Methods for treating, ameliorating, and / or preventing fibrodysplasia ossificans progressiva and heterotopic ossification and kits therefor

Abstract

Disclosed herein is a method for treating, ameliorating, and / or preventing fibrodysplasia ossificans progressiva (FOP) in a subject in need thereof.The method comprises downregulating the level and / or activity of matrix metalloproteinase 9 (MMP-9) in the subject.Also disclosed is a kit for carrying out the method disclosed herein.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 324,842, filed March 29, 2022. U.S. Provisional Patent Application No. 63 / 324,842 is incorporated herein by reference in its entirety.

[0002] Reference to sequence listing The sequence listing submitted concurrently with the present specification as an xml file entitled "046483-7360WO1.xml", created on March 28, 2023, and having a size of 4,200 bytes, is incorporated herein by reference. [Background technology]

[0003] background Fibrodysplasia ossificans progressiva (FOP; MIM#135100) is the most devastating form of extraskeletal ossification in humans. In this disease, muscle and connective tissues, such as tendons and ligaments, are gradually replaced by bone (ossification), resulting in extraskeletal bone formation (extraskeletal or heterotopic bone) that limits movement.

[0004] At birth, individuals with FOP appear normal except for a characteristic deformity of the big toe, which is present in all classically affected individuals. During the first decade of life, individuals experience sudden soft tissue swelling (or flare-ups) in the neck and back that undergo pathological degeneration into mature heterotopic bone through an endochondral pathway. Minor trauma such as intramuscular vaccination, mandibular blocks for dental work, muscle strain, blunt muscle trauma, impacts, contusions, falls, or viral illnesses such as influenza can trigger FOP flare-ups, which can lead to progressive heterotopic ossification (HO). Most patients become immobile by age 30 and require lifelong assistance with activities of daily living. The estimated median life expectancy is 56 years. Death is often due to complications of thoracic insufficiency syndrome.

[0005] No cure for FOP is yet available, and the current standard of care, medical management, is only supportive. There is a need for compositions and methods that can be used to treat, ameliorate, and / or prevent FOP. The present invention addresses this need. Summary of the Invention

[0006] overview In some aspects, the invention relates to the following non-limiting embodiments.

[0007] In some aspects, the present invention provides methods of treating, ameliorating, and / or preventing fibrodysplasia ossificans progressiva (FOP) in a subject in need thereof.

[0008] In some embodiments, the methods include downregulating matrix metalloproteinase 9 (MMP-9) levels and / or activity in the subject.

[0009] In some embodiments, downregulating MMP-9 level and / or activity in a subject comprises administering an effective amount of: Small molecule MMP-9 inhibitors, Protein MMP-9 inhibitors, A nucleic acid (and / or an expression vector expressing the nucleic acid) that downregulates MMP-9 by RNA interference; a ribozyme (and / or a vector expressing the ribozyme) that downregulates MMP-9; An expression vector comprising an expression cassette expressing a CRISPR component that downregulates MMP-9 by CRISPR knockout and / or CRISPR knockdown; and Transdominant-negative mutant protein of MMP-9 and / or expression vector expressing the transdominant-negative mutant protein of MMP-9 to a subject.

[0010] In some embodiments, the subject has a mutant ACVR1 gene.

[0011] In some aspects, the mutant ACVR1 gene encodes a constitutively active ACVR1 polypeptide.

[0012] In some embodiments, the ACVR1 polypeptide comprises at least one mutation selected from the group consisting of L196P, P197-F198 del ins L, R202I, R206H, Q207E, F246Y, R258S, R258G, G325A, G328E, G328W, G328R, G356D, R375P, and K400E.

[0013] In some embodiments, the small molecule MMP-9 inhibitor is selected from the group consisting of doxycycline, incyclinide, and minocycline, or a salt or solvate thereof.

[0014] In some embodiments, the protein MMP-9 inhibitor is an anti-MMP-9 antibody or an antigen-binding fragment thereof.

[0015] In some aspects, the method further comprises the step of surgically removing ossified tissue from the subject.

[0016] In some embodiments, the surgical removal step is performed after the level or activity of MMP-9 in the subject is downregulated.

[0017] In some aspects, the subject is a human.

[0018] In some aspects, the present invention relates to a kit for treating, ameliorating, and / or preventing fibrodysplasia ossificans progressiva (FOP) in a subject in need thereof.

[0019] In some embodiments, the kit comprises a compound for downregulating matrix metalloproteinase 9 (MMP-9) levels and / or activity in a subject, and instructions for administering an effective amount of the compound to the subject.

[0020] In some aspects, the compound is Small molecule MMP-9 inhibitors, Protein MMP-9 inhibitors, A nucleic acid (and / or an expression vector expressing the nucleic acid) that downregulates MMP-9 by RNA interference; a ribozyme (and / or a vector expressing the ribozyme) that downregulates MMP-9; An expression vector comprising an expression cassette expressing a CRISPR component that downregulates MMP-9 by CRISPR knockout and / or CRISPR knockdown; and Transdominant-negative mutant protein of MMP-9 and / or expression vector expressing the transdominant-negative mutant protein of MMP-9 The composition includes at least one selected from the group consisting of:

[0021] In some embodiments, the subject has a mutant ACVR1 gene.

[0022] In some aspects, the mutant ACVR1 gene encodes a constitutively active ACVR1 polypeptide.

[0023] In some embodiments, the ACVR1 polypeptide comprises at least one mutation selected from the group consisting of L196P, P197-F198 del ins L, R202I, R206H, Q207E, F246Y, R258S, R258G, G325A, G328E, G328W, G328R, G356D, R375P, and K400E.

[0024] In some embodiments, the small molecule MMP-9 inhibitor is selected from the group consisting of doxycycline, incyclinide, and minocycline.

[0025] In some embodiments, the protein MMP-9 inhibitor is an antibody or antigen-binding fragment thereof to MMP-9.

[0026] In some aspects, the instructions further comprise instructions for surgically removing the ossified tissue from the subject.

[0027] In some embodiments, the instructions further comprise instructions to perform the surgical removal after MMP-9 level or activity in the subject is downregulated.

[0028] In some aspects, the subject is a human. [Brief description of the drawings]

[0029] The following detailed description of the exemplary embodiments will be better understood when read in conjunction with the accompanying drawings. For purposes of illustration, non-limiting embodiments are shown in the drawings. It should be understood, however, that this description is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0030] [Figure 1] FIG. 1 illustrates the progression of FOP in a typical patient, according to some embodiments. [Figure 2A] 2A-2D show characterization of FOP in Patient-R, according to some embodiments. FIG. 2A is a photograph of the toe deformity, FIG. 2B is an X-ray of the toes, and FIG. 2C is a scout CT image (collage) showing the presence of congenital features of FOP and less HO in Patient-R. FIG. 2D is an electropherogram showing the classical ACVR1 mutation (R206H, 617G>A) in Patient-R. [Figure 2B] Please see the legend to FIG. 2A. [Figure 2C] Please see the legend to FIG. 2A. [Figure 2D] Please see the legend to FIG. 2A. [Diagram 3]3A-3C show the results of an MMP-9 assay in Patient-R, according to some embodiments. FIG. 3A shows that Patient-R had lower plasma MMP-9 (Myriad RBM assay) levels than all other FOP patients and controls (**p<0.01, n=77). FIG. 3B shows the results of a gelatin zymography assay revealing less total MMP-9 in PBMCs from Patient-R. FIG. 3C illustrates the MMP-9 enzyme activity of PBMCs from Patient-R versus control subjects and other FOP patients. The results show less MMP-9 reserve in PBMCs from Patient-R compared to PBMCs from other FOP patients. [Figure 4] 4A-4B show the assessment of MMP-9 polymorphisms in patient-R, according to some embodiments. Figure 4A: Schematic of MMP-9 protein showing various anatomical locations of A20V and D165N SNPs resulting in amino acid residue changes. Figure 4B: Sequencing of PCR amplicons confirms the presence of A20V and D165N SNPs in patient-R. [Diagram 5] Figure 5 demonstrates that MMP-9 is expressed in early lesion tissue according to some embodiments. Figure 5 shows images of early lesion tissue of FOP model mouse (Acvr1R206H / +;CreERT2- / +) 1 day, 3 days and 5 days after cardiotoxin injury. This shows that there is MMP-9 expression in inflammatory cells. [Figure 6A]Figures 6A-6N demonstrate that downregulation of MMP-9 by genetic, pharmacological, or biological means in the FOP mouse model all resulted in a reduction in HO. Figure 6A: Both MMP9- / +;Acvr1R206H / +;CreERT2- / + and MMP9- / -;Acvr1R206H / +;CreERT2- / + mice form significantly less HO than Acvr1R206H / +;CreERT2- / + mice after soft tissue injury. Figure 6B illustrates quantification of HO bone volume in the experiment of Figure 6A when analyzed by microCT (***p<0.001, n=6). Figure 6C: MMP9- / +;Acvr1R206H / +;CreERT2- / + and MMP9- / -;Acvr1R206H / +;CreERT2- / + mice produce significantly less MMP-9 than Acvr1R206H / +;CreERT2- / + control mice. Figure 6D shows histology (H&E staining) of MMP9+ / +;Acvr1+ / +, MMP9+ / +;Acvr1R206H / +;CreERT2- / +, MMP9- / -;Acvr1+ / +, and MMP9- / -;Acvr1R206H / +;CreERT2- / + mice at 14 days after cardiotoxin injury. Figure 6E: Minocycline-treated Acvr1R206H / +;CreERT2- / + mice form significantly less HO than Acvr1R206H / +;CreERT2- / + control mice after soft tissue injury. Figure 6F illustrates the quantification of HO bone volume in the experiment of Figure 6E when analyzed by micro-CT (**p<0.01, n=6). Figure 6G: Minocycline-treated Acvr1Q207D / + mice form significantly less HO than untreated Acvr1Q207D / + mice after soft tissue injury (***p<0.001, n=6). Figure 6H illustrates the quantification of HO bone volume in the experiment of Figure 6G when analyzed by micro-CT. Figure 6I shows that minocycline prevents HO when administered before or at the time of lesion activation. This indicates that MMP-9 acts to induce HO during the early inflammatory phase. Figure 6J shows the effect of various concentrations of minocycline on HO in Acvr1Q207D / + mice. Minocycline at doses of 5-100 mg / kg was effective in reducing HO.Figure 6K illustrates the results of blocking mAb MMP-9 (Gilead) on HO bone volume in Acvr1R206H / +;CreERT2- / +FOP mice after soft tissue injury. Figure 6L shows the results of gelatin zymography assay. Cell culture supernatants were derived from M1-like and M2-like macrophages 48 hours after polarizing THP-1, THP-1A20V, and THP-1D165N M0 macrophages. Figure 6M shows quantification of MMP-9 enzyme activity in gelatin zymography. MMP-9D165N enzyme activity was reduced compared to MMP-9WT and MMP-9A20V in M1-like and M2-like macrophages. Figure 6N shows ELISA for activin A in cell culture supernatants derived from M1- and M2-like macrophages 48 hours after polarization of THP-1, THP-1A20V, and THP-1D165N M0 macrophages. Compared to MMP-9WT, MMP-9A20V and MMP-9D165N dramatically reduced the levels of activin A in the supernatants. [Figure 6B] Please see the legend to FIG. 6A. [Figure 6C] Please see the legend to FIG. 6A. [Figure 6D] Please see the legend to FIG. 6A. [Figure 6E] Please see the legend to FIG. 6A. [Figure 6F] Please see the legend to FIG. 6A. [Figure 6G] Please see the legend to FIG. 6A. [Figure 6H] Please see the legend to FIG. 6A. [Figure 6I] Please see the legend to FIG. 6A. [Figure 6J] Please see the legend to FIG. 6A. [Figure 6K] Please see the legend to FIG. 6A. [Figure 6L] Please see the legend to FIG. 6A. [Figure 6M] Please see the legend to FIG. 6A. [Figure 6N] Please see the legend to FIG. 6A. [Figure 7]FIG. 7 illustrates a proposed molecular mechanism of MMP-9 involvement in FOP, according to some embodiments. [Figure 8] 8 shows protein-protein interaction (PPI) mapping results of MMP-9 connectivity according to some embodiments. PPI mapping was performed using IBM-Watson for Drug Discovery (WDD) and Ingenuity software. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] Detailed Description The following disclosure provides many different aspects or examples for implementing different features of the provided subject matter. To simplify the disclosure, specific examples of components and configurations are described below. Of course, these are merely examples and are not intended to be limiting. For example, in the following description, the formation of a first feature on or at the surface of a second feature may include an embodiment in which the first feature and the second feature are formed in direct contact, and may also include an embodiment in which an additional feature may be formed between the first feature and the second feature such that the first feature and the second feature may not be in direct contact. Furthermore, the disclosure may repeat reference numbers and / or letters in various embodiments. This repetition is for simplicity and clarity, and does not, in itself, dictate a relationship between the various aspects and / or configurations being discussed.

