm.

[0211] The nanoparticle composition can be relatively homogeneous. A polydispersity index can be used to indicate the uniformity of the nanoparticle composition, such as its particle size distribution. Small polydispersity indices (e.g., less than 0.3) typically indicate a narrow particle size distribution. The nanoparticle composition can have a polydispersity index of about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of the nanoparticle composition can be about 0.10 to about 0.20.

[0212] The zeta potential of a nanoparticle composition can be used to indicate the electrodynamic potential of the composition. For example, the zeta potential can describe the surface charge of the nanoparticle composition. Nanoparticle compositions with relatively low charges (positive or negative) are generally desirable because substances with higher charges may interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of the nanoparticle composition can be about -10mV to about +20mV, about -10mV to about +15mV, about -10mV to about +10mV, about -10mV to about +5mV, about -10mV to about 0mV, about -10mV to about -5mV, about -5mV to about +20mV, about -5mV to about +15mV, about -5mV to about +10mV, about -5mV to about +5mV, about -5mV to about 0mV, about 0mV to about +20mV, about 0mV to about +15mV, about 0mV to about +10mV, about 0mV to about +5mV, about +5mV to about +20mV, about +5mV to about +15mV, or about +5mV to about +10mV.

[0213] Encapsulation efficiency of therapeutic and / or preventative agents describes the amount of therapeutic and / or preventative agents encapsulated within or otherwise associated with the nanoparticle composition after preparation, relative to the initial amount provided. Encapsulation efficiency is ideally high (e.g., close to 100%). For example, encapsulation efficiency can be measured by comparing the amount of therapeutic and / or preventative agents in a solution containing the nanoparticle composition before and after decomposition with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free therapeutic and / or preventative agents (e.g., RNA) in solution. For the nanoparticle compositions described herein, the encapsulation efficiency for treatment and / or prevention can be at least 50%, such as 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In some embodiments, the encapsulation efficiency can be at least 90%.

[0214] The nanoparticle composition may optionally include one or more coatings. For example, the nanoparticle composition may be formulated into a coated capsule, film, or tablet. Capsules, films, or tablets comprising the compositions described herein may have any useful dimensions, tensile strength, hardness, or density.

[0215] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While methods and materials similar to or equivalent to those described herein may be used to practice or test this disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. Furthermore, materials, methods, and examples are illustrative only and are not intended to be limiting.

[0216] In some embodiments, the CRISPR gene editing system comprises one or more RNA-containing compositions. In some embodiments, the CRISPR gene editing system further comprises one or more nanoparticles. In some embodiments, the one or more RNA-containing compositions comprise guide RNA. In some embodiments, the one or more RNA-containing compositions comprise mRNA. In some embodiments, the one or more RNA-containing compositions comprise RNPs (e.g., Cas9 and guide RNA). In some embodiments, the one or more nanoparticles are lipid nanoparticles (LNPs).

[0217] In some embodiments, the CRISPR gene editing system comprises one or more LNPs, which collectively encapsulate (i) an RNA-guided nuclease or nucleic acid encoding an RNA-guided nuclease, and (ii) at least one guide RNA or nucleic acid encoding at least one guide RNA. In some embodiments, the one or more LNPs comprise a first plurality of LNPs encapsulating the RNA-guided nuclease or nucleic acid encoding an RNA-guided nuclease; and a second plurality of LNPs encapsulating the at least one guide RNA or nucleic acid encoding at least one guide RNA.

[0218] In some embodiments, the one or more LNPs comprise a component selected from the group consisting of: 3-(bisdodecylamino)-N1,N1,4-tris(dodecyl)-1-piperazinethylamine (KL10), N1-[2-(bisdodecylamino)ethyl]-N1,N4,N4-tris(dodecyl)-1,4-piperazindiethylamine (KL22), 14,25-bis(tridecyl)-15,18,21,24-tetraaza-octacosane (KL25), 1 2-Dilinolenic acid-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinolenic acid-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptadecyl-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA), 2,2-dilinolenic acid-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA) MA), 1,2-diolenyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3.β.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3.β.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethylaminopropane 3-[(9Z-,12Z)-octadec-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA(2R)), (2S)-2-({8-[(3.β.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z-,12Z)-octadec-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA(2S)), lipids including cyclic amine groups, and mixtures thereof.

[0219] In some embodiments, the one or more LNPs comprise a component selected from the group consisting of: 1,2-dilinoleoyl-sn-glycerol-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), 1,2-distearateoyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-di(undecanoyl)-sn-glycerol-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphocholine (18:0Diether) 1,2-Oleoyl-2-cholesterol hemisuccino-sn-glycerol-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphocholine (C16 Lyso PC), 1,2-Dilinoleoyl-sn-glycerol-3-phosphocholine, 1,2-disarachidanoyl-sn-glycerol-3-phosphocholine, 1,2-bis(docohexanoyl)-sn-glycerol-3-phosphocholine, 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-diphydanyl-sn-glycerol-3-phosphoethanolamine (ME) 16.0PE), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docosahexaenoyl)-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate sodium salt (DOPG), sphingomyelin (SM) and mixtures thereof.

[0220] In some embodiments, the one or more LNPs comprise 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, PEG lipids may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DMA, PEG-DSPE lipids, and mixtures thereof.

[0221] In some embodiments, the one or more LNPs comprise components selected from the group consisting of: cholesterol, coccosterol, stigmasterol, stigmasterol, sitosterol, β-sitosterol, lupeol, betulinol, ursolic acid, oleanolic acid, campesterol, fucosterol, brassosterol, ergosterol, 9,11-dehydroergosterol, tomatine, lycopene, α-tocopherol, and mixtures thereof.

[0222] In one aspect, the use of a CRISPR gene-editing system further comprising one or more LNPs targeting TGFB1, TGFBR1, and / or TGFBR2 is therapeutic. In some embodiments, the use of the system can treat fibrosis and / or scarring. In some embodiments, the use of the system can treat one or more musculoskeletal diseases, conditions, and patients, including but not limited to Loeys-Dietz syndrome, osteoarthritis, Marfan syndrome, aortic aneurysm (e.g., familial T3 aortic aneurysm), craniofacial abnormalities, and combinations thereof. In other embodiments, the use of the system can treat vegetation diseases, conditions, and patients, including but not limited to pancreatic cancer, multiple self-healing squamous cell carcinoma (Ferguson-Smith disease), gastrointestinal stromal tumor (GIST), hereditary nonpolyposis colorectal cancer (Lynch syndrome), metastatic colorectal cancer, bone vegetations, anaplastic carcinoma, spindle cell carcinoma, lung vegetations, brain vegetations, and combinations thereof.

[0223] Example LNP system

[0224] In some embodiments, LNP systems selected from those listed in the table below are used to formulate the pharmaceutical compositions described herein:

[0225] Table 5: LNP System (All values ​​are molar ratios relative to a total of 100)

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232] In some embodiments of any LNP system LNP001 to LNP240, the LNP system comprises a steroid component in a molar ratio of about 36 to about 46, or about 38 to about 42. In some embodiments of any LNP system LNP001 to LNP240, the LNP system comprises a steroid component 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 comprises a steroid component in a molar ratio of about 37, about 38, about 39, about 40, about 41, or about 42. In some embodiments of any LNP system LNP001 to LNP240, the steroid component comprises cholesterol. In some embodiments of any LNP system LNP001 to LNP240, the steroid component comprises cholesterol and one or more other steroids, wherein cholesterol comprises 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, such as hydroxyl or alkyl modified. In some embodiments, the steroid component comprises any other steroids disclosed herein. In some embodiments, the LNP system comprises a PEGylated lipid component in a molar ratio of about 1.5 to about 2.5. In some embodiments, the PEGylated lipid component comprises PEG2000 lipid. In some embodiments, the PEGylated lipid component comprises one or more of DMG-PEG and / or DMG-C-PEG. In some embodiments, the PEGylated lipid component comprises one or more of DMG-PEG2000, DMG-C-PEG2000, DSG-PEG2000, C14-PEG2000, C16-PEG2000 and / or C18-PEG2000.

[0233] The LNPs described herein typically comprise biodegradable ionizable lipids, cofactor phospholipids, cholesterol (possibly with added dexamethasone or other steroids), and a PEG2000 component. The molar lipid ratios described herein are the ratio of ionizable lipids:cofactor lipids:cholesterol [:dexamethasone]:PEG.

[0234] Modifying the molar lipid ratio: Not wanting to be bound by any particular theory, when changing the molar lipid ratio of ionizable lipids, the amount of cholesterol or co-lipids will be added or reduced. Typically, the ionizable lipids remain at a set ratio, while the co-lipids, cholesterol, and PEG content varies. If you change the amount of PEG, you will subsequently adjust the amount of primary cholesterol, and sometimes the amount of co-lipids. If you change the co-lipids, you adjust the amount of cholesterol bound to them. Due to the similar chemical structure / function in LNPs, dexamethasone or another similar steroid in the LNP structure directly replaces some of the cholesterol.

[0235] Typical ionizable lipids are: Dlin-KC2-DMA: molar ratio between 42 and 50, for example, but not limited to 44; Dlin-MC3-DMA: molar ratio between 44 and 50, for example, but not limited to 50; SM-102 (lipid H): molar ratio between 44 and 50, for example, but not limited to 50; ALC-0315: molar ratio between 44 and 50, for example, but not limited to 50; LP01 (LP000001): molar ratio between 42 and 50, for example, but not limited to 45. Co-adjuvant lipids can have molar ratios between 9 and 11, for example, but not limited to 10. Co-adjuvant lipids include DSPC, DOPE, DOTMA, and DPPC. PEG2000 lipids have a typical molar ratio of about 1.5 to about 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 molar lipid ratio of cholesterol is typically from about 36 to about 46. Dexamethasone or similar steroids supplement a portion of the cholesterol's lipid molar ratio, for example, but not limited to, a cholesterol:dexamethasone (C:D) ratio of 9:1. The molar lipid ratio of cholesterol can range from about 8 to about 10, and the molar lipid ratio of dexamethasone can range from about 0.1 to about 2, but is not limited to. Without wishing to be bound by any particular theory, modifications or substitutions to cholesterol can be considered, such as, but not limited to, hydroxyl or alkyl modifications (e.g., to improve mRNA delivery) or substitution with a potential therapeutic component such as an anti-inflammatory steroid. In some embodiments, the molar N / P ratio can be from 1 to 8. Not wanting to be bound by any details, it is believed that the ideal N / P ratio is 1-5 in order to reduce the inflammatory response of LNP in the body; however, the tested N / P ratio was 6-8.

[0236] 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, ALC-0315:DSPC:cholesterol:dexamethasone:DMG-PEG2000. In some embodiments, the molar ratio of the formulations described herein includes 50:10:38.5:1.5; 45:9:44:2.

[0237] Biophysical assays: LNP encapsulated nucleic acids efficiently, met size criteria, and exhibited homogeneity and stability after freeze-thaw cycles. Encapsulated payloads: GFP mRNA, Luciferase mRNA, and our CRISPR / Cas9 therapeutic agents (sgRNA and Cas9 mRNA).

[0238] Table 6: LNPs for various recipes created and screened.

[0239]

[0240] Additional notes regarding the LNP component:

[0241] Ionizable lipids (titrile charge): Contain tertiary amines, giving them a positive charge at acidic pH and a neutral charge at physiological pH. After being taken up into the cytosol in the endosome, they become protonated. Specifications for different types of ionizable lipids are described below.

[0242] Helper phospholipids: Anionic endosome phospholipids interact with protonated ionizable lipids to form cone-shaped ion pairs, which enhance cell membrane fusion and rupture, endosome escape, and cargo release into the cytosol.

[0243] Cholesterol: Maintains the integrity of nanoparticle membranes, assists in nucleic acid encapsulation, and enhances circulation by reducing surface-bound proteins.

[0244] Dexamethasone or similar steroids: provide the anti-inflammatory component of LNP, which reduces immunogenicity and increases transfection rate, especially in vivo.

[0245] PEGylated lipids (PEG2000): Improve circulating half-life, reduce LNP aggregation, reduce interactions with serum proteins (such as opsonins), and enhance in vivo stability. Furthermore, they contribute to particle stability, particle size, and biodistribution.

[0246] Ionizable lipids:

[0247]

[0248] (pKa = 6.41)

[0249] Cationic lipids, such as DODMA, DOTMA, and DOTAP, possess quaternary ammonium groups, which permanently give them a positive charge. In the past, using these drugs alone was shown to have adverse cycling and increased in vivo toxicity. This led to the development of ionizable lipids.

[0250] Ionizable lipids are characterized by the replacement of quaternary amines with tertiary amines, which makes them pH-dependent, i.e., they carry a neutral charge at physiological pH and a positive charge (protonation) at acidic pH.

[0251] This increases the circulating half-life and reduces toxicity in vivo.

[0252] The pKa of ionizable lipids drives in vivo performance. A pKa between 6 and 6.7 has been shown to be optimal for RNA therapeutics delivery. However, the relative pKa of the combination of all components in the LNP affects LNP transfection—tertiary amines, quaternary amino groups, and hydroxyl groups from ionizable lipids, accessory lipids, and cholesterol all alter their relative pKa due to proximity between the primates. This subsequently affects the overall surface charge.

[0253] pH dependence allows for efficient encapsulation of RNA in acidic buffer solutions, and once taken up by cells, it facilitates RNA release.

[0254] Furthermore, these lipids form an inverted hexagonal HII phase, which facilitates membrane rupture, endosome escape, and subsequent release of the payload into the cell's cytosol.

[0255] Albertson et al., 2022, "Lipid Packing Theory," elucidates the relationship between amphiphilic compounds and their self-assembled geometry. They propose a mechanism for endosome disruption mediated by ionizable lipids (also known as the molecular shape hypothesis). [https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC9250827 / ](https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC9250827 / )

[0256] MC3, ALC-0135, and SM-102 all possess tertiary amines (making them ionizable) and lack a stereocenter and ester linkage. ALC-0135 and SM-102 are improvements on MC3, incorporating additional ester bonds (which facilitate biodegradation and reduce bioaccumulation).

[0257] Unsaturation of the linear tail increases delivery efficiency and fluidity in ionizable lipids; that is, it enables bilayer lipids to form a non-bilayer phase that increases membrane rupture and payload release. Further studies have explored branching of the ionizable lipid tail, which further enhances the efficacy of delivering mRNA therapeutics due to increased protonation at endosome pH and at the cross-section of the lipid tail. This modification also leads to an increase in cone-shaped structures, which promotes membrane rupture, endosome escape, and payload release into the cytosol (Albertson et al., 2022).

[0258] LP01 combines increased ester bonds with tail branching, which has proven to be an efficient and safe delivery system for in vivo gene editing in animal models. LP01 exhibits less liver bioaccumulation and fewer safety risks.

[0259] Example LP01 LNP formulation

[0260] In some embodiments, a composition is provided comprising lipid nanoparticles (LNPs), the LNPs comprising (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease, and (ii) at least one guide RNA of a target gene or a nucleic acid encoding at least one guide RNA, wherein the LNPs comprise LP01.