[0032] Heterozygous missense mutations in the activin receptor type A I, a bone morphogenetic protein (BMP) type I receptor, have been identified in all individuals with sporadic or familial fibrodysplasia ossificans progressiva (FOP). ACVR1 mutations abolish ACVR1 autoinhibition, rendering it susceptible to dysregulated BMP pathway signaling. Activin A, a member of the transforming growth factor-β (TGF-β) family of molecules that antagonizes BMP signaling in wild-type (WT) ACVR1 background, specifically enhances BMP pathway signaling in cells harboring the ACVR1R206H mutation, driving ectopic bone formation in FOP.

[0033] The study described herein ("the study") found a special fibrodysplasia ossificans progressiva (FOP) patient with a classic R206H mutation in the Acvr1 BMP receptor. The patient had congenital features of FOP, but he developed only minor postnatal FOP features. The study found that the patient had significantly suppressed inflammatory biomarkers and had compound heterozygosity of the MMP-9 gene (one allele of the MMP-9 gene with a polymorphism results in an A20V mutation in the expressed polypeptide, and the other allele with a polymorphism results in a D165N mutation). Using protein structure modeling, the study predicted that the compound heterozygosity of MMP-9 found in this patient reduced the levels and activity of MMP-9 in the patient. The study found that MMP-9 was expressed in early diseased tissues of FOP subjects. Using several mouse FOP models, this study discovered that reducing the levels and / or activity of MMP-9 by genetic deletion (including partial deletion) of MMP-9, administration of non-limiting examples of monoclonal antibodies against MMP-9, or administration of non-limiting small molecule inhibitors of MMP-9 all resulted in reduced levels of heterotopic ossification (bone formation) in response to injury in mouse FOP models.

[0034] Accordingly, in some aspects, the present invention relates to methods of treating, ameliorating, and / or preventing fibrodysplasia ossificans progressiva (FOP) in a subject in need thereof.

[0035] In some aspects, the present invention relates to kits for treating, ameliorating, and / or preventing FOP in a subject in need thereof.

[0036] definition Each of the following terms used herein has the meaning associated with it in this section. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In general, the nomenclature used herein and the experimental procedures in animal pharmacology, pharmacology, peptide chemistry, and organic chemistry are well known and commonly used in the art. It should be understood that the order of steps or order for performing certain actions is not important so long as this disclosure remains operable. Any use of section headings is intended to aid in the reading of the document and is not intended to be construed as limiting. Information associated with a section heading may be within or outside that particular section. All publications, patents, and patent documents mentioned in this document are incorporated herein by reference in their entirety, as if each was individually incorporated by reference.

[0037] Whenever in this application an element or component is referred to as being included in and / or selected from a list of described elements or components, it should be understood that the element or component may be any one of the described elements or components, or may be selected from a group consisting of two or more of the described elements or components.

[0038] In the methods described herein, acts may be performed in any order unless a time or order of operations is expressly recited. Moreover, specified acts may be performed simultaneously unless express claim language recites the specified acts as being performed separately. For example, a claimed act of doing X and a claimed act of doing Y may be performed simultaneously in a single operation, and the resulting process is within the literal scope of the claimed process.

[0039] In this document, the terms "a," "an," or "the" are used to include one or more, unless the context clearly dictates otherwise. The term "or" is used to refer to a non-exclusive "or" unless otherwise specified. The phrases "at least one of A and B" or "at least one of A or B" have the same meaning as "A, B, or A and B."

[0040] As used herein, "about," when referring to a measurable value, e.g., amount, duration, etc., is meant to encompass variations of ±20% or ±10%, and in certain embodiments ±5%, in certain embodiments ±1%, and in certain embodiments ±0.1% from the specified value, as appropriate for practicing the disclosed methods.

[0041] A "disease" is a condition in the animal's health where the animal is unable to maintain homeostasis and where the animal's health will continue to deteriorate unless the disease is ameliorated.

[0042] A "disorder" in an animal is a health condition in which the animal is able to maintain homeostasis, but in which the animal's health condition is less favorable than it would be in the absence of the disorder. If left untreated, the disorder does not necessarily result in a further deterioration of the animal's health condition.

[0043] A disease or disorder is "alleviated" if the severity of a symptom of the disease or disorder, the frequency with which such symptoms are experienced by a patient, or both, are reduced.

[0044] In one aspect, the terms "co-administered" and "co-administration" when referring to a subject refer to administering to a subject a compound and / or composition of the present disclosure together with a compound and / or composition that can treat or prevent a disease or disorder as intended herein.In certain embodiments, the co-administered compound and / or composition is administered separately or in any kind of combination as part of a single therapeutic approach.The co-administered compound and / or composition can be formulated in any kind of combination as a mixture of solid and liquid under various solid, gel and liquid formulations, and as a solution.

[0045] The term "pharmaceutical composition" or "composition" as used herein refers to a mixture of at least one compound useful in the present disclosure with a pharmaceutically acceptable carrier.The pharmaceutical composition facilitates the administration of the compound to a patient.There are multiple techniques of administering the compound in the art, including but not limited to subcutaneous administration, intravenous administration, oral administration, aerosol administration, inhalation administration, rectal administration, vaginal administration, transdermal administration, intranasal administration, buccal administration, sublingual administration, parenteral administration, intrathecal administration, intragastric administration, ocular administration, pulmonary administration, and local administration.

[0046] As used herein, the term "pharmaceutical acceptable" refers to a relatively non-toxic material, such as a carrier or diluent, that does not abrogate the biological activity or properties of a compound, i.e., the material may be administered to an individual without causing undesired biological effects or deleteriously interacting with any of the components of the composition in which it is contained.

[0047] The term "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition, or carrier, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener, solvent, or encapsulating material, involved in carrying or transporting a compound useful in the present disclosure into or to a patient so that the compound useful in the present disclosure can perform its intended function. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation, including the compound useful in the present disclosure, and not harmful to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives. As used herein, "pharmaceutically acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, etc., that are compatible with the activity of the compound useful in the present disclosure and are physiologically acceptable to the patient. A "pharmaceutically acceptable carrier" may further include pharmaceutically acceptable salts of compounds useful in the present disclosure. Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the present disclosure are known in the art and are described, for example, in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

[0048] As used herein, the term "pharmaceutically acceptable salts" refers to salts of the compound being administered prepared from pharmaceutically acceptable non-toxic acids and bases, including inorganic acids, inorganic bases, organic acids, inorganic bases, solvates, hydrates, and clathrates thereof.

[0049] As used herein, a "pharmacologically effective amount," "therapeutically effective amount," or "effective amount" of a compound is an amount of the compound sufficient to confer a beneficial effect on the subject to which the compound is administered.

[0050] As used herein, the term "prevent" or "prevention" refers to the absence of a disorder or disease if none has occurred, or to the prevention of further development of a disorder or disease if the disorder or disease has already developed. The ability to prevent some or all of the symptoms associated with a disorder or disease is also considered.

[0051] As used herein, the terms "subject" and "individual" and "patient" can be used interchangeably and may refer to a human or non-human mammal or bird. Non-human mammals include, for example, farm animals and pets, such as sheep, cows, pigs, dogs, cats, and murine mammals. In certain embodiments, the subject is a human.

[0052] The term "treatment" or "treating" as used herein is defined as the application or administration of a therapeutic agent, i.e., a compound useful in the present disclosure (alone or in combination with another pharmaceutical agent), to a patient having a disease or disorder and / or symptoms of a disease or disorder, or to an isolated tissue or cell line derived from a patient (e.g., in the case of diagnostic or ex vivo applications), for the purpose of curing, curing, alleviating, mitigating, altering, palliating, ameliorating, improving, or affecting the disease or disorder and / or symptoms of the disease or disorder. Such treatments may be tailored and modified based on knowledge gained from the field of pharmacogenomics.

[0053] The term "fibrodysplasia ossificans progressiva" or "FOP" as used herein refers to a genetic disorder of congenital skeletal malformations and progressive heterotopic ossification (HO). In all individuals with sporadic or familial FOP, heterozygous (i.e., patients have one copy of a mutant Acvr1 gene and one copy of a wild-type Acvr1 gene) missense mutations in the bone morphogenetic protein (BMP) type I receptor activin receptor type AI / activin-like kinase 2 (ACVR1 / ALK2) have been identified. The activating R206H mutation on ACVR1 is found in most FOP patients. Details of the disease are described, for example, in Kaplan et al. (Best Practice & Research Clinical Rheumatology Volume 22, Issue 1, March 2008, Pages 191-205) and Kaplan et al. (Bone Volume 140, November 2020, 115539).

[0054] The term "heterotopic ossification" or "HO" as used herein refers to the formation of extraosseous bone in muscle and soft tissue. HO is one of the postnatal characteristics of FOP and is a common event after trauma in non-FOP patients. Details of heterotopic ossification are described, for example, in Meyers et al. (JBMR Plus 2019, 3: e10172) and Dey et al., (Transl Res. 2017 Aug; 186: 95-111).