[0261] In some embodiments, the LNP comprises the following structural components: an ionizable LP01 lipid, a neutral / auxiliary lipid component, a steroid system component, and a peg-modified lipid component. In some such embodiments of the LP01-based LNP, the neutral / auxiliary lipid component comprises DSPC. In some such embodiments of the LP01-based LNP, the steroid system component comprises cholesterol. In some such embodiments of the LP01-based LNP, the peg-modified lipid component comprises peg-modified myristate diglyceride (DMG-PEG; 1,2-dimyristoyl-sn-glycerol-3-methoxy polyethylene glycol). In some embodiments, DMG-PEG is DMG-PEG2000. In some such embodiments of the LP01-based LNP, the steroid system component comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0262] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parentheses after the components): LP01 (40-50), neutral / auxiliary lipid component (6-12), steroid system component (39-49), and peg-modified lipid component (1-3). In some such embodiments, the neutral / auxiliary lipid component comprises DSPC. In some such embodiments of the LP01-based LNP, the steroid system component comprises cholesterol. In some such embodiments of the LP01-based LNP, the peg-modified lipid component comprises peg-modified myristate diglyceride (DMG-PEG; 1,2-dimyristoyl-sn-glycerol-3-methoxy polyethylene glycol). In some embodiments, DMG-PEG is DMG-PEG2000. In some such embodiments of the LP01-based LNP, the steroid system component comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0263] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parentheses after the component): LP01 (41-49), neutral / auxiliary lipid component (6.5-11.5), steroid system component (40-48), and peg-modified lipid component (1.25-2.75). In some such embodiments, the neutral / auxiliary lipid component comprises DSPC. In some such embodiments of the LP01-based LNP, the steroid system component comprises cholesterol. In some such embodiments of the LP01-based LNP, the peg-modified lipid component comprises peg-modified myristate diglyceride (DMG-PEG; 1,2-dimyristoyl-sn-glycerol-3-methoxy polyethylene glycol). In some embodiments, DMG-PEG is DMG-PEG2000. In some such embodiments of the LP01-based LNP, the steroid system component comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0264] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parentheses after the components): LP01 (42-48), neutral / auxiliary lipid component (7-11), steroid system component (41-47), and peg-modified lipid component (1.5-2.5). In some such embodiments, the neutral / auxiliary lipid component comprises DSPC. In some such embodiments of the LP01-based LNP, the steroid system component comprises cholesterol. In some such embodiments of the LP01-based LNP, the peg-modified lipid component comprises peg-modified myristate diglyceride (DMG-PEG; 1,2-dimyristoyl-sn-glycerol-3-methoxy polyethylene glycol). In some embodiments, DMG-PEG is DMG-PEG2000. In some such embodiments of the LP01-based LNP, the steroid system component comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0265] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parentheses after the component): LP01 (43-47), neutral / accessory lipid component (7.5-10.5), steroid system component (42-46), and peg-modified lipid component (1.5-2.5). In some such embodiments, the neutral / accessory lipid component comprises DSPC. In some such embodiments of the LP01-based LNP, the steroid system component comprises cholesterol. In some such embodiments of the LP01-based LNP, the peg-modified lipid component comprises peg-modified myristate diglyceride (DMG-PEG; 1,2-dimyristoyl-sn-glycerol-3-methoxy polyethylene glycol). In some embodiments, DMG-PEG is DMG-PEG2000. In some such embodiments of the LP01-based LNP, the steroid system component comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0266] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parentheses after the components): LP01 (44-46), neutral / accessory lipid component (8-10), steroid system component (43-45), and peg-modified lipid component (1.5-2.5). In some such embodiments, the neutral / accessory lipid component comprises DSPC. In some such embodiments of the LP01-based LNP, the steroid system component comprises cholesterol. In some such embodiments of the LP01-based LNP, the peg-modified lipid component comprises peg-modified myristate diglyceride (DMG-PEG; 1,2-dimyristoyl-sn-glycerol-3-methoxy polyethylene glycol). In some embodiments, DMG-PEG is DMG-PEG2000. In some such embodiments of the LP01-based LNP, the steroid system component comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0267] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parentheses after the components): LP01 (45), a neutral / auxiliary lipid component (9), a steroid system component (44), and a peg-modified lipid component (2). In some such embodiments, the neutral / auxiliary lipid component comprises DSPC. In some such embodiments of the LP01-based LNP, the steroid system component comprises cholesterol. In some such embodiments of the LP01-based LNP, the peg-modified lipid component comprises peg-modified myristate diglyceride (DMG-PEG; 1,2-dimyristoyl-sn-glycerol-3-methoxy polyethylene glycol). In some embodiments, DMG-PEG is DMG-PEG2000. In some such embodiments of the LP01-based LNP, the steroid system component comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0268] Example SM-102LNP formulation

[0269] In some embodiments, a composition is provided comprising lipid nanoparticles (LNPs), the LNPs comprising (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease, and (ii) at least one guide RNA of a target gene or a nucleic acid encoding at least one guide RNA, wherein the LNPs comprise SM-102.

[0270] In some embodiments, the LNP comprises the following structural components: an ionizable SM-102 lipid, a neutral / accessory lipid component, a steroid system component, and a peg-modified lipid component. In some such embodiments of the SM-102-based LNP, the neutral / accessory lipid component comprises DSPC. In some such embodiments of the SM-102-based LNP, the steroid system component comprises cholesterol. In some such embodiments of the SM-102-based LNP, the peg-modified lipid component comprises peg-modified myristate diglyceride (DMG-PEG; 1,2-dimyristoyl-sn-glycerol-3-methoxy polyethylene glycol). In some embodiments, DMG-PEG is DMG-PEG2000. In some such embodiments of the SM-102-based LNP, the steroid system component comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0271] 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 / accessory lipid component (7-13), steroid system component (33.5-43.5), and peg-modified lipid component (0.5-2.5). In some such embodiments, the neutral / accessory lipid component comprises DSPC. In some such embodiments of the SM-102-based LNP, the steroid system component comprises cholesterol. In some such embodiments of the SM-102-based LNP, the peg-modified lipid component comprises peg-modified myristate diglyceride (DMG-PEG; 1,2-dimyristoyl-sn-glycerol-3-methoxy polyethylene glycol). In some embodiments, DMG-PEG is DMG-PEG2000. In some such embodiments of the SM-102-based LNP, the steroid system component comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0272] 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 / accessory lipid component (7.5-12.5), steroid system component (34.5-42.5), and peg-modified lipid component (0.75-2.25). In some such embodiments, the neutral / accessory lipid component comprises DSPC. In some such embodiments of the SM-102-based LNP, the steroid system component comprises cholesterol. In some such embodiments of the SM-102-based LNP, the peg-modified lipid component comprises peg-modified myristate diglyceride (DMG-PEG; 1,2-dimyristoyl-sn-glycerol-3-methoxy polyethylene glycol). In some embodiments, DMG-PEG is DMG-PEG2000. In some such embodiments of the SM-102-based LNP, the steroid system component comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0273] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parentheses after the components): SM-102 (47-53), neutral / auxiliary lipid component (8-12), steroid system component (35.5-41.5), and peg-modified lipid component (1-2). In some such embodiments, the neutral / auxiliary lipid component comprises DSPC. In some such embodiments of the SM-102-based LNP, the steroid system component comprises cholesterol. In some such embodiments of the SM-102-based LNP, the peg-modified lipid component comprises peg-modified myristate diglyceride (DMG-PEG; 1,2-dimyristoyl-sn-glycerol-3-methoxy polyethylene glycol). In some embodiments, DMG-PEG is DMG-PEG2000. In some such embodiments of the SM-102-based LNP, the steroid system component comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0274] 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 / accessory lipid component (8.5-11.5), steroid system component (36.5-40.5), and peg-modified lipid component (1-2). In some such embodiments, the neutral / accessory lipid component comprises DSPC. In some such embodiments of the SM-102-based LNP, the steroid system component comprises cholesterol. In some such embodiments of the SM-102-based LNP, the peg-modified lipid component comprises peg-modified myristate diglyceride (DMG-PEG; 1,2-dimyristoyl-sn-glycerol-3-methoxy polyethylene glycol). In some embodiments, DMG-PEG is DMG-PEG2000. In some such embodiments of the SM-102-based LNP, the steroid system component comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0275] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parentheses after the components): SM-102 (49-51), neutral / accessory lipid component (9-11), steroid system component (37.5-39.5), and peg-modified lipid component (1-2). In some such embodiments, the neutral / accessory lipid component comprises DSPC. In some such embodiments of the SM-102-based LNP, the steroid system component comprises cholesterol. In some such embodiments of the SM-102-based LNP, the peg-modified lipid component comprises peg-modified myristate diglyceride (DMG-PEG; 1,2-dimyristoyl-sn-glycerol-3-methoxy polyethylene glycol). In some embodiments, DMG-PEG is DMG-PEG2000. In some such embodiments of the SM-102-based LNP, the steroid system component comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0276] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parentheses after the components): SM-102 (50), a neutral / auxiliary lipid component (10), a steroid system component (38.5), and a peg-modified lipid component (1.5). In some such embodiments, the neutral / auxiliary lipid component comprises DSPC. In some such embodiments of the SM-102-based LNP, the steroid system component comprises cholesterol. In some such embodiments of the SM-102-based LNP, the peg-modified lipid component comprises peg-modified myristate diglyceride (DMG-PEG; 1,2-dimyristoyl-sn-glycerol-3-methoxy polyethylene glycol). In some embodiments, DMG-PEG is DMG-PEG2000. In some such embodiments of the SM-102-based LNP, the steroid system component comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0277] Example ALC0315 LNP formulation

[0278] In some embodiments, a composition is provided comprising lipid nanoparticles (LNPs), the LNPs comprising (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease, and (ii) at least one guide RNA of a target gene or a nucleic acid encoding at least one guide RNA, wherein the LNPs comprise ALC0315.

[0279] In some embodiments, the LNP comprises the following structural components: an ALC0315 ionizable lipid, a neutral / auxiliary lipid component, a steroid system component, and a peg-modified lipid component. In some such embodiments of the ALC0315-based LNP, the neutral / auxiliary lipid component comprises DSPC. In some such embodiments of the ALC0315-based LNP, the steroid system component comprises cholesterol. In some such embodiments of the ALC0315-based LNP, the peg-modified lipid component comprises peg-modified myristate diglyceride (DMG-PEG; 1,2-dimyristoyl-sn-glycerol-3-methoxy polyethylene glycol). In some embodiments, DMG-PEG is DMG-PEG2000. In some such embodiments of the ALC0315-based LNP, the steroid system component comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0280] 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 / auxiliary lipid component (7-13), steroid system component (33.5-43.5), and peg-modified lipid component (0.5-2.5). In some such embodiments, the neutral / auxiliary lipid component comprises DSPC. In some such embodiments of the ALC0315-based LNP, the steroid system component comprises cholesterol. In some such embodiments of the ALC0315-based LNP, the peg-modified lipid component comprises peg-modified myristate diglyceride (DMG-PEG; 1,2-dimyristoyl-sn-glycerol-3-methoxy polyethylene glycol). In some embodiments, DMG-PEG is DMG-PEG2000. In some such embodiments of the ALC0315-based LNP, the steroid system component comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0281] 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 / accessory lipid component (7.5-12.5), steroid system component (34.5-42.5), and peg-modified lipid component (0.75-2.25). In some such embodiments, the neutral / accessory lipid component comprises DSPC. In some such embodiments of the ALC0315-based LNP, the steroid system component comprises cholesterol. In some such embodiments of the ALC0315-based LNP, the peg-modified lipid component comprises peg-modified myristate diglyceride (DMG-PEG; 1,2-dimyristoyl-sn-glycerol-3-methoxy polyethylene glycol). In some embodiments, DMG-PEG is DMG-PEG2000. In some such embodiments of the ALC0315-based LNP, the steroid system component comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0282] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parentheses after the components): ALC0315 (47-53), neutral / auxiliary lipid component (8-12), steroid system component (35.5-41.5), and peg-modified lipid component (1-2). In some such embodiments, the neutral / auxiliary lipid component comprises DSPC. In some such embodiments of the ALC0315-based LNP, the steroid system component comprises cholesterol. In some such embodiments of the ALC0315-based LNP, the peg-modified lipid component comprises peg-modified myristate diglyceride (DMG-PEG; 1,2-dimyristoyl-sn-glycerol-3-methoxy polyethylene glycol). In some embodiments, DMG-PEG is DMG-PEG2000. In some such embodiments of the ALC0315-based LNP, the steroid system component comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0283] 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), a neutral / auxiliary lipid component (8.5-11.5), a steroid system component (36.5-40.5), and a peg-modified lipid component (1-2). In some such embodiments, the neutral / auxiliary lipid component comprises DSPC. In some such embodiments of the ALC0315-based LNP, the steroid system component comprises cholesterol. In some such embodiments of the ALC0315-based LNP, the peg-modified lipid component comprises peg-modified myristate diglyceride (DMG-PEG; 1,2-dimyristoyl-sn-glycerol-3-methoxy polyethylene glycol). In some embodiments, DMG-PEG is DMG-PEG2000. In some such embodiments of the ALC0315-based LNP, the steroid system component comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0284] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parentheses after the components): ALC0315 (49-51), neutral / auxiliary lipid component (9-11), steroid system component (37.5-39.5), and peg-modified lipid component (1-2). In some such embodiments, the neutral / auxiliary lipid component comprises DSPC. In some such embodiments of the ALC0315-based LNP, the steroid system component comprises cholesterol. In some such embodiments of the ALC0315-based LNP, the peg-modified lipid component comprises peg-modified myristate diglyceride (DMG-PEG; 1,2-dimyristoyl-sn-glycerol-3-methoxy polyethylene glycol). In some embodiments, DMG-PEG is DMG-PEG2000. In some such embodiments of the ALC0315-based LNP, the steroid system component comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0285] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parentheses after the components): ALC0315 (50), a neutral / auxiliary lipid component (10), a steroid system component (38.5), and a peg-modified lipid component (1.5). In some such embodiments, the neutral / auxiliary lipid component comprises DSPC. In some such embodiments of the ALC0315-based LNP, the steroid system component comprises cholesterol. In some such embodiments of the ALC0315-based LNP, the peg-modified lipid component comprises peg-modified myristate diglyceride (DMG-PEG; 1,2-dimyristoyl-sn-glycerol-3-methoxy polyethylene glycol). In some embodiments, DMG-PEG is DMG-PEG2000. In some such embodiments of the ALC0315-based LNP, the steroid system component comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0286] C. Virus-like particles

[0287] In one aspect, this disclosure covers methods 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 a VLP. Without wishing to be bound by any particular theory, in some embodiments, when nucleic acids are present in the particles, they are resistant to degradation by nucleases in aqueous solutions. In other embodiments, when proteins are present in the particles, they are protected from degradation by proteases. In some embodiments, proteins and nucleic acids encapsulated by the VLP are capable of penetrating the cell membrane.

[0288] In some embodiments, the CRISPR gene editing system comprises one or more RNA-containing compositions. In some embodiments, the CRISPR gene editing system further comprises one or more VLPs. In some embodiments, the one or more RNA-containing compositions comprise guide RNA. In some embodiments, the one or more RNA-containing compositions comprise mRNA. In some embodiments, the one or more RNA-containing compositions comprise RNPs (e.g., Cas9 and guide RNA).

[0289] In some embodiments, the CRISPR gene editing system comprises one or more virus-like particles, which collectively encapsulate (i) an RNA-guided nuclease or nucleic acid encoding an RNA-guided nuclease, and (ii) at least one guide RNA or nucleic acid encoding at least one guide RNA. In some embodiments, the one or more virus-like particles comprise: a first plurality of virus-like particles encapsulating the RNA-guided nuclease or nucleic acid encoding an RNA-guided nuclease; and a second plurality of virus-like particles encapsulating the at least one guide RNA or nucleic acid encoding at least one guide RNA.

[0290] In one aspect, the use of a CRISPR gene-editing system further comprising one or more VLPs targeting TGFB1, TGFBR1, and / or TGFBR2 is therapeutic. In some embodiments, the use of the system can treat fibrosis and / or scarring. In some embodiments, the use of the system can treat one or more musculoskeletal diseases, conditions, and patients, including but not limited to Loeys-Dietz syndrome, osteoarthritis, Marfan syndrome, aortic aneurysm (e.g., familial T3 aortic aneurysm), craniofacial abnormalities, and combinations thereof. In other embodiments, the use of the system can treat vegetation diseases, conditions, and patients, including but not limited to pancreatic cancer, multiple self-healing squamous cell carcinoma (Ferguson-Smith disease), gastrointestinal stromal tumor (GIST), hereditary nonpolyposis colorectal cancer (Lynch syndrome), metastatic colorectal cancer, bone vegetations, anaplastic carcinoma, spindle cell carcinoma, lung vegetations, brain vegetations, and combinations thereof.

[0291] D. Other delivery modes

[0292] 1. Liposomes

[0293] In some embodiments, nucleic acids encoding a CRISPR gene editing system (e.g., Cas9 or gRNA) targeting genes selected from TGFB1, TGFBR1, TGFBR2, and combinations thereof are encapsulated in positively charged liposomes (e.g., lipofectin), which may be labeled with antibodies against cell surface antigens of the target cells. These delivery mediators can also be used to deliver Cas9 protein / gRNA complexes.

[0294] 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).

[0295] In some embodiments, the composition comprises one or more liposomes that co-encapsulate (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease, and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA. In some embodiments, the one or more liposomes comprise a first plurality of liposomes that encapsulate the RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease; and a second plurality of liposomes that encapsulate the at least one guide RNA or a nucleic acid encoding at least one guide RNA.

[0296] In one aspect, the use of a CRISPR gene-editing system further comprising one or more liposomes targeting TGFB1, TGFBR1, and / or TGFBR2 is therapeutic. In some embodiments, the use of said system can treat fibrosis and / or scarring. In some embodiments, the use of said system can treat one or more musculoskeletal diseases, conditions, and patients, including but not limited to Loeys-Dietz syndrome, osteoarthritis, Marfan syndrome, aortic aneurysm (e.g., familial T3 aortic aneurysm), craniofacial abnormalities, and combinations thereof. In other embodiments, the use of said system can treat vegetation diseases, conditions, and patients, including but not limited to pancreatic cancer, multiple self-healing squamous cell carcinoma (Ferguson-Smith disease), gastrointestinal stromal tumor (GIST), hereditary nonpolyposis colorectal cancer (Lynch syndrome), metastatic colorectal cancer, bone vegetations, anaplastic carcinoma, spindle cell carcinoma, lung vegetations, brain vegetations, and combinations thereof.

[0297] 2. Lipid nanocrystals (LNC)

[0298] In one aspect, this disclosure covers methods 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 an LNC. Without wishing to be bound by any particular theory, in some embodiments, when nucleic acids are present in the nanocrystals, they are resistant to degradation by nucleases in aqueous solution. In other embodiments, when proteins are present in the nanocrystals, they are protected from degradation by proteases. In some embodiments, proteins and nucleic acids encapsulated in the nanocrystals are capable of penetrating the cell membrane.

[0299] In some embodiments, the CRISPR gene editing system comprises one or more RNA-containing compositions. In some embodiments, the CRISPR gene editing system further comprises one or more nanocrystals. In some embodiments, the one or more RNA-containing compositions comprise guide RNA. In some embodiments, the one or more RNA-containing compositions comprise mRNA. In some embodiments, the one or more RNA-containing compositions comprise RNPs (e.g., Cas9 and guide RNA). In some embodiments, the one or more nanocrystals are lipid nanocrystals (LNCs).

[0300] In some embodiments, the CRISPR gene editing system comprises one or more LNCs, which collectively encapsulate (i) an RNA-guided nuclease or nucleic acid encoding an RNA-guided nuclease, and (ii) at least one guide RNA or nucleic acid encoding at least one guide RNA. In some embodiments, the one or more LNCs comprise: a first plurality of LNCs, which encapsulate the RNA-guided nuclease or nucleic acid encoding an RNA-guided nuclease; and a second plurality of LNCs, which encapsulate the at least one guide RNA or nucleic acid encoding at least one guide RNA.

[0301] In one aspect, the use of a CRISPR gene-editing system further comprising one or more LNCs targeting TGFB1, TGFBR1, and / or TGFBR2 is therapeutic. In some embodiments, the use of the system can treat fibrosis and / or scarring. In some embodiments, the use of the system can treat one or more musculoskeletal diseases, conditions, and patients, including but not limited to Loeys-Dietz syndrome, osteoarthritis, Marfan syndrome, aortic aneurysm (e.g., familial T3 aortic aneurysm), craniofacial abnormalities, and combinations thereof. In other embodiments, the use of the system can treat vegetation diseases, conditions, and patients, including but not limited to pancreatic cancer, multiple self-healing squamous cell carcinoma (Ferguson-Smith disease), gastrointestinal stromal tumor (GIST), hereditary nonpolyposis colorectal cancer (Lynch syndrome), metastatic colorectal cancer, bone vegetations, anaplastic carcinoma, spindle cell carcinoma, lung vegetations, brain vegetations, and combinations thereof.

[0302] VI. Pharmaceutical Composition

[0303] In one aspect, this disclosure covers pharmaceutical compositions comprising a CRISPR gene-editing system for treating mammals in need. In some embodiments, the CRISPR gene-editing system targets a gene selected from TGFB1, TGFBR1, TGFBR2, and combinations thereof. In some embodiments, the mammal is selected from humans, dogs, horses, and cats.

[0304] A.TGFB1

[0305] 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 Zielińska A, et al., “Polymeric Nanoparticles: Production, Characterization, Toxicology and Ecotoxicology,” *Molecules*, 25(16):3731 (2020), the contents of which are incorporated herein by reference in their entirety for all purposes. In some embodiments, CRISPR gene editing systems targeting TGFB1 are delivered to mammalian cells via exosomes. For a review of exosomes, see Dimik M, et al., “The exosome: a review of current therapeutic roles and capabilities in human reproduction,” *DrugDeliv.Transl.Res.*, 13(2):473-502 (2023), the contents of which are incorporated herein by reference in their entirety for all purposes.

[0306] In various embodiments, the method of using a pharmaceutical composition comprising a CRISPR gene-editing system targeting the TGFB1 gene to treat mammals in need is described. In some embodiments, the method can treat fibrosis and / or scarring. In some embodiments, the method can treat one or more musculoskeletal diseases, conditions, and patients, including but not limited to Loeys-Dietz syndrome, osteoarthritis, Marfan syndrome, aortic aneurysm (e.g., familial T3 aortic aneurysm), craniofacial abnormalities, and combinations thereof. In other embodiments, the method can treat vegetation diseases, conditions, and patients, including but not limited to pancreatic cancer, multiple self-healing squamous cell carcinoma (Ferguson-Smith disease), gastrointestinal stromal tumor (GIST), hereditary nonpolyposis colorectal cancer (Lynch syndrome), metastatic colorectal cancer, bone vegetations, anaplastic carcinoma, spindle cell carcinoma, lung vegetations, brain vegetations, and combinations thereof.

[0307] 1. Treating musculoskeletal disorders by targeting the transforming growth factor β gene.

[0308] In one aspect, methods and pharmaceutical compositions are provided for treating musculoskeletal disorders using CRISPR drugs. In another aspect, methods and pharmaceutical compositions are provided for treating musculoskeletal disorders using a CRISPR pharmaceutical composition targeting the transforming growth factor β gene. 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) targeting the transforming growth factor β gene or a nucleic acid encoding at least one gRNA, wherein the gRNA specifically binds to a target sequence of a protospacer neighbor motif (PAM) sequence of a neighboring RNA-guided nuclease. In some embodiments, the CRISPR pharmaceutical composition comprises (i) mRNA encoding an RNA-guided nuclease, and (ii) at least one gRNA targeting the transforming growth factor β gene. In some embodiments, the mRNA encoding the RNA-guided nuclease and at least one gRNA are encapsulated in lipid nanoparticles (LNPs). In some embodiments, the musculoskeletal disorders treated with a CRISPR drug composition targeting the TGFB1 gene are Loeys-Dietz syndrome, osteoarthropathy, Marfan syndrome, aortic aneurysm (familial T3), or craniofacial abnormalities.

[0309] In some embodiments, the transforming growth factor β receptor gene is the TGFB1 gene. In some embodiments, the TGFB1 gene is the human TGFB1 gene, and the guide RNA contains a spacer sequence selected from SEQ ID NO:1-198.

[0310] 2. Treatment of fibrosis and / or scarring by targeting the transforming growth factor β gene.

[0311] In one aspect, methods and pharmaceutical compositions are provided for treating fibrosis and / or scarring using a CRISPR drug. In another aspect, methods and pharmaceutical compositions are provided for treating fibrosis and / or scarring using a CRISPR drug composition targeting the transforming growth factor β gene. In some embodiments, the CRISPR drug composition comprises (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease, and (ii) at least one guide RNA (gRNA) targeting the transforming growth factor β gene or a nucleic acid encoding at least one gRNA, wherein the gRNA specifically binds to a target sequence of a protospacer adjacent motif (PAM) sequence of a neighboring RNA-guided nuclease. In some embodiments, the CRISPR drug composition comprises (i) mRNA encoding an RNA-guided nuclease, and (ii) at least one gRNA targeting the transforming growth factor β gene. In some embodiments, the fibrosis and / or scarring is due to knee fibrosis, intra-articular fibrotic nodules, or epidural fibrosis, and is treated using a CRISPR drug composition targeting the TGFB1 gene. In some embodiments, the CRISPR drug composition as described herein is a post-surgical treatment for the prevention or reduction of 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), and microsurgical discectomy, followed by treatment of fibrosis and / or scarring using a CRISPR drug composition targeting the TGFB1 gene. In some embodiments, fibrosis and / or scarring are a consequence of conditions that may be induced or exacerbated by the surgical procedure.

[0312] In some embodiments, the transforming growth factor β receptor gene is the TGFB1 gene. In some embodiments, the TGFB1 gene is the human TGFB1 gene, and the guide RNA contains a spacer sequence selected from SEQ ID NO:1-198.

[0313] B.TGFBR1

[0314] 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.

[0315] In various embodiments, the method of using a pharmaceutical composition comprising a CRISPR gene-editing system targeting the TGFBR1 gene to treat mammals in need is described. In some embodiments, the method can treat fibrosis and / or scarring. In some embodiments, the method can treat one or more musculoskeletal diseases, conditions, and patients, including but not limited to Lloyd-Ditz syndrome, osteoarthritis, tendon injury, Marfan syndrome, aortic aneurysm (e.g., familial T3 aortic aneurysm), craniofacial abnormalities, and combinations thereof. In other embodiments, the method can treat vegetation diseases, conditions, and patients, including but not limited to pancreatic cancer, multiple self-healing squamous cell carcinoma (Ferguson-Smith disease), gastrointestinal stromal tumor (GIST), hereditary nonpolyposis colorectal cancer (Lynch syndrome), metastatic colorectal cancer, bone vegetations, anaplastic carcinoma, spindle cell carcinoma, lung vegetations, brain vegetations, and combinations thereof.

[0316] 1. Treating musculoskeletal disorders by targeting the transforming growth factor β receptor 1 gene.

[0317] In one aspect, methods and pharmaceutical compositions are provided for treating musculoskeletal disorders using CRISPR drugs. In another aspect, methods and pharmaceutical compositions are provided for treating musculoskeletal disorders using CRISPR pharmaceutical compositions targeting the transforming growth factor β receptor gene. 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) targeting the transforming growth factor β receptor gene or a nucleic acid encoding at least one gRNA, wherein the gRNA specifically binds to a target sequence of a protospacer neighbor motif (PAM) sequence of a neighboring 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 targeting the transforming growth factor β receptor gene. In some embodiments, the mRNA encoding the RNA-guided nuclease and at least one gRNA are encapsulated in lipid nanoparticles (LNPs). In some embodiments, the musculoskeletal disorders treated with CRISPR drug compositions targeting the TGFBR1 or TGFBR2 genes are Loeys-Dietz syndrome, osteoarthropathy, Marfan syndrome, aortic aneurysm (familial T3), or craniofacial abnormalities.

[0318] In some embodiments, the transforming growth factor β receptor gene is the TGFBR1 gene. In some embodiments, the TGFBR1 gene is the human TGFBR1 gene, and the guide RNA contains spacer sequences selected from SEQ ID NO: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 contains spacer sequences selected from SEQ ID NO:199-228. In some embodiments, editing of the TGFBR1 gene produces 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 contains spacer sequences selected from SEQ ID NO:229-308 and SEQ ID NO:317-318. In some embodiments, editing of the TGFBR1 gene produces a gene expressing a soluble receptor decoy protein. In some such embodiments, the TGFBR1 gene is the human TGFBR1 gene, and the guide RNA contains spacer sequences selected from SEQ ID NO:309-318.

[0319] 2. Treating fibrosis by targeting the transforming growth factor β receptor 1 gene.

[0320] In one aspect, methods and pharmaceutical compositions are provided for treating fibrosis and / or scarring using a CRISPR drug. In another aspect, methods and pharmaceutical compositions are provided for treating fibrosis and / or scarring using a CRISPR drug composition targeting the transforming growth factor β receptor gene. In some embodiments, the CRISPR drug composition comprises (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease, and (ii) at least one guide RNA (gRNA) targeting the transforming growth factor β receptor gene or a nucleic acid encoding at least one gRNA, wherein the gRNA specifically binds to a target sequence of a protospacer adjacent motif (PAM) sequence of a neighboring RNA-guided nuclease. In some embodiments, the CRISPR drug composition comprises (i) mRNA encoding an RNA-guided nuclease, and (ii) at least one gRNA targeting the transforming growth factor β receptor gene. In some embodiments, the fibrosis and / or scarring is caused by knee fibrosis, intra-articular fibrotic nodules, or epidural fibrosis, and is treated using a CRISPR drug composition targeting the TGFBR1 gene or the TGFBR2 gene. In some embodiments, the CRISPR pharmaceutical compositions described herein are post-surgical treatments for the prevention or reduction of fibrosis and / or scarring. For example, in some embodiments, surgical procedures selected from ligament reconstruction, anterior cruciate ligament (ACL) reconstruction, autologous ACL reconstruction, allogeneic ACL reconstruction, tendon repair, fracture repair, total knee arthroplasty (TKA), and microsurgical discectomy are followed by treatment of fibrosis and / or scarring using a CRISPR pharmaceutical composition targeting the TGFBR1 or TGFBR2 gene. In some embodiments, fibrosis and / or scarring are a consequence of conditions that may be induced or exacerbated by the surgical procedure.

[0321] In some embodiments, the transforming growth factor β receptor gene is the TGFBR1 gene. In some embodiments, the TGFBR1 gene is the human TGFBR1 gene, and the guide RNA comprises a spacer sequence selected from SEQ ID NO: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 comprises a spacer sequence selected from SEQ ID NO:199-228. In some embodiments, editing of the TGFBR1 gene produces 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 comprises a spacer sequence selected from SEQ ID NO:229-308 and SEQ ID NO:317-318. In some embodiments, editing of the TGFBR1 gene produces a gene expressing a soluble receptor decoy protein. In some such embodiments, the TGFBR1 gene is the human TGFBR1 gene, and the guide RNA comprises a spacer sequence selected from SEQ ID NO:309-318.

[0322] C.TGFBR2

[0323] 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.

[0324] In various embodiments, the method of using a pharmaceutical composition comprising a CRISPR gene-editing system targeting the TGFBR2 gene to treat mammals in need is described. In some embodiments, the method can treat fibrosis and / or scarring. In some embodiments, the method can treat one or more musculoskeletal diseases, conditions, and patients, including but not limited to Loeys-Dietz syndrome, osteoarthritis, Marfan syndrome, aortic aneurysm (e.g., familial T3 aortic aneurysm), craniofacial abnormalities, and combinations thereof. In other embodiments, the method can treat vegetation diseases, conditions, and patients, including but not limited to pancreatic cancer, multiple self-healing squamous cell carcinoma (Ferguson-Smith disease), gastrointestinal stromal tumor (GIST), hereditary nonpolyposis colorectal cancer (Lynch syndrome), metastatic colorectal cancer, bone vegetations, anaplastic carcinoma, spindle cell carcinoma, lung vegetations, brain vegetations, and combinations thereof.

[0325] 1. Treating musculoskeletal disorders by targeting the transforming growth factor β receptor 2 gene.

[0326] In one aspect, methods and pharmaceutical compositions are provided for treating musculoskeletal disorders using CRISPR drugs. In another aspect, methods and pharmaceutical compositions are provided for treating musculoskeletal disorders using a CRISPR pharmaceutical composition targeting the transforming growth factor β receptor gene. 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) targeting the transforming growth factor β receptor gene or a nucleic acid encoding at least one gRNA, wherein the gRNA specifically binds to a target sequence of a protospacer neighbor motif (PAM) sequence of a neighboring 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 targeting the transforming growth factor β receptor gene. In some embodiments, the mRNA encoding the RNA-guided nuclease and at least one gRNA are encapsulated in lipid nanoparticles (LNPs). In some embodiments, the musculoskeletal disorders treated with CRISPR drug compositions targeting the TGFBR1 or TGFBR2 genes are Loeys-Dietz syndrome, osteoarthropathy, Marfan syndrome, aortic aneurysm (familial T3), or craniofacial abnormalities.