[0055] The term "flare-up" as used herein in relation to FOP refers to the inflammatory soft tissue swelling experienced by most FOP patients, which begins in early childhood and progresses throughout life. Flare-ups include unpredictable episodes of soft tissue swelling, pain, reduced mobility, and / or stiffness. These flare-ups usually cause extra bone formation, but not always. Details of flare-ups in FOP are described, for example, in Pignolo et al. (J Bone Miner Res. 2016 Mar;31(3):650-6).

[0056] Non-limiting abbreviations used herein: FOP: fibrodysplasia ossificans progressiva. HO: heterotopic ossification. PBMC: peripheral blood mononuclear cells. SNP: single nucleotide polymorphism.

[0057] Methods for Treating, Ameliorating, and / or Preventing Fibrodysplasia Ossificans Progressiva This study found a patient with fibrodysplasia ossificans progressiva (FOP) with a classic R206H mutation in the Acvr1 BMP receptor. The patient had congenital FOP features, but only a few postnatal FOP features developed. This study found that the patient had significantly suppressed inflammatory biomarkers and had compound heterozygosity for the MMP-9 gene (one allele of the MMP-9 gene with a polymorphism results in A20V, and the other allele with a polymorphism results in D165N). Using protein structure modeling, this study predicted that the compound heterozygosity of MMP-9 found in this patient reduced the levels and activity of MMP-9 in the patient. This study found that MMP-9 is expressed in early diseased tissues of FOP subjects. Using several mouse FOP models as non-limiting examples, this study found that reducing the levels and / or activity of MMP-9 by genetic deletion (including partial deletion) of MMP-9, administration of non-limiting MMP-9 monoclonal antibodies, or administration of non-limiting small molecule inhibitors of MMP-9 all resulted in reduced levels of heterotopic ossification (bone formation) in response to soft tissue injury.

[0058] Accordingly, in some aspects, the present invention relates to a method of treating, ameliorating, and / or preventing fibrodysplasia ossificans progressiva (FOP) in a subject, hi some embodiments, the method comprises administering to the subject a compound that downregulates MMP-9 levels and / or activity in the subject.

[0059] This study found that inhibiting MMP-9 can significantly reduce the heterotopic bone formation level in model animals with Q270D mutation in Acvr1 BMP receptor. Because Q270D mutation does not cause spontaneous heterotopic bone formation, but only causes HO in response to injury, and produces very strong post-traumatic HO, Q270D model is sometimes considered as a model of HO. Therefore, in some aspects, the present invention relates to a method of treating, ameliorating, and / or preventing heterotopic bone formation in a subject in need thereof. In some embodiments, the method comprises administering to the subject a compound that downregulates the level and / or activity of MMP-9 in the subject.

[0060] In some embodiments, the subject is treated by bone marrow transplantation to replace some of the subject's hematopoietic stem cells with hematopoietic stem cells with low levels and / or activity of MMP-9. Those skilled in the art will understand that MMP-9 is expressed in multiple tissues, but is highly expressed in bone marrow and lymphoid tissues. As described elsewhere herein, this study has found that even partial reduction of MMP-9 expression by deleting only one MMP-9 allele is sufficient to counteract the heterotopic bone formation induced by soft tissue injury in FOP model animals. Thus, in certain embodiments, partial reduction and / or loss of MMP-9 expression and / or activity only in bone marrow is sufficient to mimic the partial reduction of MMP-9 expression in FOP model mice. In some embodiments, the bone marrow stem cells used for transplantation are genetically engineered to reduce levels and / or activity of MMP-9. Genetic manipulation of hematopoietic stem cells in bone marrow is described, for example, in Daniel-Moreno et al. (Bone Marrow Transplantation volume 54, pages 1940-1950 (2019)). In some embodiments, the bone marrow used for transplantation is derived from a donor with an MMP-9 polymorphism that reduces the level and / or activity of MMP-9, for example, the MMP-9 polymorphism found in patient-R described herein.

[0061] In some embodiments, the subject has a mutant ACVR1 gene. In some embodiments, the mutant ACVR1 gene encodes a constitutively active ACVR1 polypeptide. In other embodiments, the mutant ACVR1 gene comprises a mutation (e.g., one or more point mutations, deletions, and / or insertions) in the nucleic acid sequence of the ACVR1 gene encoding the ACVR1 polypeptide, compared to the wild-type gene. In some embodiments, the ACVR1 polypeptide comprises a mutation. In some embodiments, the ACVR1 polypeptide comprises one or more amino acid substitutions, deletions, and / or insertions, compared to the wild-type polypeptide. In some embodiments, the ACVR1 polypeptide comprises one or more amino acid substitutions, deletions, and / or insertions, compared to the wild-type ACVR1 polypeptide, and the wild-type ACVR1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 3. GenBank Accession No. NP_001104537, the entirety of which is incorporated herein by reference and which discloses the ACVR1 polypeptide. In some embodiments, the ACVR1 polypeptide comprises one or more mutations at amino acid residues L196, P197, F198, R202, R206, Q207, F246, R258, G325, G328, G356, R375, or K400. In some embodiments, the ACVR1 polypeptide comprises one or more mutations at amino acid residues L196, P197, F198, R202, R206, Q207, F246, R258, G325, G328, G356, R375, or K400 compared to a wild-type ACVR1 polypeptide comprising the sequence set forth in SEQ ID NO:3. In some embodiments, the ACVR1 polypeptide comprises one or more mutations selected from the group consisting of L196P, P197-F198 del ins L, R202I, R206H, Q207E, F246Y, R258S, R258G, G325A, G328E, G328W, G328R, G356D, R375P, and K400E, where del represents a deletion and ins represents an insertion.In some embodiments, the ACVR1 polypeptide comprises one or more mutations selected from the group consisting of L196P, P197-F198 del ins L, R202I, R206H, Q207E, F246Y, R258S, R258G, G325A, G328E, G328W, G328R, G356D, R375P, and K400E, compared to a wild-type ACVR1 polypeptide comprising the sequence set forth in SEQ ID NO:3, where del represents a deletion and ins represents an insertion. ACVR1 mutations responsible for FOP are also described in Haupt et al. (Bone Volume 109, April 2018, Pages 232-240) and Mukaddam et al., (Genetics of Fibrodysplasia Ossificans Progressiva, eLS, Vol 2: 1-8, 2021), each of which is incorporated by reference herein in its entirety.

[0062] In some embodiments, the method further comprises surgically removing the ossified tissue from the subject. In some embodiments, the surgical removal of the ossified tissue is performed after the level or activity of MMP-9 in the subject is downregulated.

[0063] In some aspects, the subject is a mammal, hi some aspects, the subject is a human.

[0064] Small molecule inhibitors of MMP-9 In some embodiments, compounds that downregulate MMP-9 levels and / or activity include small molecule inhibitors of MMP-9.

[0065] The term "small molecule" as used herein means that the molecule has a molecular weight of about 2000 daltons or less, e.g., about 1800 daltons or less, about 1600 daltons or less, about 1400 daltons or less, about 1200 daltons or less, about 1000 daltons or less, or about 900 daltons or less. Non-limiting examples of small molecules that inhibit MMP-9 include actinonin, ageladine A TFA, apigenin-7-glucuronide, ARP 100, astragaloside IV, BR351, chlorhexidine dihydrochloride, sipemastat, CMC2.24, CP-471474, CP-544439, cyclic CTTHWGFTLC, cyclic CTTHWGFTLC TFA, FSL-1 TFA, Ginkgolide C, Ilomastat (also known as GM6001), JNJ0966, Luteolin 7-O-glucuronide, Marimastat, MMP-2 / MMP-9 inhibitor I, MMP-2 / MMP-9 inhibitor II, MMP3 inhibitor 1, MMP-9-IN-1, MMP-9 inhibitor I, MMP-9 inhibitor II, MMP-9 / MMP-13 inhibitor I, MMP inhibitor II, MMP13-IN-3, MMPI-1154, Morroniside, ND-336, NNGH, (R)-ND-336, PF-00356231 hydrochloride, PD-166793, Prinomastat, Prinomastat hydrochloride, Salvianolic acid A, S 3304, SB-3CT, SM-7368, tanomastat, tetracycline derivatives such as doxycycline, incyclinide, and minocycline, UK 356618, UK-370106, XL-784, etc. MMP-9 inhibitors are described, for example, in Fields (Cells. 2019 Sep; 8(9): 984.).

[0066] In some embodiments, small molecule inhibitors of MMP-9 include selective MMP-9 small molecule inhibitors. As used herein, the term "selective MMP-9 inhibitor" refers to a small molecule inhibitor that has an IC50 or IC60 inhibitor or a small molecule inhibitor or a selective MMP-9 ... 50 IC50 or greater than, for example, IC50 for any other matrix metalloproteinase 50IC for MMP-9 greater than 0.1, greater than 0.2, greater than 0.3, greater than 0.5, greater than 0.8, or greater than 1.0 50 Non-limiting examples of selective MMP-9 small molecule inhibitors include apigenin-7-glucuronide, FFAGLDD, FSL-1 TFA, ginkgolide C, isoliquiritin apiosid, JNJ0966, luteolin 7-O-glucuronide, MMP-9-IN-1, (R)-ND-336, SM-7368, FFAGLDD TFA, and the like.

[0067] In some embodiments, the small molecule inhibitor of MMP-9 includes compounds that are used in the medical field for purposes other than inhibiting MMP-9 and are generally known to be safe.Non-limiting examples of such compounds include tetracyclines, such as doxycycline, incyclinide, and minocycline.Examples of tetracyclines that can inhibit MMP-9 are described, for example, in Griffin et al. (Am J Physiol Cell Physiol. 2010 Sep; 299(3): C539-C548.), the entire references of which are incorporated herein by reference.

[0068] Protein inhibitors of MMP-9 In some embodiments, compounds that downregulate MMP-9 levels and / or activity include protein inhibitors of MMP-9.

[0069] In some embodiments, the protein inhibitor of MMP-9 includes an antibody against MMP-9 or an antigen-binding fragment thereof. Examples of antibodies against MMP-9 include anti-Ac-MMP-9 antibody (4A3), anti-MMP-9 antibody (E-11), anti-MMP-9 antibody (2C3), and anti-MMP-9 antibody (6-6B) from Santa Cruz Biotechnology, MMP9 monoclonal antibody (5G3), MMP9 recombinant rabbit monoclonal antibody (JA80-73), and MMP9 monoclonal antibody (5C3) from Invitrogen, etc.

[0070] In some embodiments, protein inhibitors of MMP-9 include non-antibody protein or peptide inhibitors of MMP-9. Non-limiting examples of non-antibody protein or peptide inhibitors of MMP-9 include proteins from the tissue inhibitor of metalloproteinases (TIMP) family, such as TIMP-1, TIMP-3, FFAGLDD peptide, FFAGLDD TFA, cyclic CTTHWGFTLC, cyclic CTTHWGFTLC TFA, and the like.

[0071] RNA interference nucleic acid for downregulating MMP-9 and vector expressing same In some embodiments, compounds that downregulate MMP-9 levels and / or activity include nucleic acids that downregulate MMP-9 levels by RNA interference, and / or expression vectors that express the nucleic acids.