[0327] In some embodiments, the transforming growth factor β receptor gene is the TGFBR2 gene. In some embodiments, the TGFBR2 gene is the human TGFBR2 gene, and the guide RNA contains spacer sequences selected from SEQ ID NO: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 contains spacer sequences selected from SEQ ID NO:321-354. In some embodiments, editing of the TGFBR2 gene produces 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 contains spacer sequences selected from SEQ ID NO:355-498 and SEQ ID NO:503-508. In some embodiments, editing of the TGFBR2 gene produces a gene expressing a soluble receptor decoy protein. In some such embodiments, the TGFBR2 gene is the human TGFBR2 gene, and the guide RNA contains spacer sequences selected from SEQ ID NO:499-508.

[0328] 2. Treatment of fibrosis and / or scarring by targeting the transforming growth factor β receptor 2 gene.

[0329] In one aspect, methods and pharmaceutical compositions are provided for treating fibrosis and / or scarring using a CRISPR drug. In another aspect, methods and pharmaceutical compositions are provided for treating fibrosis and / or scarring using a CRISPR drug composition targeting the transforming growth factor β receptor gene. In some embodiments, the CRISPR drug composition comprises (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease, and (ii) at least one guide RNA (gRNA) targeting the transforming growth factor β receptor gene or a nucleic acid encoding at least one gRNA, wherein the gRNA specifically binds to a target sequence of a protospacer adjacent motif (PAM) sequence of a neighboring RNA-guided nuclease. In some embodiments, the CRISPR drug composition comprises (i) mRNA encoding an RNA-guided nuclease, and (ii) at least one gRNA targeting the transforming growth factor β receptor gene. In some embodiments, the fibrosis and / or scarring is caused by knee fibrosis, intra-articular fibrotic nodules, or epidural fibrosis, and is treated using a CRISPR drug composition targeting the TGFBR1 gene or the TGFBR2 gene. In some embodiments, the CRISPR pharmaceutical compositions described herein are post-surgical treatments for the prevention or reduction of fibrosis and / or scarring. For example, in some embodiments, surgical procedures selected from ligament reconstruction, anterior cruciate ligament (ACL) reconstruction, autologous ACL reconstruction, allogeneic ACL reconstruction, tendon repair, fracture repair, total knee arthroplasty (TKA), and microsurgical discectomy are followed by treatment of fibrosis and / or scarring using a CRISPR pharmaceutical composition targeting the TGFBR1 or TGFBR2 gene. In some embodiments, fibrosis and / or scarring are a consequence of conditions that may be induced or exacerbated by the surgical procedure.

[0330] In some embodiments, the transforming growth factor β receptor gene is the TGFBR2 gene. In some embodiments, the TGFBR2 gene is the human TGFBR2 gene, and the guide RNA contains spacer sequences selected from SEQ ID NO: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 contains spacer sequences selected from SEQ ID NO:321-354. In some embodiments, editing of the TGFBR2 gene produces 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 contains spacer sequences selected from SEQ ID NO:355-498 and SEQ ID NO:503-508. In some embodiments, editing of the TGFBR2 gene produces a gene expressing a soluble receptor decoy protein. In some such embodiments, the TGFBR2 gene is the human TGFBR2 gene, and the guide RNA contains spacer sequences selected from SEQ ID NO:499-508.

[0331] VII. Method of application

[0332] The methods and compositions described herein cover the use of pharmaceutical compositions comprising CRISPR gene editing systems as active ingredients.

[0333] Depending on the method / route of administration, there are several types of drug dosage forms. These include various types of liquid, solid, and semi-solid dosage forms. Common drug dosage forms include pills, tablets or capsules, decoctions or syrups, and natural or herbal forms, such as a wide variety of plants or foods, as well as many other forms. It is worth noting that the route of administration (ROA) for drug delivery depends on the dosage form of the substance in question. Liquid drug dosage forms are liquid forms of compounds intended for administration or consumption in a given dose as a drug or medicine.

[0334] As described below, the compositions of this disclosure can be delivered to a subject subcutaneously (e.g., intra-articular or intervertebral disc injection), dermally (e.g., via a patch), and / or via an implant. Exemplary drug dosage forms include, for example, pills, permeation delivery systems, elixirs, emulsions, hydrogels, suspensions, syrups, capsules, tablets, orally dissolving tablets (ODTs), gel capsules, films, adhesive topical patches, lollipops, tablets, chewing gum, dry powder inhalers (DPIs), vaporizers, nebulizers, metered-dose inhalers (MDIs), ointments, transdermal patches, and intradermal implants.

[0335] As used herein, “dermal delivery” or “dermal administration” can refer to a route of administration in which a drug dosage form is delivered to or through the dermis (i.e., the layer of skin between the epidermis (which constitutes the skin) and the subcutaneous tissue). “Subcutaneous delivery” can refer to a route of administration in which a drug dosage form reaches or is below the subcutaneous tissue layer.

[0336] Methods for preparing suitable pharmaceutical compositions are known in the art, see, for example, *Remington: The Science and Practice of Pharmacy*, 21st edition, 2005; and books in the *Drugs and the Pharmaceutical Sciences: A Series of Textbooks and Monographs* (Dekker, NY). For example, solutions or suspensions intended for parenteral, intradermal, or subcutaneous administration may include the following components: sterile diluents such as water for injection, saline solution, non-volatile oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial 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 pH, such as sodium chloride or glucose. pH may be adjusted using acids or bases (such as hydrochloric acid or sodium hydroxide). Parenteral preparations can be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0337] Suitable injectable pharmaceutical compositions may include sterile aqueous solutions (if water-soluble) or dispersions and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, Cremophor EL... TM(BASF, Parsippany, NJ) or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and should be fluid enough to be easily injectable. It should be stable under the conditions of manufacture and storage and must be preservative-resistant against microbial contamination such as bacteria and fungi. The carrier can 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. For example, by using a coating such as lecithin, the desired particle size can be maintained in the dispersed state, and by using surfactants, appropriate flowability can be maintained. Prevention of microbial action can be achieved by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be preferred to include an isotonic agent in the composition, such as sugars, polyols such as mannitol, sorbitol, sodium chloride. Prolonged absorption of injectable compositions can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin, in the composition.

[0338] Pharmaceutical compositions suitable for injectable use may also include cryoprotectants, which improve the stability of the formulation, especially when frozen. For further discussion on conditions suitable for storing LNPs, see Kim B et al., “Optimization of storage conditions for lipid nanoparticle-formulated self-replicating RNA vaccines,” J. Control Release, 353:241-53 (2023), the contents of which are incorporated herein by reference in their entirety for all purposes.

[0339] Sterile injectable solutions can be prepared by incorporating the desired amount of the active compound into a suitable solvent, which, if desired, contains one or a combination of the components listed above, followed by filtration sterilization. Typically, dispersions are prepared by incorporating the active compound into a sterile medium containing a basic dispersion medium and any other desired components from those listed above. In the case of sterile powders used to prepare sterile injectable solutions, preferred methods of preparation include vacuum drying and freeze-drying, which produce a powder of the active ingredient plus any other desired components from its previously sterile filtered solution.

[0340] As a nucleic acid or a therapeutic compound comprising a nucleic acid, it can be administered by any method suitable for the administration of nucleic acid agents such as DNA vaccines. These methods include gene guns, bio-injectors, and skin patches, as well as needle-free methods, such as the microparticle DNA vaccine technology disclosed in U.S. Patent No. 6,194,389 and the transdermal needle-free vaccination of mammals using a powder form of vaccine as described in U.S. Patent No. 6,168,587. Furthermore, intranasal delivery is possible, as described, particularly in Hamajima et al., *Clinical Immunology and Immunopathology*, 88(2), 205-10 (1998). Liposomes (e.g., as described in U.S. Patent No. 6,472,375) and microencapsulation can also be used. Biodegradable, targeted microparticle delivery systems (e.g., as described in U.S. Patent No. 6,471,996) can also be used.

[0341] Therapeutic compounds can be prepared using a carrier that protects the therapeutic compound from rapid elimination from the body, such as controlled-release formulations, including implants and microencapsulated delivery systems. Biodegradable, 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-glycolic acid)), polyglycolic acid, collagen, polyorthoesters, polyethylene glycol-coated liposomes, hyaluronic acid, and polylactic acid can be used. Such formulations can be prepared using standard techniques or are commercially available, for example, from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (including liposomes targeting selected cells with monoclonal antibodies against cellular antigens) can also be used as pharmaceutically acceptable carriers. They can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811. Semi-solid, gel, soft gel, or other formulations (including controlled-release formulations) can be used, for example, when application to surgical sites is required. Methods for preparing such formulations are known in the art and may include the use of biodegradable, biocompatible polymers. See, for example, Sawyer et al., Yale J Biol Med., Dec 2006; 79(3-4): 141-152.

[0342] The pharmaceutical compositions described herein may be included in containers, kits, packages, or dispensers along with the instructions for use.

[0343] A. Systemic application

[0344] In some embodiments, the pharmaceutical composition comprising the CRISPR gene-editing system is administered systemically to a mammal in need. 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.

[0345] B. Topical application

[0346] In some embodiments, the pharmaceutical composition comprising a CRISPR gene-editing system is administered topically to a mammal in need. In some embodiments, the topical administration is an intra-articular injection. In some embodiments, the composition is formulated for intra-discal 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 perispinal injection. In some embodiments, the composition is formulated for administration to the facet joints of the spine.

[0347] In some embodiments, a pharmaceutical composition comprising a CRISPR gene-editing system is locally administered to a mammal in need during a surgical procedure. In some embodiments, a pharmaceutical composition comprising a CRISPR gene-editing system is locally administered to a mammal in need 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or 30 days after a surgical procedure.

[0348] 1. Application after musculoskeletal trauma

[0349] In some embodiments, the compositions and methods disclosed herein are applied to a subject following musculoskeletal trauma. In some embodiments, musculoskeletal trauma is the result of a surgical procedure (i.e., a surgical incision). In some embodiments, musculoskeletal trauma is a wound.

[0350] In some embodiments, the composition containing RNA-guided 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 other embodiments, the patch is a bioabsorbable patch. In some embodiments, the composition is a sieve agent. In some embodiments, the composition is a hydrogel.

[0351] Other exemplary compositions comprising RNA-guided nucleases and sgRNA applied to wounds or incisions include the following patents (each of which is incorporated herein by reference in its entirety for all purposes): U.S. Patents 8,574,627, 9,204,953, 9,272,073, 9,592,324, 9,597,426, 9,993,298, 10,471,181, 10,765,423, 10,835,235, 11,338,062, and 11,426,156; and U.S. Patent Applications US20080039877A1, US20200345366A1, and US20200069478A1.

[0352] In some embodiments, this disclosure provides pharmaceutical compositions for treating or preventing conditions having symptoms at least partially caused by intercellular signaling mediated by the transforming growth factor β (TGFβ) signaling pathway, the compositions comprising: (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease; and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA, said at least one guide RNA or the nucleic acid encoding at least one guide RNA targeting the TGFB1 gene, the TGFBR1 gene, the TGFBR2 gene, or a combination thereof.

[0353] In some embodiments, the condition is fibrosis.

[0354] In some embodiments, fibrosis is postoperative fibrosis.

[0355] In some embodiments, fibrosis is fibrosis following ligament reconstruction.

[0356] In some embodiments, fibrosis is fibrosis following anterior cruciate ligament (ACL) reconstruction, fibrosis following autologous ACL reconstruction, fibrosis following allogeneic ACL reconstruction, or fibrosis following tendon repair.

[0357] In some embodiments, fibrosis is knee joint fibrosis.

[0358] In some embodiments, fibrosis is caused by intra-articular fibrous nodules.

[0359] In some embodiments, fibrosis is fibrosis following joint replacement surgery.

[0360] In some embodiments, fibrosis is fibrosis following total knee arthroplasty (TKA).

[0361] In some embodiments, fibrosis is associated with tendon injury or surgery.

[0362] In some embodiments, fibrosis is fibrosis following fracture repair.

[0363] In some embodiments, fibrosis is fibrosis following microdiscectomy.

[0364] In some embodiments, fibrosis is epidural fibrosis following microdiscectomy.

[0365] In some embodiments, fibrosis is fibrosis following lumbar laminectomy.

[0366] In some embodiments, fibrosis is epidural fibrosis following lumbar laminectomy.

[0367] In some embodiments, fibrosis is fibrosis following anterior acromioplasty.

[0368] In some embodiments, fibrosis is fibrosis following subacromial decompression surgery.

[0369] In some embodiments, fibrosis is soft tissue fibrosis following anterior acromioplasty.

[0370] In some embodiments, fibrosis is joint contracture following anterior acromioplasty.

[0371] In some embodiments, fibrosis is arthroscopic fibrosis.

[0372] In some embodiments, fibrosis is arthroscopic fibrosis of the joint.

[0373] In some embodiments, fiberization is trabecular mesh fiberization.

[0374] In some embodiments, trabecular meshwork fibrosis is fibrosis following glaucoma surgery.

[0375] In some embodiments, fibrosis is epidural fibrosis.

[0376] In some embodiments, fibrosis is atrial fibrosis, cardiac fibrosis, myocardial fibrosis, and / or fibrosis following open-heart surgery.

[0377] In some embodiments, fibrosis is fibrosis following liposuction.

[0378] In some embodiments, fibrosis is idiopathic pulmonary fibrosis (IPF).

[0379] In some embodiments, fibrosis is fibrosis in the knee, shoulder, or elbow joint.

[0380] In some embodiments, the condition includes fibrosis in the kidney tissue.

[0381] In some embodiments, the condition is chronic kidney disease.

[0382] In some embodiments, the condition includes fibrosis in the skin tissue.

[0383] In some embodiments, the condition is post-wound scarring, keloid scarring, renal systemic fibrosis, or scleroderma / systemic sclerosis.

[0384] In some embodiments, the condition includes fibrosis in the lung tissue.

[0385] In some embodiments, the condition is fibrothorax, pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, radiation-induced lung injury, progressive massive fibrosis, or scleroderma / systemic sclerosis.

[0386] In some embodiments, the condition includes fibrosis in the liver tissue.

[0387] In some embodiments, the condition is cirrhosis or bridging fibrosis.

[0388] In some embodiments, the condition includes fibrosis in the heart tissue.

[0389] In some embodiments, fibrosis is interstitial fibrosis.

[0390] In some embodiments, the condition is congestive heart failure or hypertension.

[0391] In some embodiments, fibrosis is alternative fibrosis.

[0392] In some embodiments, the condition is myocardial infarction.

[0393] In some embodiments, the condition includes fibrosis in the brain tissue.

[0394] In some embodiments, fibrosis is a gelatinous scar.

[0395] In some embodiments, the condition includes fibrosis in the intestinal tissue.

[0396] In some embodiments, the condition is Crohn's disease.

[0397] In some embodiments, the condition includes fibrosis in the hands or fingers.

[0398] In some embodiments, the condition is palmar aponeurosis contracture.

[0399] In some embodiments, the condition includes fibrosis in the lymphoid tissue.

[0400] In some embodiments, fibrosis is mediastinal fibrosis.

[0401] In some embodiments, the condition includes fibrosis in the bone marrow tissue.

[0402] In some embodiments, fibrosis is myelofibrosis.

[0403] In some embodiments, the condition includes fibrosis in the penile tissue.

[0404] In some embodiments, the condition is Peroni disease.

[0405] In some embodiments, the condition includes fibrosis in the soft tissues of the retroperitoneal space.

[0406] In some embodiments, fibrosis is retroperitoneal fibrosis.

[0407] In some embodiments, the condition includes fibrosis in the musculoskeletal tissue.

[0408] In some embodiments, the condition is a musculoskeletal disorder.

[0409] In some embodiments, musculoskeletal disorders are hereditary musculoskeletal diseases.

[0410] In some embodiments, musculoskeletal disorders are muscular dystrophy.

[0411] In some embodiments, the musculoskeletal disorders are selected from Duchenne muscular dystrophy (DMD), myotonic dystrophy (DM), facioscapulohumeral muscular dystrophy (FSHD), and limb-girdle muscular dystrophy (LGMD).

[0412] In some embodiments, musculoskeletal disorders are type II collagen diseases.

[0413] In some embodiments, musculoskeletal disorders are selected from achondroplasia type II, achondroplasia, Stickler syndrome, and Czech dysplasia.

[0414] In some embodiments, the musculoskeletal disorder is osteogenesis imperfecta (OI).

[0415] In some embodiments, musculoskeletal disorders are autoimmune musculoskeletal diseases.