[0072] In some embodiments, the nucleic acid that downregulates MMP-9 level by RNA interference includes isolated nucleic acid. In other embodiments, the modulator is an RNAi molecule (including but not limited to siRNA and / or shRNA and / or miRNA) or an antisense molecule that inhibits MMP-9 expression and / or activity. In still other embodiments, the nucleic acid preferably includes a promoter / regulatory sequence that can direct the expression of the nucleic acid. Thus, the present specification provides expression vectors and methods for introducing exogenous DNA into cells and concomitantly expressing exogenous DNA in the cells, such as those described in, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York), and those described elsewhere herein.

[0073] In certain embodiments, siRNAs are used to reduce MMP-9 levels. RNA interference (RNAi) is a phenomenon in which double-stranded RNA (dsRNA) is introduced into a diverse range of organisms and cell types, resulting in degradation of complementary mRNA. In cells, long dsRNAs are cleaved into short 21-25 nucleotide small interfering RNAs, or siRNAs, by a ribonuclease known as Dicer. The siRNAs then assemble with protein components into RNA-induced silencing complexes (RISCs) that unwind in the process. The activated RISC then binds to the complementary transcripts through base-pairing interactions between the siRNA antisense strand and the mRNA. The bound mRNA is cleaved, and gene silencing occurs through sequence-specific degradation of the mRNA. See, for example, U.S. Patent No. 6,506,559; Fire et al., 1998, Nature 391(19):306-311; Timmons et al., 1998, Nature 395:854; Montgomery et al., 1998, TIG 14 (7):255-258; Engelke, Ed., RNA Interference (RNAi) Nuts & Bolts of RNAi Technology, DNA Press, Eagleville, PA (2003); and Hannon, Ed., RNAi A Guide to Gene Silencing, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2003). Soutschek et al. (2004, Nature 432:173-178) describe chemical modifications to siRNA that aid in intravenous systemic delivery. Optimization of siRNA involves consideration of overall G / C content, C / T content at the ends, Tm, and nucleotide content of the 3' overhangs. See, e.g., Schwartz et al., 2003, Cell, 115:199-208 and Khvorova et al., 2003, Cell 115:209-216.Thus, the present specification also includes methods for decreasing MMP-9 levels using RNAi technology.

[0074] In certain embodiments, the present disclosure provides a vector comprising an siRNA or antisense polynucleotide. In other embodiments, the siRNA or antisense polynucleotide inhibits the expression of MMP-9. The incorporation of a desired polynucleotide into a vector and the selection of the vector are well known in the art.

[0075] In certain embodiments, the expression vector described herein encodes a short hairpin RNA (shRNA) inhibitor.shRNA inhibitors are well known in the art and are directed to target mRNA, thereby reducing the expression of the target.In certain embodiments, the encoded shRNA is expressed by cells, and then processed into siRNA.For example, in certain cases, cells have a natural enzyme (e.g., dicer) that cuts shRNA to form siRNA.

[0076] siRNA, shRNA, or antisense polynucleotides can be cloned into many types of vectors as described elsewhere herein. For expression of siRNA or antisense polynucleotides, at least one module in each promoter functions to position the start point for RNA synthesis.

[0077] To evaluate the expression of siRNA, shRNA or antisense polynucleotide, the expression vector to be introduced into cells may also contain either a selection marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from the population of cells to be transfected or infected with the viral vector.In certain embodiments, the selection marker may be carried on a separate DNA fragment and used in a co-transfection procedure.Both the selection marker and the reporter gene may be flanked with appropriate control sequences to allow expression in the host cell.Useful selection markers are known in the art and include, for example, antibiotic resistance genes, such as neomycin resistance.

[0078] After generating a siRNA polynucleotide, one of skill in the art will appreciate that the siRNA polynucleotide has certain characteristics that can be modified to improve the siRNA as a therapeutic compound. Thus, in some embodiments, siRNA polynucleotides are further designed to resist degradation by modification to include phosphorothioates, or other linkages, methylphosphonates, sulfones, sulfates, ketyl, phosphorodithioates, phosphoramidates, phosphate esters, and the like (see, e.g., Agrwal et al., 1987, Tetrahedron Lett. 28:3539-3542; Stec et al., 1985 Tetrahedron Lett. 26:2191-2194; Moody et al., 1989 Nucleic Acids Res. 12:4769-4782; Eckstein, 1989 Trends Biol. Sci. 14:97-100; Stein, In: Oligodeoxynucleotides. Antisense Inhibitors of Gene Expression, Cohen, ed., Macmillan Press, London, pp. 97-117). (1989)).

[0079] Any polynucleotide can be further modified to enhance in vivo stability.Possible modifications include, but are not limited to, adding flanking sequences at 5'-end and / or 3'-end; using phosphorothioate or 2'O-methyl in backbone instead of phosphodiester linkage; and / or containing non-traditional bases such as inosine, queosine, and eubutosine, and acetyl-, methyl-, thio-, and other modifications of adenine, cytidine, guanine, thymine, and uridine.

[0080] In certain embodiments, antisense nucleic acid sequences expressed by plasmid vectors are used to inhibit MMP-9 protein expression. Antisense expression vectors are used to transfect mammalian cells or whole mammals, thereby reducing the endogenous expression of MMP-9.

[0081] Antisense molecules and their use for inhibiting gene expression are well known in the art (see, for example, Cohen, 1989, In: Oligodeoxyribonucleotides, Antisense Inhibitors of Gene Expression, CRC Press).Antisense nucleic acid, as this term is defined elsewhere herein, is a DNA or RNA molecule that is complementary to at least a portion of a specific mRNA molecule (Weintraub, 1990, Scientific American 262:40).In cells, antisense nucleic acid hybridizes with corresponding mRNA to form a double-stranded molecule, thereby inhibiting the translation of genes.

[0082] The use of antisense methods to inhibit gene translation is known in the art and is described, for example, in Marcus-Sakura (1988, Anal. Biochem. 172:289).Such antisense molecules can be provided to cells through gene expression using DNA encoding the antisense molecule, as disclosed by Inoue, 1993, U.S. Patent No. 5,190,931.

[0083] Alternatively, the antisense molecules herein may be synthetically produced and then provided to cells. Antisense oligomers of about 10 to about 30, more preferably about 15 nucleotides can be used as they are easily synthesized and introduced into target cells. Synthetic antisense molecules contemplated by the present specification include oligonucleotide derivatives known in the art that have improved biological activity compared to unmodified oligonucleotides (see U.S. Pat. No. 5,023,243).

[0084] Ribozyme that downregulates MMP-9 and vector expressing same In some embodiments, compounds that downregulate MMP-9 levels and / or activity include ribozymes and / or vectors that express ribozymes that downregulate MMP-9.

[0085] Ribozymes are used to inhibit MMP-9 protein expression. Ribozymes useful for inhibiting expression of target molecules can be designed by incorporating a target sequence that is complementary, for example, to the mRNA sequence encoding MMP-9, into the basic ribozyme structure. Ribozymes are antisense RNAs with catalytic sites that can specifically cleave complementary RNA. Thus, ribozymes with sequences complementary to MMP-9 mRNA sequences can downregulate MMP-9 expression by reducing MMP-9 mRNA levels. Ribozymes targeting MMP-9 can be synthesized using commercially available reagents (Applied Biosystems, Inc., Foster City, CA) or genetically expressed from DNA that codes for them. In some embodiments, DNA that codes for ribozymes is incorporated into a vector as described elsewhere herein.

[0086] Compounds that downregulate MMP-9 by CRISPR knockout / knockdown and other knockout / knockdown methods In some embodiments, the compound that downregulates the level and / or activity of MMP-9 includes compounds that downregulate MMP-9 by CRISPR knockout / knockdown and other knockout / knockdown methods. In some embodiments, the compound includes an expression vector that includes an expression cassette, which expresses a CRISPR component that downregulates MMP-9 by CRISPR knockout or CRISPR knockdown.

[0087] In some embodiments, compounds that downregulate the activity or levels of MMP-9 include the CRISPR / Cas9 system for knocking out MMP-9.

[0088] The CRISPR / Cas9 system is a facile and efficient system for inducing targeted gene changes. Target recognition by the Cas9 protein requires a "seed" sequence within the guide RNA (gRNA) and a conserved dinucleotide containing a protospacer adjacent motif (PAM) sequence upstream of the gRNA binding region. Thereby, the CRISPR / Cas9 system can be engineered to cleave virtually any DNA sequence by redesigning the gRNA in cell lines (e.g., 293 T cells), primary cells, and CAR T cells. The CRISPR / Cas9 system can simultaneously target multiple genomic loci by co-expressing one Cas9 protein and two or more gRNAs, making this system uniquely suitable for editing multiple genes or synergistic activation of target genes.

[0089] The Cas9 protein and guide RNA form a complex that locates and cleaves the target sequence. Cas9 is composed of six domains: REC I, REC II, Bridge Helix, PAM interacting, HNH, and RuvC. The RecI domain binds to the guide RNA, while the Bridge Helix binds to the target DNA. The HNH and RuvC domains are nuclease domains. The guide RNA is engineered to have a 5' end that is complementary to the target DNA sequence. Binding of the guide RNA to the Cas9 protein causes a conformational change that activates the protein. Once activated, Cas9 searches the target DNA by binding to a sequence that matches the protospacer adjacent motif (PAM) sequence of the target DNA. The PAM is a di- or tri-nucleotide sequence that is within one nucleotide downstream of the region complementary to the guide RNA. In one non-limiting example, the PAM sequence is 5'-NGG-3'. When the Cas9 protein finds its target sequence with the appropriate PAM, it melts the bases upstream of the PAM and pairs them with the complementary region on the guide RNA. The RuvC and HNH nuclease domains then cleave the target DNA three nucleotide bases upstream of the PAM.

[0090] One non-limiting example of a CRISPR / Cas system used to inhibit gene expression, CRISPRi, is described in US Patent Application Publication No. US2014 / 0068797. CRISPRi induces permanent gene disruption utilizing the RNA-guided Cas9 endonuclease to introduce DNA double-strand breaks that trigger error-prone repair pathways resulting in frameshift mutations. Catalytically dead Cas9 lacks endonuclease activity. When co-expressed with guide RNA, a DNA recognition complex is generated that specifically interferes with transcription elongation, RNA polymerase binding, or transcription factor binding. This CRISPRi system efficiently silences the expression of target genes.

[0091] CRISPR / Cas gene disruption occurs when a guide nucleic acid sequence specific to a target gene and a Cas endonuclease are introduced into a cell, forming a complex that allows the Cas endonuclease to introduce a double-strand break into the target gene. In certain embodiments, the CRISPR / Cas system comprises an expression vector, such as, but not limited to, the pAd5F35-CRISPR vector. In other embodiments, the Cas expression vector induces the expression of Cas9 endonuclease. Other endonucleases may also be used, including, but not limited to, T7, Cas3, Cas8a, Cas8b, Cas10d, Cse1, Csy1, Csn2, Cas4, Cas10, Csm2, Cmr5, Fok1, other nucleases known in the art, and any combination thereof.