[0416] In some embodiments, musculoskeletal disorders are autoimmune musculoskeletal diseases with polygenic susceptibility.

[0417] In some embodiments, musculoskeletal disorders are spondyloarthritis (SA).

[0418] In some embodiments, the musculoskeletal condition is ankylosing spondylitis.

[0419] In some embodiments, musculoskeletal disorders are rheumatoid arthritis.

[0420] In some embodiments, musculoskeletal disorders are autoimmune osteoarthritis.

[0421] In some embodiments, musculoskeletal disorders are osteoarthritis (OA).

[0422] In some embodiments, musculoskeletal disorders are progressive diaphyseal dysplasia (Camurati-Engelmanndisease).

[0423] In some embodiments, the musculoskeletal disorder is Sjogren's Syndrome.

[0424] In some embodiments, musculoskeletal disorders are mechanical musculoskeletal injuries.

[0425] In some embodiments, musculoskeletal disorders are selected from chronic muscle injury, tendon rupture, post-traumatic osteoarthritis, and post-traumatic joint fibrosis.

[0426] In some embodiments, the condition is cancer. In some embodiments, the cancer is musculoskeletal cancer. In some embodiments, the cancer is cancer in which TGF-β or TGFBR is involved.

[0427] In some embodiments, the at least one guide RNA or the nucleic acid encoding at least one guide RNA targets the mammalian TGFB1 gene.

[0428] In some embodiments, the mammalian TGFB1 gene is the canine, equine, or cat TGFB1 gene.

[0429] In some embodiments, the mammalian TGFB1 gene is the human TGFB1 gene.

[0430] In some embodiments, the at least one guide RNA contains selectable... Figure 1A-1D The spacer subsequences of the groups formed by the sequences shown (SEQ ID NO: 1-198).

[0431] In some embodiments, the at least one guide RNA contains selectable... Figure 12A The spacer subsequences of the group consisting of those sequences (SEQ ID NO: 520-527) shown.

[0432] In some embodiments, the at least one guide RNA comprises a spacer sequence of OHTG03 (SEQ ID NO:522) or OHTG04 (SEQ ID NO:523).

[0433] In some embodiments, the at least one guide RNA or the nucleic acid encoding at least one guide RNA targets the mammalian TGFBR1 gene.

[0434] In some embodiments, the mammalian TGFBR1 gene is the canine, equine, or cat TGFBR1 gene.

[0435] In some embodiments, the mammalian TGFBR1 gene is the human TGFBR1 gene.

[0436] In some embodiments, the at least one guide RNA contains selectable... Figure 2A-2C The spacer subsequences of the group consisting of those sequences (SEQ ID NO:199-320) shown.

[0437] In some embodiments, the at least one guide RNA contains selectable... Figure 12B The spacer subsequences of the group consisting of those sequences (SEQ ID NO: 528-552) shown.

[0438] In some embodiments, the at least one guide RNA contains selectable... Figure 5B The interval subsequences that form the groups of those sequences shown.

[0439] In some embodiments, the at least one guide RNA comprises a spacer subsequence of OHTIR04 (SEQ ID NO:532) or OHTIR08 (SEQ ID NO:539).

[0440] In some embodiments, the at least one guide RNA or the nucleic acid encoding at least one guide RNA targets the mammalian TGFBR2 gene.

[0441] In some embodiments, the mammalian TGFBR2 gene is the canine, equine, or cat TGFBR2 gene.

[0442] In some embodiments, the mammalian TGFBR2 gene is the human TGFBR2 gene.

[0443] In some embodiments, the at least one guide RNA contains selectable... Figures 3A-3D The spacer subsequences of the group consisting of those sequences (SEQ ID NO:321-519) shown.

[0444] In some embodiments, the at least one guide RNA contains selectable... Figure 12C The spacer subsequences of the group consisting of those sequences (SEQ ID NO: 553-604) shown.

[0445] In some embodiments, the at least one guide RNA contains selectable... Figure 6B The interval subsequences that form the groups of those sequences shown.

[0446] In some embodiments, the at least one guide RNA comprises a spacer subsequence of OHTIIR04 (SEQ ID NO:563).

[0447] In some embodiments, the RNA-guided nuclease or the nucleic acid encoding the RNA-guided nuclease is the RNA-guided nuclease.

[0448] In some embodiments, the RNA-guided nuclease or the nucleic acid encoding the RNA-guided nuclease is DNA encoding the RNA-guided nuclease.

[0449] In some embodiments, the RNA-guided nuclease or the nucleic acid encoding the RNA-guided nuclease is mRNA encoding the RNA-guided nuclease.

[0450] In some embodiments, the RNA-guided nuclease is a Cas protein.

[0451] In some embodiments, the Cas protein is the Cas9 protein.

[0452] In some embodiments, the Cas9 protein is the Streptococcus pyogenes Cas9 polypeptide.

[0453] In some embodiments, the Cas9 protein is selected from the group consisting of: ESCas9, HFCas9, PECas9, and ARCas9.

[0454] In some embodiments, the at least one guide RNA or the nucleic acid encoding the at least one guide RNA is the at least one guide RNA.

[0455] In some embodiments, the at least one guide RNA or the nucleic acid encoding the at least one guide RNA is DNA encoding the at least one guide RNA.

[0456] In some embodiments, the pharmaceutical composition comprises nucleic acids encoding both the RNA-guided nuclease and the at least one guide RNA.

[0457] In some embodiments, the at least one guide RNA is a single guide RNA (sgRNA).

[0458] In some embodiments, the composition comprises one or more viral vectors, the one or more viral vectors collectively comprising (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease, and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA that targets a gene encoding a transmembrane receptor.

[0459] In some embodiments, the one or more viral vectors comprise a recombinant virus selected from the following: retrovirus, adenovirus, adeno-associated virus, lentivirus, and herpes simplex virus-1.

[0460] In some embodiments, the one or more viral vectors comprise recombinant adeno-associated virus (AAV).

[0461] In some embodiments, recombinant AAV belongs to serotype 5 (AAV5).

[0462] In some embodiments, recombinant AAV belongs to serotype 6 (AAV6).

[0463] In some embodiments, the composition comprises one or more lipid nanoparticles (LNPs), the one or more LNPs collectively comprising (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease, and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA that targets a gene encoding a transmembrane receptor.

[0464] In some embodiments, the one or more LNPs comprise: a first plurality of LNPs encapsulating the RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease; and a second plurality of LNPs encapsulating the at least one guide RNA or a nucleic acid encoding at least one guide RNA.

[0465] In some embodiments, the one or more LNPs comprise a plurality of LNPs, the plurality of LNPs encapsulating (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease, and (ii) both a guide RNA targeting a gene encoding a transmembrane receptor or a nucleic acid encoding at least one guide RNA.

[0466] In some embodiments, the one or more LNPs comprise a component selected from the group consisting of: 3-(bisdodecylamino)-N1,N1,4-tris(dodecyl)-1-piperazinethylamine (KL10), N1-[2-(bisdodecylamino)ethyl]-N1,N4,N4-tris(dodecyl)-1,4-piperazindiethylamine (KL22), 14,25-bis(tridecyl)-15,18,21,24-tetraaza-octacosane (KL25), 1 2-Dilinolenic acid-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinolenic acid-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptadecyl-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA), 2,2-dilinolenic acid-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA) MA), 1,2-diolenyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3.β.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3.β.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethylaminopropane 3-[(9Z-,12Z)-octadec-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA(2R)), (2S)-2-({8-[(3.β.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z-,12Z)-octadec-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA(2S)), lipids including cyclic amine groups, and mixtures thereof.

[0467] In some embodiments, the LNP comprises a component selected from the group consisting of: 1,2-dilinoleoyl-sn-glycerol-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), 1,2-dispalmitoyl-sn-glycerol-3-phosphocholine (DPPC), 1,2-distearateoyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-di(undecanoyl)-sn-glycerol-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphocholine (18:0Diether) 1,2-Oleoyl-2-cholesterol hemisuccino-sn-glycerol-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphocholine (C16 Lyso PC), 1,2-Dilinoleoyl-sn-glycerol-3-phosphocholine, 1,2-disarachidanoyl-sn-glycerol-3-phosphocholine, 1,2-bis(docohexanoyl)-sn-glycerol-3-phosphocholine, 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-diphydanyl-sn-glycerol-3-phosphoethanolamine (ME) 16.0PE), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docosahexaenoyl)-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate sodium salt (DOPG), sphingomyelin (SM) and mixtures thereof.

[0468] In some embodiments, the LNP comprises a component selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. For example, the PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DMA, PEG-DSPE lipids, and mixtures thereof.

[0469] In some embodiments, the LNP comprises a component selected from the group consisting of: cholesterol, coccosterol, stigmasterol, stigmasterol, sitosterol, β-sitosterol, lupeol, betulinol, ursolic acid, oleanolic acid, campesterol, fucosterol, brassosterol, ergosterol, 9,11-dehydroergosterol, tomatine, lycopene, α-tocopherol, and mixtures thereof.

[0470] In some embodiments, the composition comprises one or more liposomes, the one or more liposomes collectively comprising (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease, and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA that targets a gene encoding a transmembrane receptor.

[0471] In some embodiments, the one or more liposomes comprise: a first plurality of liposomes encapsulating the RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease; and a second plurality of liposomes encapsulating the at least one guide RNA or a nucleic acid encoding at least one guide RNA.

[0472] In some embodiments, the one or more liposomes comprise a plurality of liposomes that encapsulate (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease, and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA that targets a gene encoding a transmembrane receptor.

[0473] In some embodiments, the composition comprises one or more virus-like particles, the one or more virus-like particles collectively comprising (i) an RNA-guided nuclease or nucleic acid encoding an RNA-guided nuclease, and (ii) at least one guide RNA or nucleic acid encoding at least one guide RNA that targets a gene encoding a transmembrane receptor.

[0474] In some embodiments, the one or more virus-like particles comprise:

[0475] In some embodiments, a first plurality of virus-like particles encapsulate an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease; and a second plurality of virus-like particles encapsulate the at least one guide RNA or a nucleic acid encoding at least one guide RNA.

[0476] In some embodiments, the one or more virus-like particles comprise a plurality of virus-like particles that encapsulate (i) the RNA-guided nuclease or nucleic acid encoding the RNA-guided nuclease, and (ii) both the at least one guide RNA or nucleic acid encoding the at least one guide RNA that targets and encapsulates the gene encoding the transmembrane receptor.

[0477] In some embodiments, the composition is formulated for parenteral administration.

[0478] In some embodiments, the composition is formulated for intra-articular injection into the joint of a subject.

[0479] In some embodiments, the composition is formulated for intradiscal injection.

[0480] In some embodiments, the composition is formulated for peridiscal injection.

[0481] In some embodiments, the composition is formulated for intraspinal injection.

[0482] In some embodiments, this disclosure provides a method for treating or preventing oxygen free radicals in a subject of need by administering a therapeutically effective amount of a composition, wherein the composition comprises: (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease; and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA, wherein the at least one guide RNA or the nucleic acid encoding at least one guide RNA targets the TGFB1 gene, the TGFBR1 gene, the TGFBR2 gene, or a combination thereof.

[0483] In some embodiments, this disclosure provides a method for treating or preventing a condition in a subject of need that has symptoms at least in part caused by intercellular signaling mediated by the transforming growth factor β (TGFβ) signaling pathway, the method comprising: administering a therapeutically effective amount of a composition, wherein the composition comprises: (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease; and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA, said at least one guide RNA or the nucleic acid encoding at least one guide RNA targeting the TGFB1 gene, the TGFBR1 gene, the TGFBR2 gene, or a combination thereof.

[0484] In some embodiments, the condition is fibrosis.

[0485] In some embodiments, fibrosis is postoperative fibrosis.

[0486] In some embodiments, fibrosis is fibrosis following ligament reconstruction.

[0487] In some embodiments, fibrosis is fibrosis following anterior cruciate ligament (ACL) reconstruction, fibrosis following autologous ACL reconstruction, or fibrosis following allogeneic ACL reconstruction.

[0488] In some embodiments, fibrosis is knee joint fibrosis.

[0489] In some embodiments, fibrosis is caused by intra-articular fibrous nodules.

[0490] In some embodiments, fibrosis is fibrosis following joint replacement surgery.

[0491] In some embodiments, fibrosis is fibrosis following total knee arthroplasty (TKA).

[0492] In some embodiments, fibrosis is associated with tendon injury or surgery.

[0493] In some embodiments, fibrosis is fibrosis following fracture repair.

[0494] In some embodiments, fibrosis is fibrosis following microdiscectomy.

[0495] In some embodiments, fibrosis is epidural fibrosis following microdiscectomy.

[0496] In some embodiments, fibrosis is fibrosis following lumbar laminectomy.

[0497] In some embodiments, fibrosis is epidural fibrosis following lumbar laminectomy.

[0498] In some embodiments, fibrosis is fibrosis following anterior acromioplasty.

[0499] In some embodiments, fibrosis is fibrosis following subacromial decompression surgery.

[0500] In some embodiments, fibrosis is soft tissue fibrosis following anterior acromioplasty.

[0501] In some embodiments, fibrosis is joint contracture following anterior acromioplasty.

[0502] In some embodiments, fibrosis is arthroscopic fibrosis.

[0503] VIII. Examples

[0504] The embodiments covered herein are now described with reference to the following examples. These embodiments are provided for illustrative purposes only, and the disclosure covered herein should in no way be construed as limiting to these embodiments, but rather as covering any and all variations that become apparent as a result of the teachings provided herein.

[0505] Example 1: Bioinformatics design of a guide for blocking TGF-β signaling

[0506] We have developed a computer-based and in vitro screening pipeline to identify candidate CRISPR-Cas guides for gene editing. This pipeline is used to identify guides that effectively edit the human TGFB1 gene (hTGFB1), human TGFBR1 gene (hTGFRB1), and human TGFBR2 gene (hTGFRB2), thereby blocking TGF-β signaling in vivo to treat musculoskeletal fibrosis and / or scar formation. The first step of the pipeline is to identify all possible spacer sequences of crRNA or sgRNA targeting specific CRISPR-Cas proteins in the coding regions of hTGFB1, hTGFRB1, and hTGFRB2. Many algorithms for identifying such sequences are known in the art. Generally, these algorithms function by identifying the protospacer neighbor motif (PAM) sequence of a specific CRISPR-Cas protein and then locating the spacer sequence (typically at the 5' direct position of the PAM site) according to the requirements of the specific Cas protein. For example, the *S. pyogenes* Cas9 (SpCas9) protein used in this embodiment recognizes the 5'-NGG-3' PAM sequence. Therefore, all sequences at the direct 5' of the NGG trinucleotide are possible spacer sequences for crRNA or sgRNA. Spacer sequences are identified in this manner, along with biographical information about them, as shown in Figure 27.

[0507] Next, each identified spacer subsequence was evaluated across three different metrics (see Figures 1-3): potential off-target editing at locations in the genome other than the target gene, efficiency of mid-target editing, and the likelihood of editing leading to frameshift mutations. Multiple algorithms were used for each metric, such as... Figure 4B , 5B As shown in 6B, the combined approach used is based on the assumption that each model has a blind spot that may distort the fitness of a particular guide RNA. Weighting these scores to obtain a consistent score for each of these properties provides a better predictor of sgRNA fitness.

[0508] Off-target editing effects are predicted by averaging scores generated by the MIT, CFD, and Elevation (human only) models. The MIT algorithm, also known as the Hsu-Zhang score, is described in Hsu, PD, et al., “DNA targeting specificity of RNA-guided Cas9 nucleases.” *Nature Biotechnology* 31, 827–832 (2013). The model is based on a position penalty matrix (1x20) generated from 15 EMX1 sgRNA libraries that mismatch with the target at each position. The CFD algorithm (cutting frequency determination) is based on a threat matrix (12x20) that considers both position and mismatch type, as well as PAM integrity (27,897 'CD33' sgRNAs + 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). Elevation scores were obtained using a machine learning algorithm trained on genome-wide (GUIDE-Seq) and other aggregated off-target profile analysis 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 marked “OFF” show the average scores from two or three models, respectively. Columns marked “OffTarget#” show the number of potential off-targets calculated by CRISPOR, up to four mismatches.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).