[0092] In certain embodiments, inducing the Cas expression vector comprises exposing the cell to an agent that activates an inducible promoter in the Cas expression vector. In such embodiments, the Cas expression vector comprises an inducible promoter, such as one that is induced by exposure to an antibiotic (e.g., tetracycline or a derivative of tetracycline, such as doxycycline). However, it should be understood that other inducible promoters can be used. The inducing agent can be a selective condition that induces the inducible promoter (e.g., exposure to an agent, such as an antibiotic). This causes the Cas expression vector to be expressed.

[0093] In certain embodiments, the guide RNA and Cas9 can be delivered to cells as a ribonucleoprotein (RNP) complex. RNPs are composed of purified Cas9 protein complexed with gRNA and are known in the art to be efficiently delivered to multiple types of cells, including but not limited to neurons, stem cells, and immune cells (Addgene, Cambridge, MA; Mirus Bio LLC, Madison, WI).

[0094] Guide RNA is specific to the target, which is the genomic region of interest and the region of double-strand break induced by Cas endonuclease.The target sequence of guide RNA sequence can be inside the locus of gene or inside the non-coding region of genome.In certain embodiments, guide nucleic acid sequence is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more nucleotides in length.

[0095] Guide RNAs (gRNAs), also called "short guide RNAs" or "sgRNAs", provide both target specificity and scaffolding / binding capabilities for the Cas9 nuclease. gRNAs can be synthetic RNAs composed of targeting and scaffolding sequences derived from endogenous bacterial crRNAs and tracrRNAs. gRNAs are used to target Cas9 to specific genomic loci in genome engineering experiments. Guide RNAs can be designed using standard tools well known in the art.

[0096] In the context of CRISPR complex formation, "target sequence" refers to a sequence that guide sequence is designed to have some complementarity with. In this case, hybridization between target sequence and guide sequence promotes the formation of CRISPR complex. Full complementarity is not necessarily required, as long as there is sufficient complementarity to cause hybridization and promote the formation of CRISPR complex. Target sequence can include any polynucleotide, such as DNA or RNA polynucleotide. In certain embodiments, target sequence is located in the nucleus or cytoplasm of a cell. In other embodiments, target sequence can be in an organelle of eukaryotic cell, such as mitochondria or nucleus. Typically, in the context of endogenous CRISPR systems, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands at or near the target sequence (e.g., within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs of the target sequence). As with the target sequence, perfect complementarity is not believed to be required, provided this is sufficient for function.

[0097] In certain embodiments, one or more vectors are introduced into the host cell to drive the expression of one or more elements of the CRISPR system, such that the expression of the elements of the CRISPR system directs the formation of a CRISPR complex at one or more target sites. For example, the Cas enzyme, the guide sequence linked to the tracr-mate sequence, and the tracr sequence may each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements may be combined in one vector, and one or more additional vectors may provide any components of the CRISPR system that are not included in the first vector. The CRISPR system elements combined in one vector may be arranged in any suitable direction, for example, an element may be arranged 5' ("upstream") or 3' ("downstream") relative to the second element. The coding sequence of an element may be arranged on the same or opposite strand of the coding sequence of the second element, and may be oriented in the same or opposite direction. In certain embodiments, one promoter drives expression of transcripts encoding a CRISPR enzyme and one or more of a guide sequence, a tracr mate sequence (optionally operably linked to a guide sequence), and a tracr sequence embedded within one or more intron sequences (e.g., each in a different intron, two or more in at least one intron, or all in one intron).

[0098] In certain embodiments, the CRISPR enzyme is part of a fusion protein that includes one or more heterologous protein domains (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition to the CRISPR enzyme). The CRISPR enzyme fusion protein may include any additional protein sequence, and may optionally include a linker sequence between any two domains. Examples of protein domains that can be fused to the CRISPR enzyme include, but are not limited to, epitope tags, reporter gene sequences, and protein domains that have one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Additional domains that can be part of fusion proteins that include CRISPR enzymes are described in U.S. Patent Application Publication No. US20110059502, which is incorporated herein by reference. In certain embodiments, a tagged CRISPR enzyme is used to localize the target sequence.

[0099] Conventional virus-based and non-virus-based gene transfer methods can be used to introduce nucleic acid into mammalian and non-mammalian cells or target tissues. Such methods can be used to administer nucleic acid encoding components of CRISPR system to cells in culture or in host organisms. Non-viral vector delivery systems include DNA plasmids, RNA (e.g., transcripts of vectors described herein), naked nucleic acid, and nucleic acid complexed with delivery vehicles such as liposomes. Viral vector delivery systems include DNA and RNA viruses that have episomal or integrated genomes after delivery to cells (Anderson, 1992, Science 256:808-813; and Yu, et al., 1994, Gene Therapy 1:13-26).

[0100] In certain embodiments, the CRISPR / Cas is derived from a type II CRISPR / Cas system. In other embodiments, the CRISPR / Cas system is derived from a Cas9 protein. The Cas9 protein may be derived from Streptococcus pyogenes, Streptococcus thermophilus, or other species.

[0101] Generally, Cas proteins contain at least one RNA recognition and / or RNA binding domain. The RNA recognition and / or RNA binding domain interacts with the guiding RNA. Cas proteins may also contain nuclease domains (i.e., DNase or RNase domains), DNA binding domains, helicase domains, RNase domains, protein-protein interaction domains, dimerization domains, and other domains. Cas proteins can be modified to increase nucleic acid binding affinity and / or specificity, to modify enzymatic activity, and / or to change other properties of the protein. In certain embodiments, the Cas-like protein of the fusion protein may be derived from a wild-type Cas9 protein or a fragment thereof. In other embodiments, the Cas may be derived from a modified Cas9 protein. For example, the amino acid sequence of the Cas9 protein can be modified to modify one or more properties of the protein (e.g., nuclease activity, affinity, stability, etc.). Alternatively, the Cas9 protein domains that are not involved in RNA-guided cleavage can be removed from the modified Cas9 protein, such that the modified Cas9 protein is smaller than the wild-type Cas9 protein. Generally, Cas9 proteins contain at least two nuclease (i.e., DNase) domains. For example, Cas9 proteins may contain a RuvC-like nuclease domain and an HNH-like nuclease domain. The RuvC and HNH domains work together to cleave a single strand and generate a double-stranded break in DNA (Jinek, et al., 2012, Science, 337:816-821). In certain embodiments, Cas9-derived proteins can be modified to contain only one functional nuclease domain (either the RuvC-like or HNH-like nuclease domain). For example, Cas9-derived proteins can be modified to delete or mutate one of the nuclease domains such that the nuclease domain is no longer functional (i.e., there is no nuclease activity).In some embodiments in which one of the nuclease domains is inactive, the Cas9 derived protein can introduce nicks into double stranded nucleic acids (such proteins are called "nickases") but cannot cleave double stranded DNA. In any of the above embodiments, any or all of the nuclease domains can be inactivated by one or more deletion, insertion and / or substitution mutations using well known methods such as site-directed mutagenesis, PCR-mediated mutagenesis, and whole gene synthesis, as well as other methods known in the art.

[0102] In a non-limiting embodiment, the vector drives the expression of CRISPR system. The art is rich in suitable vectors that are useful herein. The vector to be used is suitable for replication, and optionally integration, in eukaryotic cells. Representative vectors contain transcription and translation terminators, initiation sequences, and promoters that are useful for regulating the expression of desired nucleic acid sequences. The vectors herein may also be used in standard gene delivery protocols for nucleic acids. Methods for gene delivery are known in the art (U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety).

[0103] Additionally, the vector may be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. thThe vectors are described in the following publications: Viruses, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 2012 (Eds. Edition, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 2012) and other virology and molecular biology manuals. Viruses are useful as vectors, including but not limited to retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, Sindbis viruses, gamma retroviruses, and lentiviruses. In general, suitable vectors contain an origin of replication that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers (e.g., WO01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193).

[0104] In some embodiments, the compound that downregulates the activity or expression level of MMP-9 comprises a nucleic acid that downregulates the expression level of MMP-9 by CRISPR knockdown, including but not limited to CRISPR Cas13 knockdown (see, for example, Mendez-Mancilla et al., Cell Chemical Biology 29, 1-7, 2021 Jul 27, and Kushawah et al., Dev Cell. 2020 Sep 28;54(6):805-817, which are incorporated herein by reference in their entirety).

[0105] In some aspects, the present invention includes any other methods for effecting gene knockdown and / or editing that allow for deletion and / or inactivation of MMP-9, such as, but not limited to, those described in WO2018 / 236840 (herein incorporated by reference in its entirety).

[0106] Compounds that downregulate MMP-9 by inactivation and / or sequestration In some embodiments, the compound that downregulates MMP-9 activity or expression level comprises a protein that downregulates MMP-9 activity by inactivating and / or sequestering MMP-9. In some embodiments, the compound comprises a nucleic acid that expresses a protein that downregulates MMP-9 activity by inactivating and / or sequestering MMP-9. In some embodiments, the compound comprises an expression vector that expresses a protein that downregulates MMP-9 activity by inactivating and / or sequestering MMP-9 (see the "Vectors" section for a non-limiting description of vectors).

[0107] In some embodiments, the compound that downregulates the expression level of MMP-9 is a transdominant negative mutant of MMP-9 and / or a nucleic acid or vector expressing a transdominant negative mutant of MMP-9.

[0108] Kits for treating, ameliorating, and / or preventing fibrodysplasia ossificans progressiva In some aspects, the present invention relates to a kit for treating, ameliorating, and / or preventing fibrodysplasia ossificans progressiva (FOP) in a subject in need thereof.

[0109] In some aspects, the present invention relates to kits for treating, ameliorating, and / or preventing heterotopic ossification in a subject in need thereof.

[0110] In some embodiments, the kit comprises a compound that downregulates MMP-9 levels and / or activity in a subject and a manual instructing that an effective amount of the compound is administered to the subject. In some embodiments, the compound that downregulates MMP-9 levels and / or activity in a subject is the same or similar as those described elsewhere herein, e.g., in the section "Methods of Treating, Ameliorating, and / or Preventing Fibrodysplasia Ossificans Progressiva."

[0111] vector The vector may enhance the stability of the nucleic acid, may facilitate easier delivery, or may enable expression of the nucleic acid or its protein product in a cell.

[0112] Thus, in some embodiments, a protein inhibitor or a nucleic acid that downregulates the activity or expression level of MMP-9 is incorporated into the vector.

[0113] In some aspects, the present specification relates to a vector comprising the nucleic acid sequence of the present specification or the construct of the present specification. The choice of vector depends on the host cell into which the vector is subsequently introduced. In certain aspects, the vector of the present specification is an expression vector. Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. In certain aspects, the expression vector is selected from the group consisting of viral vectors, bacterial vectors, and mammalian cell vectors. For use with the present specification, a system based on prokaryotic and / or eukaryotic vectors can be used to produce polynucleotides or their cognate polypeptides. Many such systems are commercially and widely available.

[0114] In some embodiments, vector is a viral vector.Viral vector technology is well known in the art and described in, for example, virology and molecular biology manuals.Viruses are useful as vectors, including but not limited to retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.Generally, suitable vectors contain a replication origin that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers (see, for example, WO01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193).