[0509] On-target editing efficiency was predicted by averaging scores generated by the Azimuth model, the DeepSpCas9 model, and the CrisprScan model. The Azimuth model is an enhanced regression tree model trained using 881 sgRNAs delivered via lentiviral delivery (MOLM13 / NB4 / TF1 cells and other unpublished data). Doench, JG et al., “Optimized sgRNA design to maximize CRISPR-Cas9 activity and minimize off-target effects.” 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 marked “ON” show the average scores provided by the three models.

[0510] The potential for frameshift mutations in a putative guide was predicted by averaging scores generated by the Lindel and InDelphi models. Lindel is a machine learning model trained on profiling data of 1.16 million independent mutations triggered by CRISPR / Cas9-mediated cleavage and non-homologous end joining 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 Acid Research*, 47, 7989–8003 (2019). InDelphi is a machine learning model trained on insertions and deletions generated from 1872 sgRNAs.) Shen, MW, et al., “Predictable and precise template-free CRISPR editing of pathogenic variants”. *Nature*, 563, 646–651 (2018). The column labeled “Frameshift” shows the average score provided by the two models. The column labeled “Precision” shows the frequency distribution of insertions and deletions estimated by inDelphi. High precision close to 100 represents sgRNAs characterized by one or very few repair outcomes.

[0511] Then, candidate spacer sequences were evaluated based on the presence of Graf motifs and whether TT or GCC was present in the four PAM proximal bases of the spacer sequence, as shown in Figure 26-42 for example, with TT or GCC. Graf, R. et al., “sgRNA sequence motifs effectively block CRISPR / Cas9-mediated gene editing”, Cell Reports, 26(5), 1098-103 (2019). Graf et al. reported that TT and GCC motifs are markers of inefficient sgRNAs. Spacer sequences with Graf motifs, especially GCC motifs, should be avoided if possible. In contrast to TT motifs, GCC motifs remain crucial if the sgRNA is synthesized de novo rather than through transcription.

[0512] As shown in Figures 1-3, for example, three consensus scores were then calculated for each spacer subsequence. 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 total score represents the average of the “ON”, “OFF”, and “Frameshift” scores. Additionally, for humans only, the VBC Bioscore was used to predict whether possible in-frame mutations would interfere with protein function. This is more likely to occur in conserved genomic DNA sequences encoding key protein domains. Therefore, the Bioscore is based on protein domain annotation, phylogenetic conservation, amino acid identity, and exon size. (Michlits, G. et al., “Multilayered VBC score predicts sgRNAs that efficiently generate loss-of-function alleles”, *Nature Methods*, 17, 708-716 (2020).) The final criteria for selecting candidates are primarily based on the total score (>70 in most cases), a relatively low number of potential off-targets (<200 in most cases), the absence of Graf motifs (if possible), and genomic cut-off locations within the coding sequence (to produce knockout, or, in the case of hTGFBR1 and hTGFBR2, to produce truncated proteins with reduced function (e.g., decoy receptors)).

[0513] Finally, the CRISPR-targeted domains were determined based on the nucleotide positions in the gene. Figure 4A , 5A 6A). Use domain location to predict whether editing will produce wild-type-like proteins (edits in the sequence encoding the extreme C-terminus of the receptor that are unlikely to block receptor-mediated signaling), complete knockout of any functional protein (edits in the sequence encoding the N-terminus and / or essential functional domains), and (if applicable) soluble decoy receptors (edits in the sequence encoding the transmembrane domain of the receptor) or membrane-bound decoy receptors (edits in the sequence encoding the intracellular signaling domain of the receptor).

[0514] In fact, this computational analysis shows that several candidate sgRNAs are predicted to generate genome editing, resulting in dominant-negative (DN) TGFB receptors that influence intracellular pro-inflammatory signaling. These predictive studies provide a high-throughput method for validating candidates that does not require time-consuming and resource-intensive brute-force approaches, and demonstrate the feasibility of predicting the induction of genetic knockout (or dominant-negative TGFB receptor) as a unique approach to treating musculoskeletal fibrosis and / or scarring.

[0515] Example 2: Validation of TGFB1 editing in human cells

[0516] After designing and analyzing a large number of sgRNAs predicted to edit TGFB1 to produce genetic knockout using the bioinformatics methods described in Example 1 (see Figure 1), the next step is to assess their ability to function in vitro. Of particular interest is whether a preferred edit will be observed.

[0517] To test this, 80 pmol of the sgRNA candidate was introduced into THP-1 human monocytes via electroporation as part of the ribonucleoprotein (i.e., pre-assembled with 25 pmol of Cas9 protein at room temperature for at least 5 minutes). Briefly, approximately 400,000 cells were resuspended in 20 μl of SG buffer (Lonza), added to the pre-assembled Cas9 ribonucleoprotein complex, transferred to 16-well nuclear transfection dishes, and electroporated using a 4D nuclear transfection instrument (Lonza) with pulse code FF-100. Electroporated cells were held in SG buffer for approximately 10 minutes, then transferred to 12-well tissue culture plates containing complete culture medium. After 2–5 days of culture, cells were dissociated using the DNeasy Blood & Tissue Kit (Qiagen) to extract genomic DNA. The sgRNA target region was amplified by PCR, and editing efficacy was deduced from Sanger sequencing traces using the Inference of CRISPR Edits (ICE v1.2). See Conant, D. et al. (2022). The CRISPR Journal, 5(1), 123-130.

[0518] Although several sgRNA candidates were able to efficiently induce mutations in the hTGFB1 gene, with several candidates showing frequencies exceeding 50%, only one candidate—OHTG03 (SEQ ID NO: 682)—showed the ability to induce the same editing in a large number of cells when paired with wild-type (WT) Cas9 or high-fidelity AR-Cas9 (IDT, Cat#IDT, catalog #1081060; Vakulskas, CA et al. (2018). Nature Medicine, 24(8):1216-1224). Figure 7A (B). Compared to WT-Cas9, the use of AR-Cas9 is expected to improve edit specificity (i.e., reduce off-target editing). These results generally confirm the computer analysis described in Example 1.

[0519] Example 3: Validation of TGFBR1 editing in human cells

[0520] After designing and analyzing a large number of sgRNAs predicted to edit the TGFBR1 receptor to generate knockout or (soluble or membrane-bound) dominant-negative decoys using the bioinformatics methods described in Example 1 (see Figure 2), the next step is to evaluate their ability to function in vitro. Of particular interest is whether a preferred mid-target editing will be observed in any of the candidates.

[0521] To test this, sgRNA candidates were complexed with Cas9 nuclease to form a ribonucleoprotein (RNP) complex, and then human THP-1 monocytes were electroporated to introduce the RNP into the cells, as previously described above. Editing efficacy was then measured by Sanger sequencing. While several candidates were able to efficiently induce hTGFBR1 gene mutations, several of them showed a WT-Cas9 frequency exceeding 50% ( Figure 8A However, the use of AR-Cas9 generally reduces this effectiveness. Figure 8B These results generally confirm the computer analysis and demonstrate that several sgRNA candidates, including OHTIR04, can effectively edit TGFBR1 in human cells at a certain frequency.

[0522] Example 4: Validation of TGFBR2 editing in human cells

[0523] After designing and analyzing a large number of sgRNAs predicted to edit the TGFBR2 receptor to generate knockout or (soluble or membrane-bound) dominant-negative decoys using the bioinformatics methods described in Example 1 (see Figure 3), the next step is to evaluate their ability to function in vitro. Of particular interest is whether a preferred edit will be observed in any of the candidates.

[0524] To test this, sgRNA candidates were complexed with Cas9 nuclease to form a ribonucleoprotein (RNP) complex, and then human THP-1 monocytes were electroporated to introduce the RNP into the cells, as described above. Editing efficiency was then measured by Sanger sequencing. While several candidates were able to efficiently induce hTGFBR2 gene mutations, several of them showed WT-Cas9 frequencies approaching or exceeding 50%. Figure 9A However, the use of AR-Cas9 generally reduces this effectiveness. Figure 9B Notably, pairing AR-Cas9 with OHTIIR11 (SEQ ID NO: 1065) increases editing primacy (i.e., the proportion of edits included in the desired intermediate-target edit), although the editing efficacy is lower than that of WT-Cas9. These results generally confirm the computational analysis and demonstrate that several sgRNA candidates, including OHTIIR11, can effectively edit TGFBR2 in human cells at a certain frequency.

[0525] Example 5: Editing of TGFBR1 or TGFBR2 resulted in human monocytes being unresponsive to TGF-β stimulation.

[0526] After designing and validating a large number of guides targeting the TGF-β pathway, the next step involved functional testing of the edited cells. As part of this test, approximately 400,000 edited human THP-1 monocytes were treated with 1 μg / ml LPS or 20 ng / μl recombinant TGF-β for 6 hours. Total cellular RNA was reverse transcribed and then subjected to quantitative PCR (RT-qPCR) to verify whether human LPS or TGF-β could stimulate the transcription of TGFB1, as previously reported. See Hall, MC, et al. (2003). Journal of Biological Chemistry, 278(12), 10304-10313; Xiang Yin et al., 2017, J Atheroscler Thromb, 2017; 24:55-67. doi:10.5551 / jat.35204. LPS treatment moderately increased TGFB1 transcription in control cells (injected with WT-Cas9 only). Figure 10A However, compared with the unedited control, TGFBR1-edited cells showed a five-fold reduction in TGFB1 transcription levels, while TGFBR2-edited cells showed a two-fold reduction.

[0527] As an additional test, TIMP1 induction was measured on the same cellular RNA, which is typically upregulated via TGF-β signaling via SMAD protein (see Hall, MC et al., (2003). Journal of Biochemistry, 278(12), 10304-10313.). Strong TIMP1 induction was observed in the unedited control compared to untreated cells in LPS-treated cells. Figure 10B Compared to the unedited control, TGFBR1-edited monocytes showed a 50% reduction in TIMP1 induction. TGFBR2-edited cells showed no change.

[0528] The results after treatment with natural ligands were even more striking. TGFB1 induction as a response to TGF-β treatment was significantly stronger in cells injected with only unedited WTCas9, while editing TGFBR1 or TGFBR2 reduced this response by a factor of three. Figure 10C These results are similar to those observed after TGF-β treatment for TIMP1 induction. Figure 10D These results collectively indicate that the edited cells were unresponsive to TGF-β treatment because their TGFB1 levels remained significantly lower than those of the control. In summary, these results suggest that both TGF-β positive feedback (TGFB1-induced) and downstream signaling (TIMP1-induced) are reduced in TGFBR1- and TGFBR2-edited cells.

[0529] Example 6: Validation of TGFB1 editing in human cells

[0530] In designing and analyzing a large number of sgRNA candidates predicted to edit the human TGFB1 gene and generate genetic knockout (see...) Figure 11A Following this, the next step is to evaluate their ability to function in vitro. Of particular interest is whether preferred (i.e., mid-target) editing will be observed in the edited cells.

[0531] To test this, the sgRNA candidate (SEQ ID NO:1-198) was introduced into human THP-1 mononuclear cells via electroporation as part of a ribonucleoprotein, as described in Example 2 above.

[0532] The results of these studies are expected to further confirm the robustness of the computer analysis and identify preferred candidates that can indeed induce mutations in the TGFB1 gene with high efficacy. Compared to WT-Cas9, the use of high-fidelity AR-Cas9 is also expected to improve editing specificity (i.e., reduce off-target editing).

[0533] Example 7: In vitro delivery of guide RNA for editing TGFB1 into human cells via lipid nanoparticles

[0534] Additional experiments will evaluate the suitability of using lipid nanoparticles (LNPs) for mRNA delivery to human cells. LNPs containing sgRNA (SEQ ID NO: 1-198) and WT- or AR-Cas9 mRNA will be exposed to THP-1 monocytes for 8 to 24 hours under typical cell culture conditions. The editing will be validated using Sanger sequencing as described in Example 2. Following confirmation of the editing, various functional assays as described in Example 5 will be performed.

[0535] Example 8: In vitro delivery of guide RNA for editing TGFB1 into human cells via adeno-associated virus

[0536] Since all validation experiments will be performed via electroporation of cell cultures to introduce RNA-dependent nucleases and sgRNA, additional experiments will evaluate the suitability of using an adeno-associated virus (AAV) vector to deliver sgRNA into human cells.

[0537] Table 2. Exemplary AAV vectors for introducing sgRNA into human cells.

[0538]

[0539] Exemplary AAV vectors are shown in Table 2. After preparation according to the manufacturer's instructions to integrate one or more of the sgRNAs containing SEQ ID NO: 1-198 into the viral vector, the vector was incubated with approximately 500,000 U2OS cells stably transfected with Cas9 (MOI = 0.1 to 10) (see, for example, Rojas-Fernandez, A. et al., (2015). Scientific Reports, 5(1), 1-6.). The edits were validated using Sanger sequencing as described in Example 2 2–7 days post-infection. After confirmation of the edits, various functional assays as described in Example 5 were performed.

[0540] Example 9: Validation of TGFBR1 editing in human cells

[0541] In designing and analyzing a large number of sgRNA candidates predicted to edit the human transforming growth factor β receptor 1 protein to produce knockout or dominant-negative receptors (see [link to study]). Figure 11B Following this, the next step is to evaluate their ability to function in vitro. Of particular interest is whether preferred (i.e., mid-target) editing will be observed in the edited cells.

[0542] To test this, the sgRNA candidate (SEQ ID NO:199-320) was introduced into human THP-1 mononuclear cells via electroporation as part of a ribonucleoprotein, as described in Example 2 above.

[0543] The results of these studies are expected to further confirm the robustness of the computer analysis and identify several preferred candidates that can indeed induce mutations in the TGFBR1 gene with high efficacy. Compared to WT-Cas9, the use of high-fidelity AR-Cas9 is also expected to improve editing specificity (i.e., reduce off-target editing).

[0544] Example 10: In vitro delivery of guide RNA that edits TGFBR1 to human cells via lipid nanoparticles

[0545] Since all validation experiments will be performed via electroporation of cell cultures to introduce RNA-dependent nucleases and sgRNA, additional experiments will evaluate the suitability of using lipid nanoparticles (LNPs) for mRNA delivery to human cells. LNPs containing sgRNA (SEQ ID NO: 199-320) or Cas9 mRNA will be incubated with THP-1 monocytes and then validated as described in Example 7. Following confirmation of the edits, various functional assays will be performed as described in Example 5.

[0546] Example 11: In vitro delivery of guide RNA that edits TGFBR1 into human cells via adeno-associated virus

[0547] Since all validation experiments will be performed via electroporation of cell cultures to introduce RNA-dependent nucleases and sgRNAs, additional experiments will evaluate the suitability of using an adeno-associated virus (AAV) vector to deliver sgRNAs into human cells. Exemplary AAV vectors are shown in Table 2. After preparation according to the manufacturer's instructions to integrate one or more of the sgRNAs containing SEQ ID NO:199-320 into the viral vector, the vector is introduced into human U2OS cells and the editing is validated, as described in Example 8 above. After confirming the editing, various functional assays as described in Example 5 are performed.

[0548] Example 12: Validation of TGFBR2 editing in human cells

[0549] In designing and analyzing a large number of sgRNA candidates predicted to edit the human transforming growth factor β receptor 2 protein to produce knockout or dominant-negative receptors (see [link to study]). Figure 11C Following this, the next step is to evaluate their ability to function in vitro. Of particular interest is whether preferred (i.e., mid-target) editing will be observed in the edited cells.

[0550] To test this, the sgRNA candidate (SEQ ID NO:321-519) was introduced into human THP-1 mononuclear cells via electroporation as part of a ribonucleoprotein, as described in Example 2 above.

[0551] The results of these studies are expected to further confirm the robustness of the computer analysis and identify several preferred candidates that can indeed induce mutations in the TGFBR2 gene with high efficacy. Compared to WT-Cas9, the use of high-fidelity AR-Cas9 is also expected to improve editing specificity (i.e., reduce off-target editing).

[0552] Example 13: In vitro delivery of guide RNA that edits TGFBR2 into human cells via lipid nanoparticles

[0553] Since all validation experiments will be performed via electroporation of cell cultures to introduce RNA-dependent nucleases and sgRNA, additional experiments will evaluate the suitability of using lipid nanoparticles (LNPs) for mRNA delivery to human cells. LNPs containing sgRNA (SEQ ID NO:321-519) or Cas9 mRNA will be incubated with THP-1 monocytes and then validated as described in Example 7. Following confirmation of the edits, various functional assays will be performed as described in Example 5.

[0554] Example 14: In vitro delivery of guide RNA that edits TGFBR2 into human cells via adeno-associated virus

[0555] Since all validation experiments will be performed via electroporation of cell cultures to introduce RNA-dependent nucleases and sgRNAs, additional experiments will evaluate the suitability of using an adeno-associated virus (AAV) vector to deliver sgRNAs into human cells. Exemplary AAV vectors are shown in Table 2. After preparation according to the manufacturer's instructions to integrate one or more of the sgRNAs containing SEQ ID NO:321-519 into the viral vector, the vector is introduced into human U2OS cells and the editing is validated, as described in Example 8 above. After confirming the editing, various functional assays as described in Example 5 will be performed.

[0556] Example 15: Surgical treatment of joint fibrosis

[0557] Further experiments will be conducted to demonstrate the efficacy of in vivo treatment of joint fibrosis in the post-surgical environment. In short, a composition comprising a pre-assembled RNP complex targeting the rabbit TGFB1, TGFBR1, and TGFBR2 genes and the Cas9 enzyme will be prepared according to Example 2. These RNPs will be injected intra-articularly into rabbits exhibiting joint fibrosis.

[0558] In summary, rabbits in two cohorts underwent contracture-plasty and knee fixation, followed by remobilization surgery at eight weeks. During remobilization, one cohort underwent capsular release surgery, while the other did not. Each cohort was then divided into sub-cohorts. For each cohort, one sub-cohort received an injection of the prepared RNPs described above, while the other sub-cohort did not. Clinical signs of joint fibrosis were then evaluated at several time points following the injection (e.g., 10, 16, and 24 weeks), and fibrosis of the amputated limb was evaluated at the end of the study. An example of a study using this rabbit model of joint fibrosis is described in Trousdale et al., BoneJoint Res., 2022, 11(1):32-39, doi:10.1302 / 2046-3758.111.BJR-2021-0546.R1.

[0559] Example 16: Editing of TGFB1, TGFBR1, or TGFBR2 resulted in unresponsive human monocytes to the driven expression of multiple TGF-β reactive gene products.

[0560] Additional functional studies were conducted on edited and control THP-1 monocytes, with TGF-β added for 6 hours. Total cellular RNA was then collected for RT-qPCR analysis, as described in Example 5 above. Compared to unedited control cells, SERPINE1 showed strong induction after TGF-β treatment (…). Figure 11A Induction was significantly reduced in all edited cells, especially in monocytes edited with TGFBR1 and TGFBR2. Similarly, Col1A2 was also induced by TGF-β treatment, and all edited monocytes showed an approximately two-fold reduction in induction. Figure 11B It is worth noting that FN1 ( Figure 11C ) and CTGF( Figure 11D The induction intensity of TGF-β was relatively low. Therefore, the relative induction was not very pronounced in the edited cells. However, it should be noted that the FN1 and CTGF levels exhibited by TGFBR1 and TGFBR2-edited monocytes were comparable to those in the unedited control. This result indicates that even after 6 hours of TGF-β treatment, these cells were unable to induce RNA levels exceeding the background level. Overall, these results suggest that, following exposure to TGF-β, several additional TGF-β-responsive gene products were barely expressed or completely absent in the background in TGFBR1 and TGFBR2-edited cells, while TGFB1-edited cells also showed a significant reduction in the induction of these TGF-β-responsive gene products.

[0561] The examples set forth above are provided to offer a complete disclosure and description of embodiments of how to manufacture and use the compositions, systems, and methods of this disclosure to those skilled in the art, and are not intended to limit the scope of what the inventors consider to be their invention. Modifications to the above-described patterns for carrying out embodiments of this disclosure, which will be apparent to those skilled in the art, are intended to fall within the scope of the appended claims. All patents and publications mentioned in the specification demonstrate the level of skill of those skilled in the art to which this disclosure pertains.

[0562] All headings and section titles are for clarity and reference purposes only and should not be construed as limiting in any way. For example, those skilled in the art will appreciate the usefulness of appropriately combining various aspects from different headings and sections in accordance with the spirit and scope of the disclosure described herein.

[0563] It should be understood that the methods described herein are not limited to the specific methods, protocols, subjects, and sequencing technologies described herein, and therefore can be varied. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the methods and compositions described herein, which is defined only by the appended claims. While some embodiments of this disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, alterations, and substitutions will occur to those skilled in the art without departing from this disclosure. It should be understood that various alternatives to the embodiments of this disclosure described herein may be employed in the practice of this disclosure. The appended claims are intended to define the scope of this disclosure and thereby cover the methods and structures within the scope of these claims and their equivalents.

[0564] The examples set forth above are provided to offer a complete disclosure and description of embodiments of how to manufacture and use the compositions, systems, and methods of this disclosure to those skilled in the art, and are not intended to limit the scope of what the inventors consider to be their invention. Modifications to the above-described patterns for carrying out embodiments of this disclosure, which will be apparent to those skilled in the art, are intended to fall within the scope of the appended claims. All patents and publications mentioned in the specification demonstrate the level of skill of those skilled in the art to which this disclosure pertains.

[0565] All headings and section titles are for clarity and reference purposes only and should not be construed as limiting in any way. For example, those skilled in the art will appreciate the usefulness of appropriately combining various aspects from different headings and sections in accordance with the spirit and scope of the disclosure described herein.

[0566] It should be understood that the methods described herein are not limited to the specific methods, protocols, subjects, and sequencing technologies described herein, and therefore can be varied. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the methods and compositions described herein, which is defined only by the appended claims. While some embodiments of this disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, alterations, and substitutions will occur to those skilled in the art without departing from this disclosure. It should be understood that various alternatives to the embodiments of this disclosure described herein may be employed in the practice of this disclosure. The appended claims are intended to define the scope of this disclosure and thereby cover the methods and structures within the scope of these claims and their equivalents.

[0567] For illustrative purposes, several aspects are described with reference to exemplary applications. Unless otherwise stated, any embodiment can be combined with any other embodiment. It should be understood that numerous specific details, relationships, and methods are set forth to provide a comprehensive understanding of the features described herein. However, those skilled in the art will readily recognize that the features described herein can be implemented without one or more of the specific details or by other methods. The features described herein are not limited to the illustrated order of actions or events, as some actions may occur in a different order and / or simultaneously with other actions or events. Furthermore, not all actions or events shown are necessary for implementing the methods according to the features described herein.

[0568] While some embodiments have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided merely as examples. This is not to imply that this disclosure is limited to the specific examples provided in the specification. Although this disclosure has been described with reference to the foregoing specification, the description and illustrations of the embodiments herein are not intended to be construed as limiting. Many variations, modifications, and substitutions will occur to those skilled in the art without departing from this disclosure.

[0569] Furthermore, it should be understood that all aspects of this disclosure are not limited to the specific descriptions, configurations, or relative proportions set forth 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 employed in the practice of this disclosure. Therefore, this disclosure is also intended to cover any such substitutions, modifications, variations, or equivalents. The appended claims are intended to define the scope of this disclosure and thereby cover the methods and structures within the scope of these claims and their equivalents.

[0570] All publications, patents, and patent applications cited herein are incorporated by reference to the same extent that each individual publication, patent, or patent application expressly and individually indicates its inclusion by reference. In the event of any conflict between terminology used herein and terminology found in the incorporated references, the terminology used herein shall prevail.

Claims

1. A pharmaceutical composition for treating or preventing a condition having symptoms at least partially caused by intercellular signaling mediated by the transforming growth factor β (TGFβ) signaling pathway, said composition comprising: (i) RNA-guided nucleases or nucleic acids encoding RNA-guided nucleases; and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA, wherein the at least one guide RNA or the nucleic acid encoding at least one guide RNA targets the TGFB1 gene, the TGFBR1 gene, the TGFBR2 gene, or a combination thereof.

2. The pharmaceutical composition according to claim 1, wherein the condition 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 post-ligament reconstruction fibrosis.

5. The pharmaceutical composition according to claim 4, wherein the fibrosis is fibrosis following anterior cruciate ligament (ACL) reconstruction, fibrosis following autologous ACL reconstruction, or fibrosis following 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 fibrosis following total knee arthroplasty (TKA).

9. The pharmaceutical composition of claim 2, wherein the fibrosis is associated with tendon injury or surgery.

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

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

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

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

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

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

16. The pharmaceutical composition of claim 2, wherein the fibrosis is subacromial decompression fibrosis.

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

18. The pharmaceutical composition of claim 2, wherein the fibrosis is anterior acromioplasty-induced joint contracture.

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

20. The pharmaceutical composition according to claim 2, wherein the fibrosis is arthroscopic joint fibrosis.

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

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

23. The pharmaceutical composition of 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 following open-heart surgery.

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 of claim 2, wherein the fibrosis is fibrosis in the knee, shoulder, or elbow joint.

28. The pharmaceutical composition of claim 1, wherein the condition includes fibrosis in the kidney tissue.

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

30. The pharmaceutical composition of claim 1, wherein the condition includes fibrosis in the skin tissue.

31. The pharmaceutical composition of claim 30, wherein the condition is post-wound scarring, keloid scarring, renal systemic fibrosis, or scleroderma / systemic sclerosis.

32. The pharmaceutical composition of claim 1, wherein the condition includes fibrosis in lung tissue.

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

34. The pharmaceutical composition of claim 1, wherein the condition includes fibrosis in liver tissue.

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

36. The pharmaceutical composition of claim 1, wherein the condition includes fibrosis in the heart tissue.

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

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

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

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

41. The pharmaceutical composition of claim 1, wherein the condition includes fibrosis in brain tissue.

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

43. The pharmaceutical composition of claim 1, wherein the condition includes fibrosis in intestinal tissue.

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

45. The pharmaceutical composition of claim 1, wherein the condition includes fibrosis in the hand or fingers.

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

47. The pharmaceutical composition of claim 1, wherein the condition comprises fibrosis in lymphoid tissue.

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

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

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

51. The pharmaceutical composition of claim 1, wherein the condition includes fibrosis in penile tissue.

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

53. The pharmaceutical composition of claim 1, wherein the condition comprises fibrosis in the soft tissues of the retroperitoneal space.

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

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

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

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

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

59. The pharmaceutical composition of claim 58, wherein the musculoskeletal disorder is a hereditary musculoskeletal disease.

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

61. The pharmaceutical composition of 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 of claim 58, wherein the musculoskeletal disorder is type II collagen disease.

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

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

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

66. The pharmaceutical composition of claim 58, wherein the musculoskeletal disorder is an autoimmune musculoskeletal disease with polygenic susceptibility.

67. The pharmaceutical composition of claim 58, wherein the musculoskeletal disorder is spondyloarthritis (SA).

68. The pharmaceutical composition of claim 58, wherein the musculoskeletal condition is ankylosing spondylitis.

69. The pharmaceutical composition of claim 58, wherein the musculoskeletal condition is rheumatoid arthritis.

70. The pharmaceutical composition of claim 58, wherein the musculoskeletal condition is autoimmune osteoarthritis.

71. The pharmaceutical composition of claim 58, wherein the musculoskeletal condition is osteoarthritis (OA).

72. The pharmaceutical composition of claim 58, wherein the musculoskeletal disorder is Camurati-Engelmann disease.

73. The pharmaceutical composition of claim 58, wherein the musculoskeletal disorder is Sjogren's Syndrome.

74. The pharmaceutical composition of claim 58, wherein the musculoskeletal condition is a mechanical musculoskeletal injury.

75. The pharmaceutical composition of claim 58, wherein the musculoskeletal condition is selected from chronic muscle injury, tendon rupture, post-traumatic osteoarthritis, and post-traumatic joint fibrosis.

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

77. The pharmaceutical composition of claim 76, wherein the mammalian TGFB1 gene is a canine, equine, or cat TGFB1 gene.

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

79. The pharmaceutical composition of claim 78, wherein the at least one guide RNA comprises a spacer subsequence selected from the group consisting of those sequences (SEQ ID NO: 1-198) shown in Figures 1A-1D.

80. The pharmaceutical composition of claim 78, wherein the at least one guide RNA comprises a spacer subsequence selected from the group consisting of those sequences shown in Figure 12A (SEQ ID NO: 520-527).

81. The pharmaceutical composition of claim 78, wherein the at least one guide RNA comprises a spacer sequence of 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 the nucleic acid encoding at least one guide RNA targets the mammalian TGFBR1 gene.

83. The pharmaceutical composition of claim 82, wherein the mammalian TGFBR1 gene is a canine, equine, 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 of claim 84, wherein the at least one guide RNA comprises a spacer subsequence selected from the group consisting of those sequences (SEQ ID NO: 199-320) shown in Figures 2A-2C.

86. The pharmaceutical composition of claim 84, wherein the at least one guide RNA comprises a spacer subsequence selected from the group consisting of those sequences shown in Figure 12B (SEQ ID NO: 528-552).

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

88. The pharmaceutical composition of claim 84, wherein the at least one guide RNA comprises a spacer sequence of 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 the nucleic acid encoding at least one guide RNA targets the mammalian TGFBR2 gene.

90. The pharmaceutical composition of claim 89, wherein the mammalian TGFBR2 gene is a canine, equine, 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 of claim 91, wherein the at least one guide RNA comprises a spacer subsequence selected from the group consisting of those sequences (SEQ ID NO: 321-519) shown in Figures 3A-3D.

93. The pharmaceutical composition of claim 91, wherein the at least one guide RNA comprises a spacer subsequence selected from the group consisting of those sequences shown in Figure 12C (SEQ ID NO: 553-604).

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

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

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

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

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

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

100. The pharmaceutical composition of claim 99, wherein the Cas protein is the Cas9 protein.

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

102. The pharmaceutical composition of 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 the 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 the nucleic acid encoding the at least one guide RNA is DNA encoding the at least one guide RNA.

105. The pharmaceutical composition according to any one of claims 1 to 102, comprising a nucleic acid encoding both the RNA-guided 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 one or more viral vectors collectively comprising (i) the RNA-guided nuclease or nucleic acid encoding the RNA-guided nuclease, and (ii) the at least one guide RNA or nucleic acid encoding at least one guide RNA that targets a gene encoding a transmembrane receptor.

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

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

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

111. The pharmaceutical composition of claim 109, wherein the recombinant AAV belongs to 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), the one or more LNPs collectively comprising (i) the RNA-guided nuclease or nucleic acid encoding the RNA-guided nuclease, and (ii) the at least one guide RNA or nucleic acid encoding at least one guide RNA that targets the gene encoding the transmembrane receptor.

113. The pharmaceutical composition of claim 112, wherein the one or more LNPs comprise: The first plurality of LNPs, wherein the first plurality of LNPs encapsulate the RNA-guided nuclease or the nucleic acid encoding the RNA-guided nuclease; and The second or more LNPs encapsulate the at least one guide RNA or a nucleic acid encoding at least one guide RNA.

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

115. The pharmaceutical composition according to any one of claims 112 to 114, wherein the one or more LNPs comprise a component selected from the group consisting of: 3-(bis(dodecylamino)-N1,N1,4-tris(dodecyl)-1-piperazine ethylamine (KL10), N1-[2-(bis(dodecylamino)ethyl]-N1,N4,N4-tris(dodecyl)-1,4-piperazine diethylamine (KL22), 14,25-bis(tridecyl)-15,18,21,24-tetraaza-octacosandecane (KL... 25), 1,2-diolenoyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-diolenoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptadecyl-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA), 2,2-diolenoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-diolenoyloxy- N,N-Dimethylaminopropane (DODMA), 2-({8-[(3.β.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3.β.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z-,12Z)-octadec- 9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA(2R)(Octyl-CLinDMA(2R))), (2S)-2-({8-[(3.β.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z-,12Z)-octadec-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA(2S)(Octyl-CLinDMA(2S))), lipids including cyclic amine groups, and mixtures thereof.