[0115] In some embodiments, the viral vector is a suitable adeno-associated virus (AAV), such as the AAV1-AAV8 family of adeno-associated viruses. In some embodiments, the viral vector is a viral vector capable of infecting humans. The desired nucleic acid sequence, such as the nucleic acid that downregulates MMP-9, can be inserted between inverted terminal repeats (ITRs) in the AAV. In various embodiments, the viral vector is AAV2 or AAV8. The promoter can be a thyroxine-binding globulin (TBG) promoter. In various embodiments, the promoter is a human promoter sequence that allows for desired nucleic acid expression in bone marrow, lymphoid tissue, connective tissue, kidney, bladder, and other tissues in which MMP-9 is expressed. In some embodiments, the promoter is a neuron-selective or neuron-specific promoter. The AAV can be a recombinant AAV, where the capsid is from one AAV serotype and the ITR is from another AAV serotype. In various embodiments, the AAV capsid is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and AAV8 capsid. In various embodiments, the ITR in the AAV is at least one ITR selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and AAV8 ITR. In various embodiments, the present specification contemplates an AAV8 viral vector (recombinant or non-recombinant) that contains a desired nucleic acid expression sequence and at least one promoter sequence that causes high systemic expression of the desired nucleic acid when administered to a subject. In some embodiments, the viral vector is a recombinant or non-recombinant AAV2 or AAV5 that contains any of the desired nucleic acid expression sequences described herein.

[0116] In some embodiments, the vector into which the nucleic acid sequence is introduced is a plasmid that may or may not be integrated into the genome of the host cell when introduced into the cell. Illustrative non-limiting examples of vectors into which the nucleotide sequence herein or the genetic construct herein may be inserted include tet-on inducible vectors for expression in eukaryotic cells.

[0117] The vector can be obtained by conventional methods known to those skilled in the art (Sambrook et al., 2012). In a particular embodiment, the vector is a vector useful for transforming animal cells.

[0118] In certain aspects, the recombinant expression vector may also contain a nucleic acid molecule encoding a peptide or peptidomimetic inhibitor herein, as described elsewhere herein.

[0119] A promoter may be a promoter naturally associated with a gene or polynucleotide sequence, as may be obtained by isolating 5' non-coding sequences located upstream of a coding segment and / or exon. Such promoters are sometimes referred to as "endogenous". Similarly, an enhancer may be an enhancer naturally associated with a polynucleotide sequence, located downstream or upstream of the polynucleotide sequence. Alternatively, certain benefits are obtained by placing a coding polynucleotide segment under the control of a recombinant or heterologous promoter. A recombinant or heterologous promoter refers to a promoter that is not naturally associated with a polynucleotide sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer that is not naturally associated with a polynucleotide sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, as well as promoters or enhancers that are not "natural", i.e., contain different elements of different transcriptional regulatory regions and / or mutations that alter expression. In addition to producing promoter and enhancer nucleic acid sequences synthetically, the sequences may be produced using nucleic acid amplification techniques, including recombinant cloning and / or PCR™, in conjunction with the compositions disclosed herein (U.S. Patent No. 4,683,202; U.S. Patent No. 5,928,906).Furthermore, it is contemplated that control sequences that direct transcription and / or expression of sequences in non-nuclear organelles, e.g., mitochondria, chloroplasts, etc., may also be used.

[0120] It is important to utilize a promoter and / or enhancer that effectively induces the expression of the DNA segment in the cell type, organelle, and organism selected for expression. Those skilled in the art of molecular biology usually know how to use a combination of promoters, enhancers, and cell types for protein expression. The promoter used may be a constitutive promoter, a tissue-specific promoter, an inducible promoter, and / or a promoter that is useful under appropriate conditions to induce high-level expression of the introduced DNA segment, for example, a promoter that is advantageous in large-scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous.

[0121] Recombinant expression vector may also contain a selection marker gene that facilitates the selection of transformed or transfected host cells.Suitable selection marker genes are proteins that confer resistance to certain drugs, such as G418 and hygromycin, β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or genes that code for immunoglobulins or parts thereof, such as the Fc part of immunoglobulins, preferably IgG.Selection markers may be introduced on separate vectors from the nucleic acid of interest.

[0122] Combination therapy In some embodiments, methods of treating, ameliorating, and / or preventing a disease and / or disorder contemplated herein include administering to a subject an effective amount of at least one compound and / or composition contemplated in the present disclosure.

[0123] In some embodiments, the subject is further administered at least one additional drug that treats, alleviates, and / or prevents the disease and / or disorder intended herein.In other embodiments, the compound and at least one additional drug are co-administered to the subject.In still other embodiments, the compound and at least one additional drug are co-formulated.

[0124] It is intended that the compounds contemplated in the present disclosure are useful in combination with one or more additional compounds.These additional compounds may include the compounds of the present disclosure, and / or at least one additional agent for treating a neurodegenerative condition, and / or at least one additional agent for treating one or more diseases or disorders contemplated herein.

[0125] The synergistic effect is e.g., Sigmoid-E max The drug combination effect can be calculated using appropriate methods such as the Loewe additivity equation (Holford & Scheiner, 1981, Clin. Pharmacokinet. 6:429-453), the Loewe additivity equation (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 114:313-326), and the median-effect equation (Chou & Talalay, 1984, Adv. Enzyme Regul. 22:27-55). Each of the above-mentioned equations can be applied to the experimental data to generate a corresponding graph that aids in the evaluation of the effect of the drug combination. The corresponding graphs associated with the above-mentioned equations are the concentration-effect curve, the isobologram curve, and the combination index curve, respectively.

[0126] Administration / Dosage / Formulation Dosage schedules may affect what constitutes an effective amount. The therapeutic formulations contemplated in this disclosure may be administered to a subject before or after the onset of a disease or disorder contemplated herein. Furthermore, several divided doses as well as staggered doses may be administered daily or continuously. Alternatively, the dose may be continuously infused or bolus injected. Furthermore, the dosage of the therapeutic formulations contemplated in this disclosure may be increased or decreased proportionately as indicated by the exigencies of the therapeutic or prophylactic situation.

[0127] The compositions contemplated in the present disclosure can be administered to a patient, preferably a mammal, more preferably a human, using known procedures at dosages and for periods of time effective to treat the disease and / or disorder contemplated herein in the patient. The effective amount of the therapeutic compound required to achieve a therapeutic effect may vary according to factors such as the status of the disease or disorder in the patient; the age, sex, and weight of the patient; and the ability of the therapeutic compound contemplated in the present disclosure to treat the disease and / or disorder contemplated herein in the patient. Dosage regimens may be adjusted to provide an optimal therapeutic response. For example, several divided doses may be administered daily. Alternatively, the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dosage range for the therapeutic compound contemplated in the present disclosure is about 1-5,000 mg / kg body weight / day. One of ordinary skill in the art would be able to study the relevant factors and make a determination regarding the effective amount of the therapeutic compound without undue experimentation.

[0128] Actual dosage levels of the active ingredients in the pharmaceutical formulations contemplated within this disclosure may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and method of administration without toxicity to the patient.

[0129] In particular, the selected dosage level will depend on a variety of factors, including the activity of the particular compound used, the time of administration, the rate of excretion of the compound, the duration of treatment, other drugs, compounds, or materials used in combination with the compound, the age, sex, weight, condition, general health, and prior medical history of the patient being treated, and similar factors well known in the medical arts.

[0130] A medical doctor, e.g., a physician or veterinarian, having ordinary skill in the art can readily determine the effective amount of the pharmaceutical composition required and prescribe an effective amount of the pharmaceutical composition. For example, the physician or veterinarian could begin dosing with a compound contemplated in the present disclosure used in a pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

[0131] In certain embodiments, it is particularly advantageous to formulate the compound in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form, as used herein, refers to a physically discrete unit suitable as a unitary dosage to the patient to be treated. Each unit contains a predetermined amount of therapeutic compound calculated to combine with the required pharmaceutical vehicle to produce the desired therapeutic effect. The unit dosage form contemplated in this disclosure is determined by and directly influenced by (a) the unique characteristics of the therapeutic compound and the specific therapeutic effect to be achieved, and (b) the constraints inherent in the field of compounding / formulating such therapeutic compounds to treat the disease or disorder contemplated herein.

[0132] In certain embodiments, the compositions of the present disclosure are formulated with one or more pharma- ceutically acceptable excipients or carriers.In certain embodiments, the pharmaceutical compositions of the present disclosure comprise a therapeutically effective amount of the compounds of the present disclosure and a pharma- ceutically acceptable carrier.

[0133] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. The activity of microorganisms can be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols, for example, mannitol and sorbitol, in the composition. Prolonged absorption of the injectable composition can be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0134] In certain embodiments, the compositions of the present disclosure are administered to patients in dosages of 1 to 5 or more times per day. In other embodiments, the compositions of the present disclosure are administered to patients in a range of dosages, including but not limited to, once per day, once every 2 days, once every 3 days to once per week, and once every 2 weeks. It is readily apparent to those skilled in the art that the frequency of administration of the various combination compositions of the present disclosure will vary from individual to individual, depending on many factors, including but not limited to, age, disease or disorder being treated, sex, general health, and other factors. Thus, the present disclosure should not be construed as being limited to any particular dosing regimen, and the exact dosage and composition administered to any patient will be determined by the attending physician, taking into account all other factors for the patient.

[0135] The compounds of the present disclosure for administration may be administered in the form of a single dose in the range of about 1 μg to about 10,000 mg, about 20 μg to about 9,500 mg, about 40 μg to about 9,000 mg, about 75 μg to about 8,500 mg, about 150 μg to about 7,500 mg, about 200 μg to about 7,000 mg, about 3050 μg to about 6,000 mg, about 500 μg to about 5,000 mg, about 750 μg to about 4,000 mg, about 1 mg to about 3,000 mg, or about 2 mg to about 4,000 mg. g, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg to about 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800 mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80 mg to about 500 mg, and any and all total or partial increments therebetween.

[0136] In some embodiments, the dosage of the disclosed compound is about 1 mg to about 2,500 mg. In some embodiments, the dosage of the disclosed compound used in the compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, the dose of the second compound described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all full or partial increments thereof.

[0137] In certain embodiments, the present disclosure relates to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the present disclosure, alone or in combination with a second pharmaceutical agent, and instructions for using the compound to treat, prevent, or reduce one or more symptoms of a neurodegenerative condition in a patient.

[0138] The formulations may be used in admixture with conventional excipients, i.e., pharma- ceutically acceptable organic or inorganic carrier substances, suitable for intracranial, intrathecal, oral, parenteral, intranasal, intravenous, subcutaneous, enteral, or any other suitable administration method known in the art. Pharmaceutical preparations may be sterilized and, if desired, may be mixed with auxiliary substances, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring substances, flavoring substances, and / or aromatic substances. These may be combined with other active agents, such as other analgesics, if desired.