116. The pharmaceutical composition according to any one of claims 112 to 115, wherein the LNP comprises a component selected from the group consisting of: 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-distearateoyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-di(undecanoyl)-sn-glycerol-3-phosphate choline (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline (18:0 Diether) 1,2-Oleoyl-2-cholesterol hemisuccino-sn-glycerol-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphocholine (C16LysoPC), 1,2-Dilinolenoyl-sn-glycerol-3-phosphocholine, 1,2-disarachidanoyl-sn-glycerol-3-phosphocholine, 1,2-bis(docohexanoyl)-sn-glycerol-3-phosphocholine, 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-diphydanyl-sn-glycerol-3-phosphoethanolamine (ME) 16.0PE), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docosahexaenoyl)-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate 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 pharmaceutical 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, stigmasterol, sitosterol, β-sitosterol, lupeol, betulinol, ursolic acid, oleanolic acid, campesterol, fucosterol, brassosterol, ergosterol, 9,11-dehydroergosterol, tomatine, lycopene, α-tocopherol, and mixtures thereof.

119. The pharmaceutical composition according to any one of claims 112 to 114, wherein the one or more LNPs comprise LP01 ionizable lipids, neutral / auxiliary lipid components, steroid system components, and pegized lipid components.

120. The pharmaceutical composition according to any one of claims 112 to 114, wherein the one or more LNPs comprise SM-102 ionizable lipids, neutral / auxiliary lipid components, steroid system components, and peg-modified lipid components.

121. The pharmaceutical composition according to any one of claims 112 to 114, wherein the one or more LNPs comprise an ALC0315 ionizable lipid, a neutral / auxiliary lipid component, a steroid system component, and a pegized lipid component.

122. The pharmaceutical composition according to any one of claims 119 to 121, wherein the neutral / auxiliary 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 pegified lipid component comprises DMG-PEG.

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

128. The pharmaceutical composition according to any one of claims 119 to 127, wherein the molar ratio of the LPO1, neutral / auxiliary lipid component, steroid system component and pegged lipid component comprising the one or more LNPs is (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 molar ratio of SM-102, neutral / auxiliary lipid component, steroid system component and peg-modified lipid component comprising the one or more LNPs is (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 molar ratio of ALC0315, neutral / auxiliary lipid component, steroid system component and pegged lipid component comprising the one or more LNPs is (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, the one or more liposomes collectively comprising (i) the RNA-guided nuclease or nucleic acid encoding the RNA-guided nuclease, and (ii) the at least one guide RNA or nucleic acid encoding at least one guide RNA that targets the gene encoding the transmembrane receptor.

132. The pharmaceutical composition of claim 131, wherein the one or more liposomes comprise: A first plurality of liposomes, wherein the first plurality of liposomes encapsulate the RNA-guided nuclease or nucleic acid encoding the RNA-guided nuclease; and A second plurality of liposomes, wherein the second plurality of liposomes encapsulate the at least one guide RNA or a nucleic acid encoding at least one guide RNA.

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

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

135. The pharmaceutical composition of claim 134, wherein the one or more virus-like particles comprise: A first plurality of virus-like particles, wherein the first plurality of virus-like particles encapsulate the RNA-guided nuclease or nucleic acid encoding the RNA-guided nuclease; and The second plurality of virus-like particles encapsulate the at least one guide RNA or a nucleic acid encoding at least one guide RNA.

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

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 a subject's joint.

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 condition in a subject of need, having symptoms at least partially caused by intercellular signaling mediated through the transforming growth factor β (TGFβ) signaling pathway, the method comprising: Administer a therapeutically effective amount of the composition, wherein the composition comprises: (i) RNA-guided nucleases or nucleic acids encoding RNA-guided nucleases; and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA, wherein the at least one guide RNA or the nucleic acid encoding at least one guide RNA targets the TGFB1 gene, the TGFBR1 gene, the TGFBR2 gene, or a combination thereof.

143. The method of claim 142, wherein the condition is fibrosis.

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

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

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

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

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

149. The method of claim 143, wherein the fibrosis is fibrosis following total knee arthroplasty (TKA).

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

151. The method of claim 143, wherein the fibrosis is fibrosis of the knee joint after TKA.

152. The method of claim 143, wherein the fibrosis is fibrosis following microsurgical discectomy.

153. The method of claim 143, wherein the fibrosis is epidural fibrosis following microsurgical discectomy.

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

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

156. The method of claim 143, wherein the fibrosis is fibrosis following anterior acromioplasty.

157. The method of claim 143, wherein the fibrosis is subacromial decompression fibrosis.

158. The method of claim 143, wherein the fibrosis is soft tissue fibrosis following anterior acromioplasty.

159. The method of claim 143, wherein the fibrosis is anterior acromioplasty followed by joint contracture.

160. The method of claim 143, wherein the fibrosis is arthroscopic fibrosis.

161. The method of claim 143, wherein the fibrosis is arthroscopic joint fibrosis.

162. The method of claim 143, wherein the fiberization is trabecular mesh fiberization.

163. The method of claim 162, wherein the trabecular meshwork fibrosis is postoperative fibrosis following glaucoma surgery.

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

165. The method of claim 143, wherein the fibrosis is atrial fibrosis, cardiac fibrosis, myocardial fibrosis, and / or fibrosis following open-heart surgery.

166. The method of claim 143, wherein the fibrosis is post-liposuction fibrosis.

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

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

169. The method of claim 142, wherein the condition includes fibrosis in the kidney tissue.

170. The method of claim 142, wherein the condition is chronic kidney disease.

171. The method of claim 142, wherein the condition includes fibrosis in the skin tissue.

172. The method of claim 142, wherein the condition is post-wound scarring, keloid scarring, renal systemic fibrosis, or scleroderma / systemic sclerosis.

173. The method of claim 142, wherein the condition includes fibrosis in the lung tissue.

174. The method of claim 142, wherein the condition is fibrothorax, pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, radiation-induced lung injury, progressive massive fibrosis, or scleroderma / systemic sclerosis.

175. The method of claim 142, wherein the condition includes fibrosis in liver tissue.

176. The method of claim 142, wherein the condition is cirrhosis or bridging fibrosis.

177. The method of claim 142, wherein the condition includes fibrosis in the heart tissue.

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

179. The method of claim 142, wherein the condition is congestive heart failure or hypertension.

180. The method of claim 143, wherein the fiberization is alternative fiberization.

181. The method of claim 142, wherein the condition is myocardial infarction.

182. The method of claim 142, wherein the condition includes fibrosis in brain tissue.

183. The method of claim 143, wherein the fibrosis is a gelatinous scar.

184. The method of claim 142, wherein the condition includes fibrosis in intestinal tissue.

185. The method of claim 142, wherein the condition is Crohn's disease.

186. The method of claim 142, wherein the condition includes fibrosis in the hand or fingers.

187. The method of claim 142, wherein the condition is palmar aponeurosis contracture.

188. The method of claim 142, wherein the condition includes fibrosis in lymphoid tissue.

189. The method of claim 143, wherein the fiberization is mediastinal fiberization.

190. The method of claim 142, wherein the condition includes fibrosis in bone marrow tissue.

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

192. The method of claim 142, wherein the condition includes fibrosis in the penile tissue.

193. The method of claim 142, wherein the condition is Peronie's disease.

194. The method of claim 142, wherein the condition includes fibrosis in the soft tissues of the retroperitoneal space.

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

196. The method of claim 142, wherein the condition is cancer.

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

198. The method of claim 142, wherein the condition includes fibrosis in musculoskeletal tissue.

199. The method of claim 142, wherein the condition is a musculoskeletal disorder.

200. The method of claim 199, wherein the musculoskeletal disorder is a hereditary musculoskeletal disease.

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

202. The method of 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 of claim 199, wherein the musculoskeletal disorder is type II collagen disease.

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

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

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

207. The method of claim 199, wherein the musculoskeletal disorder is an autoimmune musculoskeletal disease with polygenic susceptibility.

208. The method of claim 199, wherein the musculoskeletal disorder is spondyloarthritis (SA).

209. The method of claim 199, wherein the musculoskeletal condition is ankylosing spondylitis.

210. The method of claim 199, wherein the musculoskeletal condition is rheumatoid arthritis.

211. The method of claim 199, wherein the musculoskeletal condition is autoimmune osteoarthritis.

212. The method of claim 199, wherein the musculoskeletal condition is osteoarthritis (OA).

213. The method of claim 199, wherein the musculoskeletal disorder is progressive diaphyseal dysplasia.

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

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

216. The method of claim 199, wherein the musculoskeletal condition is selected from chronic muscle injury, tendon rupture, post-traumatic osteoarthritis, and post-traumatic joint fibrosis.

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

218. The method of claim 217, wherein the mammalian TGFB1 gene is a canine, equine, or cat TGFB1 gene.

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

220. The method of claim 219, wherein the at least one guide RNA comprises a spacer subsequence selected from the group consisting of those sequences (SEQ ID NO: 1-198) shown in Figures 1A-1D.

221. The method of claim 219, wherein the at least one guide RNA comprises a spacer subsequence selected from the group consisting of those sequences shown in Figure 12A (SEQ ID NO: 520-527).

222. The method of 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 the nucleic acid encoding at least one guide RNA targets the mammalian TGFBR1 gene.

224. The method of claim 223, wherein the mammalian TGFBR1 gene is a canine, equine, or cat TGFBR1 gene.

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

226. The method of claim 225, wherein the at least one guide RNA comprises a spacer subsequence selected from the group consisting of those sequences (SEQ ID NO: 199-320) shown in Figures 2A-2C.

227. The method of claim 225, wherein the at least one guide RNA comprises a spacer subsequence selected from the group consisting of those sequences shown in Figure 12B (SEQ ID NO: 528-552).

228. The method of claim 225, wherein the at least one guide RNA comprises a spacer subsequence selected from the group consisting of those sequences shown in Figure 5B.

229. The method of 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 the nucleic acid encoding at least one guide RNA targets the mammalian TGFBR2 gene.

231. The method of claim 230, wherein the mammalian TGFBR1 gene is a canine, equine, or cat TGFBR1 gene.

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

233. The method of claim 232, wherein the at least one guide RNA comprises a spacer subsequence selected from the group consisting of those sequences (SEQ ID NO: 321-519) shown in Figures 3A-3D.

234. The method of claim 232, wherein the at least one guide RNA comprises a spacer subsequence selected from the group consisting of those sequences shown in Figure 12C (SEQ ID NO: 553-604).

235. The method of claim 232, wherein the at least one guide RNA comprises a spacer subsequence selected from the group consisting of those sequences shown in Figure 6B.

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

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

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

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

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

241. The method of claim 240, wherein the Cas protein is the Cas9 protein.

242. The method of claim 241, wherein the Cas9 protein is a Streptococcus pyogenes Cas9 polypeptide.

243. The method of 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 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-guided 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, the one or more viral vectors collectively comprising (i) the RNA-guided nuclease or nucleic acid encoding the RNA-guided nuclease, and (ii) the at least one guide RNA or nucleic acid encoding at least one guide RNA that targets a gene encoding a transmembrane receptor.

249. The method of claim 248, wherein the one or more viral vectors comprise a recombinant virus selected from the group consisting of: retrovirus, adenovirus, adeno-associated virus, lentivirus, and herpes simplex virus-1.

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

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

252. The method of claim 250, wherein the recombinant AAV belongs to 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), the one or more LNPs collectively comprising (i) the RNA-guided nuclease or nucleic acid encoding the RNA-guided nuclease, and (ii) the at least one guide RNA or nucleic acid encoding at least one guide RNA that targets the gene encoding the transmembrane receptor.

254. The method of claim 253, wherein the one or more LNPs comprise: The first plurality of LNPs, wherein the first plurality of LNPs encapsulate the RNA-guided nuclease or the nucleic acid encoding the RNA-guided nuclease; and The second or more LNPs encapsulate the at least one guide RNA or a nucleic acid encoding at least one guide RNA.

255. The method of claim 253, wherein the one or more LNPs comprise a plurality of LNPs, the plurality of LNPs encapsulating (i) the RNA-guided nuclease or nucleic acid encoding the RNA-guided nuclease, and (ii) both the at least one guide RNA or nucleic acid encoding at least one guide RNA that targets the gene encoding the transmembrane receptor.

256. The method according to any one of claims 253 to 255, wherein the one or more LNPs comprise a component selected from the group consisting of: 3-(bisdodecylamino)-N1,N1,4-tris(dodecyl)-1-piperazine ethylamine (KL10), N1-[2-(bisdodecylamino)ethyl]-N1,N4,N4-tris(dodecyl)-1,4-piperazine diethylamine (KL22), 14,25-bis(tridecyl)-15,18,21,24- Tetraaza-octacosane (KL25), 1,2-dilinolenicooxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinolenico-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptadecyl-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA), 2,2-dilinolenico-4-(2-dimethylaminoethyl)-[1,3]-dioxolane ( DLin-KC2-DMA), 1,2-diolenyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3.β.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3.β.)-cholest-5-en-3-yloxy]octyl}oxy)-N N,N-dimethyl-3-[(9Z-,12Z)-octadec-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA(2R)), (2S)-2-({8-[(3.β.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z-,12Z)-octadec-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA(2S)), lipids including cyclic amine groups, and mixtures thereof.

257. The method according to any one of claims 253 to 256, wherein the LNP comprises a component selected from the group consisting of: 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-distearateoyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-di(undecanoyl)-sn-glycerol-3-phosphate choline (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline (18:0 Diether) 1,2-Oleoyl-2-cholesterol hemisuccino-sn-glycerol-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphocholine (C16 Lyso PC), 1,2-Dilinoleoyl-sn-glycerol-3-phosphocholine, 1,2-disarachidanoyl-sn-glycerol-3-phosphocholine, 1,2-bis(docohexanoyl)-sn-glycerol-3-phosphocholine, 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-diphydanyl-sn-glycerol-3-phosphoethanolamine (ME) 16.0PE), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docosahexaenoyl)-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate 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, coccosterol, stigmasterol, stigmasterol, sitosterol, β-sitosterol, lupeol, betulinol, ursolic acid, oleanolic acid, campesterol, fucosterol, brassosterol, ergosterol, 9,11-dehydroergosterol, tomatine, lycopene, α-tocopherol, and mixtures thereof.

260. The method according to any one of claims 253 to 255, wherein the one or more LNPs comprise LP01 ionizable lipids, neutral / auxiliary lipid components, steroid system components, and pegized lipid components.

261. The method according to any one of claims 253 to 255, wherein the one or more LNPs comprise SM-102 ionizable lipids, neutral / auxiliary lipid components, steroid system components, and peg-modified lipid components.

262. The method according to any one of claims 253 to 255, wherein the one or more LNPs comprise ALC0315 ionizable lipids, neutral / auxiliary lipid components, steroid system components, and pegized lipid components.

263. The method according to any one of claims 260 to 262, wherein the neutral / auxiliary 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 glucocorticoid steroids.

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

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

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

269. The method according to any one of claims 260 to 268, wherein the molar ratio of the LPO1, neutral / auxiliary lipid component, steroid system component and pegized lipid component comprising the one or more LNPs is (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 molar ratio of SM-102, neutral / auxiliary lipid component, steroid system component and peg-modified lipid component comprising the one or more LNPs is (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 molar ratio of ALC0315, neutral / auxiliary lipid component, steroid system component and peg-modified lipid component comprising the one or more LNPs is (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, the one or more liposomes collectively comprising (i) the RNA-guided nuclease or nucleic acid encoding the RNA-guided nuclease, and (ii) the at least one guide RNA or nucleic acid encoding at least one guide RNA that targets the gene encoding the transmembrane receptor.

273. The method of claim 272, wherein the one or more liposomes comprise: A first plurality of liposomes, wherein the first plurality of liposomes encapsulate the RNA-guided nuclease or nucleic acid encoding the RNA-guided nuclease; and A second plurality of liposomes, wherein the second plurality of liposomes encapsulate the at least one guide RNA or a nucleic acid encoding at least one guide RNA.

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

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

276. The method of claim 275, wherein the one or more virus-like particles comprise: A first plurality of virus-like particles, wherein the first plurality of virus-like particles encapsulate the RNA-guided nuclease or nucleic acid encoding the RNA-guided nuclease; and The second plurality of virus-like particles encapsulate the at least one guide RNA or a nucleic acid encoding at least one guide RNA.

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

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

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

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

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

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

283. The method of any one of claims 142 to 277, wherein the application comprises application to the cells or tissues of interest.

284. The method according to any one of claims 142 to 277, wherein the application is performed during surgical procedures.

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

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

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

288. The method of claim 284, wherein the composition is applied to a stent implanted during surgery.

289. The method of claim 284, wherein the composition is dip-coated onto the implant.

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