[0139] The route of administration of any composition of the present disclosure includes oral, nasal, rectal, vaginal, parenteral, buccal, sublingual, or local administration. The compounds for use in the present disclosure may be formulated for administration by any suitable route, such as oral or parenteral administration, for example, transdermal, transmucosal (e.g., sublingual, lingual, (buccal), (urethral), vaginal (e.g., vaginal and perivaginal), nasal (intranasal), and rectal), intravesical, intrapulmonary, intraduodenal, intragastric, intrathecal, subcutaneous, intramuscular, intradermal, intraarterial, intravenous, intrabronchial, inhalation, and local administration.

[0140] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration, etc. It should be understood that the formulations and compositions useful in the present disclosure are not limited to the specific formulations and compositions described herein.

[0141] Oral route For oral application, tablets, dragees, liquids, drops, suppositories, or capsules, caplets and gelcaps are particularly suitable. Compositions intended for oral use can be prepared according to any method known in the art, and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutical excipients suitable for the manufacture of tablets. Such excipients include, for example, inert diluents, such as lactose; granulating and disintegrating agents, such as corn starch; binding agents, such as starch; and lubricants, such as magnesium stearate. Tablets may be uncoated or may be coated using known methods for accurate or delayed release of the active ingredient. Preparations for oral use may also be presented as hard gelatin capsules in which the active ingredient is mixed with an inert diluent.

[0142] For oral administration, the compounds of the present disclosure may take the form of tablets or capsules prepared by conventional means with pharma- ceutically acceptable excipients, such as binders (e.g., polyvinylpyrrolidone, hydroxypropylcellulose, or hydroxypropylmethylcellulose); fillers (e.g., corn starch, lactose, microcrystalline cellulose, or calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). If desired, tablets may be coated using suitable methods and coating materials, such as the OPADRY™ film coating system available from Colorcon, West Point, Pa (e.g., OPADRY™ OY Type, OYC Type, Organic Enteric OY-P Type, Aqueous Enteric OY-A Type, OY-PM Type, and OPADRY™ White, 32K18400). Liquid preparations for oral administration may take the form of solutions, syrups, or suspensions. Liquid preparations may be prepared by conventional means with pharma- ceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methylcellulose, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxybenzoates or sorbic acid).

[0143] The present disclosure also includes multi-layer tablets that include a layer that provides delayed release of one or more compounds of the present disclosure and an additional layer that provides immediate release of another drug. The use of wax / pH-sensitive polymer mixtures may result in gastric insoluble compositions in which the active ingredient is entrapped and ensures its delayed release.

[0144] Parenteral Administration For parenteral administration, the compounds of the present disclosure may be formulated for injection or infusion, e.g., intravenous, intramuscular, or subcutaneous, and may be formulated for administration in a bolus dose and / or continuous infusion. Suspensions, solutions, or emulsions in oily or aqueous vehicles, optionally containing other formulatory agents such as suspending, stabilizing, and / or dispersing agents, may be used.

[0145] Further dosage forms Further dosage forms of the present disclosure include those described in U.S. Patent Nos. 6,340,475; 6,488,962; 6,451,808; 5,972,389; 5,582,837; and 5,007,790. Further dosage forms of the present disclosure also include those described in U.S. Patent Application Nos. 20030147952; 20030104062; 20030104053; 20030044466; 20030039688; and 20020051820. Further dosage forms of the present disclosure include those described in PCT application numbers WO03 / 35041; WO03 / 35040; WO03 / 35029; WO03 / 35177; WO03 / 35039; WO02 / 96404; WO02 / 32416; WO01 / 97783; WO01 / 56544; WO01 / 32217; WO98 / 55107; WO98 / 11879; WO97 / 47285; WO93 / 18755; and WO90 / 11757.

[0146] Sustained Release Formulations and Drug Delivery Systems In certain aspects, the formulations of the present disclosure may be, but are not limited to, short-acting formulations, rapid-offset formulations, and sustained release formulations, including sustained release formulations, delayed release formulations, and pulsatile release formulations.

[0147] The term sustained release is used in the conventional sense to refer to a drug formulation that gradually releases drug over an extended period of time, which may, but does not necessarily, result in a substantially constant blood concentration of drug over an extended period of time, which may be as long as a month or more and should result in a longer release than an equivalent amount of drug administered in bolus form.

[0148] For sustained release, the compound may be formulated with a suitable polymeric or hydrophobic material that imparts sustained release properties to the compound. Thus, the compound for use in the disclosed method may be administered in the form of microparticles, for example, by injection, or may be administered by implantation in the form of a cachet or disk.

[0149] In certain aspects of the present disclosure, the compounds of the present disclosure are administered to a patient using sustained release formulations, either alone or in combination with another pharmaceutical agent.

[0150] The term delayed release is used herein in its conventional sense to refer to a drug formulation that first releases the drug after some delay after the drug is administered, which may, but is not necessarily, include a delay from about 10 minutes to about 12 hours.

[0151] The term pulsatile release is used herein in the conventional sense to refer to a drug formulation that releases drug in a manner that results in a pulsatile plasma profile of the drug after the drug is administered.

[0152] The term immediate release is used in the conventional sense to refer to a drug formulation that releases the drug immediately after it is administered.

[0153] Short term, as used herein, refers to any period of time after the drug is administered, up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any or all full or partial increments thereof.

[0154] As used herein, rapid off-action refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all full or partial increments thereof, after the drug is administered.

[0155] dosage The therapeutically effective amount or dose of the compounds of the present disclosure will depend on the age, sex, weight of the patient, the current medical condition of the patient, and the progression of the neurodegenerative condition in the patient being treated. Those skilled in the art will be able to determine the appropriate dosage depending on these and other factors.

[0156] A suitable dose of the compound of the present disclosure may range from about 0.01 mg to about 5,000 mg per day, for example, from about 0.1 mg to about 1,000 mg, for example, from about 1 mg to about 500 mg, for example, from about 5 mg to about 250 mg per day. The dose may be administered in a single dose or in multiple doses, for example, 1 to 4 or more times per day. When multiple doses are used, the amount of each dose may be the same or different. For example, a 1 mg dose per day may be administered as two 0.5 mg doses with about 12 hours between doses.

[0157] It is understood that the amount of compound administered daily may be, in non-limiting examples, administered every day, every other day, every second day, every third day, every fourth day, or every fifth day. For example, when administered every other day, a dose of 5 mg per day may be administered starting on Monday, a first dose of 5 mg may be administered on Wednesday the following day, and a second dose of 5 mg may be administered on Friday the following day.

[0158] If the subject's condition improves, optionally, the modulators of the present disclosure are administered continuously at the discretion of the physician. Alternatively, the dose of the administered drug is temporarily reduced or temporarily suspended for a certain period of time (i.e., a "drug holiday"). Optionally, the length of the drug holiday is from 2 days to 1 year, including, by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. Dose reductions during drug holidays include, by way of example only, 10% to 100%, including 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%.

[0159] Once improvement of the patient's condition has occurred, a maintenance dose is administered as needed. Thereafter, the dosage or frequency of administration, or both, is reduced as a function of the patient's condition until a level at which the improved disease persists. In certain embodiments, patients require intermittent treatment on a long-term basis upon recurrence of symptoms and / or infection.

[0160] The compounds for use in the disclosed methods may be formulated in unit dosage form. The term "unit dosage form" refers to a physically discrete unit suitable as a unitary dosage to a patient undergoing treatment. Each unit contains a predetermined amount of active material calculated to produce a desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier. The unit dosage form may be a unit dosage form for a single daily dose, or a unit dosage form for multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.

[0161] The toxicity and therapeutic efficacy of such a treatment regimen may optionally be evaluated using LD 50 (the dose that causes death in 50% of the population) and ED 50These methods can be used to determine the therapeutic index, including, but not limited to, determining the dose that is therapeutically effective in 50% of a population (LD50). The dose ratio between toxic and therapeutic effects is the therapeutic index, and the LD 50 and ED 50 The therapeutic index is expressed as a ratio of ED to ED. Capsid assembly modulators that exhibit large therapeutic indices are preferred. Data obtained from cell culture assays and animal studies are optionally used in formulating a range of dosages for human use. The dosage of such capsid assembly modulators is preferably within the range of ED with minimal toxicity. 50 Optionally, the dosage varies within this range depending upon the dosage form employed and the route of administration utilized.

[0162] Those skilled in the art will recognize, or can recognize using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents are considered to be within the scope of this disclosure and are covered by the appended claims. For example, variations in assay and / or reaction conditions according to art-recognized alternatives and using no more than routine experimentation should be understood to be within the scope of this application.

[0163] Whenever values ​​and ranges are provided herein, it should be understood that all values ​​and ranges encompassed by these values ​​and ranges are intended to be included within the scope of the disclosure. Moreover, all values ​​that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by this application. EXAMPLES

[0164] The present specification will be further described by referring to the following experimental examples.These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified.Therefore, the present specification should not be interpreted as being limited to the following examples, but rather as embracing any and all variations that become apparent as a result of the disclosure provided herein.

[0165] Example 1: Related information and summary The progressive transformation of muscles, tendons, ligaments, aponeuroses, and fascia into extraosseous bone tissue is a cardinal feature of fibrodysplasia ossificans progressiva (FOP; MIM#135100), the most devastating form of extraosseous bone formation in humans. At birth, individuals with FOP appear normal except for a characteristic deformity of the big toe, which is present in all classically affected individuals. During the first decade of life, they experience sudden soft tissue swellings (or flare-ups) in the neck and back that undergo pathological degeneration into mature heterotopic bone through the endochondral pathway. Minor trauma such as intramuscular vaccination, mandibular blocks for dental work, muscle overexertion, blunt muscle trauma, impacts, contusions, falls, or viral illnesses such as influenza can trigger FOP flare-ups, which can lead to progressive heterotopic ossification (HO).

[0166] Most patients become immobile by age 30 and require lifelong assistance with activities of daily living. Median life expectancy is 56 years. Death frequently results from complications of thoracic insufficiency syndrome. No cure for FOP is yet available, and there is an unmet need for effective therapies. Standard of care, medical management, is currently supportive.

[0167] Heterozygous missense mutations in the bone morphogenetic protein (BMP) type I receptor, activin receptor type AI / activin-like kinase 2 (ACVR1 / ALK2), have been identified in all individuals with sporadic or familial FOP. ACVR1 mutations abolish ACVR1 autoinhibition, rendering it susceptible to dysregulated BMP pathway signaling. Importantly, activin A, a member of the transforming growth factor-β (TGF-β) family of molecules that antagonize BMP signaling in a wild-type (WT) ACVR1 background, is downregulated by ACVR1. R206H BMP pathway signaling is specifically enhanced within cells carrying the mutation, driving ectopic bone formation in FOP.

[0168] Although loss of mutant receptor autoinhibition is necessary for the myriad developmental features of FOP, it does not appear to be sufficient to induce the sudden flare-ups that lead to disabling postnatal HO.FOP flare-ups have a strong influence on the underlying inflammatory triggers.

[0169] This study identified Patient-R, a 22-year-old with classical developmental features of FOP and a canonical ACVR1 (R206H) mutation, but extreme resilience to FOP postnatal features, including the absence of disabling flare-ups and severe progressive HO. Patient-R is now 35 years old. The presence of classical developmental abnormalities and the contradictory paucity of FOP postnatal features led us to hypothesize that Patient-R lacks an inflammatory trigger for flare-up initiation and subsequent heterotopic ossification. Biomarker analysis and genetic studies in Patient-R revealed low basal activity of matrix metalloproteinase-9 (MMP-9) and compound heterozygosity for MMP-9. These observations prompted studies in FOP mice, which revealed that even partial inhibition of MMP-9 activity by genetic, pharmacological, and biological means potently inhibited HO, revealing unexpected molecular targets in FOP and, potentially, the more common form of HO.

[0170] Example 2: Case report Patient-R, a 22-year-old asymptomatic individual, was admitted to the FOP clinic. He was the product of a full-term pregnancy and cesarean section delivery. Short, malformed toes and a thumb with absent interphalangeal joints were noted at birth. He had received routine childhood vaccinations without any flare-ups. At age 3 years, he was bitten by a Loxosceles spider in his left thigh. There was no dermonecrosis, but he developed a 1-centimeter bony nodule in his left quadriceps that did not affect movement. He enjoyed an active childhood playing contact sports and experienced no flare-ups or loss of movement. There was no family history of skeletal anomalies or HO.

[0171] At age 21 years, he developed a firm nodule on the left side of his neck after multiple episodes of vomiting secondary to suspected viral gastroenteritis. Excisional biopsy of the nodule revealed a fibroproliferative lesion. A 2-centimeter nonrestrictive ribbon-like HO developed at the surgical site. Skeletal examination revealed classic developmental features of FOP, including malformed toes and thumbs, ankylosis of several facet joints in the subaxial cervical vertebrae, short and wide femoral necks, hip dysplasia, proximal medial tibial osteochondroma, and a small amount of asymptomatic HO in the lower lumbar spine (Figure 2A). Genetic testing confirmed the classic FOP mutation (ACVR1 c.617G>A; R206H). There was no evidence of chimerism.

[0172] Physical examination revealed a healthy 22-year-old male with absent interphalangeal joints of the thumb and big toe, bilateral proximal medial tibial osteochondromas, and normal range of motion of all other joints of the axial and appendicular skeleton, except for slightly reduced motion of the cervical spine and hip joints. His Cumulative Analogue Joint Involvement Scale (CAJIS) score, a validated tool to assess FOP disease burden, was 2 / 30 (age-related range: 12–22; median: 18). Patient-R participated in a funded natural history study of classically affected FOP patients. In this natural history study, a baseline whole-body computed tomography (WBCT) was performed to assess the whole-body burden of HO. Patient-R's WBCT volume of HO was 47,000 mm 3 (average of 13 subjects aged 25-35: 434,000 mm 3 ).

[0173] Patient R had the mildest form of classic FOP reported to date in a patient his age, with one mild post-traumatic flare-up of FOP and approximately 90 percent less heterotopic bone compared to age-matched controls. The presence of classic developmental features of FOP (Figure 2A) suggested that the mutant receptor was active during embryogenesis. However, the fewer postnatal inflammatory flare-ups of FOP and subsequent HO, and the exceptional preservation of mobility in FOP adults, suggested that he may lack inflammatory triggers for flare-ups and HO.

[0174] Efforts were made to identify factors that contribute to the resilience to postnatal flare-ups and HO in this unique, now 35-year-old FOP individual (Patient-R), and to ascertain whether this factor was causally associated with HO in an accurate mouse model of FOP. If such a factor were identified, it may uncover previously unknown protective mechanisms in FOP and herald new therapeutic strategies to prevent and treat the relentless progression of this disabling condition.

[0175] Example 3: Methods and Materials Patient Study Approval Samples from patient-R and unaffected controls were obtained with informed consent. The study protocol was approved by the Investigational Review Board of the Perelman School of Medicine at the University of Pennsylvania. All human biopsies were obtained before FOP diagnosis, as tissue trauma in FOP frequently induces episodes of heterotopic ossification.

[0176] Animal Testing Approval All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania.

[0177] Venous blood collection Venous blood samples were collected from FOP patients and their families as part of routine clinic visits at the Department of Orthopaedic Surgery at the Perelman School of Medicine of the University of Pennsylvania. Blood was collected in 10 ml K2 EDTA tubes (BD Catalog No. 366643) for adults and children over 2 years of age, or in 4 ml K2 EDTA tubes (BD Catalog No. 367861) for children under 2 years of age. Samples were kept at room temperature until processing 2-24 hours later.

[0178] Peripheral blood mononuclear cells (PBMCs) were obtained as follows: the buffy coat was diluted 1:2 with phosphate buffered saline (PBS), gently layered on top of an equal volume of Ficoll-Paque, and centrifuged at 400g for 30 minutes to separate the PBMCs from the red blood cells. The mononuclear cell layer was carefully collected and rinsed twice with PBS. The PBMCs were stored at -80°C.

[0179] Preparation of venous blood samples Plasma was separated by one of two methods depending on whether PBMCs were isolated from the sample for other reasons. Undiluted blood was layered on top of Ficoll-Paque (GE Healthcare catalogue no. 17-1440-02) in one or two 15 ml conical tubes and centrifuged at 800 x g for 20 min at room temperature without the brake. Plasma was collected from the top layer (leaving PBMCs, red blood cells and Ficoll behind) and then transferred to a new 15 ml tube. Plasma was centrifuged at 1400 x g for 10 min with the brake to remove remaining cells and the supernatant was aliquoted into a 1.5 ml cryovial. Samples were stored below -80°C. Alternatively, undiluted blood was centrifuged in the original collection tube at 1400 x g for 20 min at room temperature. Plasma was transferred to a new 15 ml tube and centrifuged at 1400 x g for 10 min to remove remaining cells. The resulting supernatant was aliquoted into 1.5 ml cryovials and stored below -80°C.

[0180] One cryovial for each selected sample was thawed on ice, the tube was mixed briefly by tapping with a finger, and then 500 μl aliquots were distributed into pre-labeled and refrigerated 1.5 ml screw-top microfuge tubes. These were flash frozen in a dry ice / ethanol bath and then packed into a freezer box. They were stored at -80°C and shipped overnight on dry ice to Myriad Rules Based Medicine (RBM; Salt Lake City, UT) for multiplex analysis.

[0181] Multiplexed Luminex Analysis Multiplex Luminex analysis was performed with Myriad RBM (Pignolo et al., J Bone Miner Res., 2021). All samples were stored below -70°C until testing. Samples were thawed at room temperature, vortexed, spun at 3700xg for 5 min for clarification, and transferred to a master microtiter plate. Using automated pipetting, an aliquot of each sample was added to individual microsphere multiplexes of the selected Multi Analyte Profile and blocker. This mixture was mixed thoroughly and incubated at room temperature for 1 h. A multiplexed cocktail of biotinylated reporter antibodies was added by the robot and thoroughly mixed before being incubated for an additional hour at room temperature. The multiplexes were labeled with an excess of streptavidin-phycoerythrin solution, mixed thoroughly, and incubated at room temperature for 1 hour. The volume of each multiplex reaction was reduced by vacuum filtration and washed three times. After the final wash, buffer was added to increase the volume for analysis using the Luminex instrument, and the resulting data was interpreted using proprietary software developed by Myriad RBM.

[0182] Calibrators and controls were included on each microtiter plate for each multiplex. Eight-point calibrators to generate a standard curve were run in the first and last columns of each plate, and controls at three concentration levels were run in duplicate. Standard curves, controls, and sample QC were performed to ensure proper assay performance. Study sample values ​​for each analyte were determined using four- and five-parameter logistics and weighted and unweighted curve-fitting algorithms included in the data analysis package.

[0183] cell culture Thaw PBMCs, suspend them in AIM-V medium, and plate 2.5 x 10 cells in a 24-well plate. 6 Cells were plated at a density of 1000 cells / ml and incubated at 37°C in a 5% CO2 atmosphere. PBMCs were incubated with 20ng / ml TNF-α for 72 hours. Cells were lysed with RIPA Lysis and Extraction buffer (ThermoFisher Scientific, Grand Island, NY, USA; # 89901) containing 1x proteinase inhibitor cocktail (ThermoFisher Scientific, Grand Island, NY, USA; #78429) and collected for analysis as cell culture supernatants.

[0184] Clinical assessment of flare-up status At the time of sample collection, 40 patient samples and, whenever possible, age- and sex-matched controls were stratified based on flare-up status (Pignolo et al., J Bone Miner Res, 2021). The study measured levels of 113 analytes in plasma samples from four subject groups: unaffected individuals and individuals with FOP by flare-up status (active, remote, and quiescent). Flare-up status was arbitrarily defined at the time of sample collection by the time from the appearance of clinically confirmed symptoms and signs consistent with the last flare-up. Flare-up status was defined as follows: (1) active flare-up is a current / ongoing clinical flare-up at the time of sample collection; (2) remote when a flare-up has occurred within 1–2 years of sample collection; and (3) quiescent status occurs when the last flare-up has occurred more than 2 years prior to sample collection.

[0185] Protein-Protein Interaction (PPI) Mapping PPI mapping was performed using IBM-Watson for Drug Discovery (WDD) and Ingenuity software, which was used to generate association networks from biomarker predictions. Based on previously described methods for analyzing pathway interactions between differentially expressed proteins, biological association network extraction was applied to proteins significantly associated with FOP genotype and flare-up status. The confidence level was set to >95%. The captured links were supported by at least two published documents.

[0186] FOP mice (cardiotoxins - skeletal muscle damage caused by minocycline and doxycycline treatment) Conditional-on knock-in mouse model Acvr1 [R206H] FlEx (Acvr1 R206H / + ) was used to generate tamoxifen-inducible global R206H mutant allele expression following recombination with Cre recombinase (Hatsell et al., Sci Transl Med. 2015 Sep 2;7(303):303ra137). R206H / + and MMP-9 - / - (The Jackson Laboratory, Bar Harbor, ME, USA; Stock #007084) mice were crossed with CreERT2 mice (The Jackson Laboratory, Bar Harbor, ME, USA; Stock #008463) to express Acvr1 R206H / + ;CreERT2 - / + , Acvr1 R206H / + ;CreERT2 - / + MMP-9 - / + and Acvr1 R206H / + ;CreERT2 - / + MMP-9 - / - Mice were generated.

[0187] To induce reversal of the R206H mutant allele, mice were intraperitoneally injected five times over a 2-week period (starting at 4 weeks of age) with tamoxifen (100 mg / kg body weight, 10 mg / ml stock solution, Sigma, St. Louis, MO, USA; #T5648) dissolved in corn oil.

[0188] The quadriceps muscle (7 weeks old) was injured by injecting 50 μL of 10 μM Naja mossambica cardiotoxin (Sigma-Aldrich, St. Louis, MO, USA; #C9759).

[0189] Mice were treated intraperitoneally daily with minocycline 100 mg / kg (Cayman Chemical, Ann Arbor, Michigan, USA; #14454) dissolved in pharmaceutical grade saline or appropriate controls for 3 days before and 14 days after induction of HO.

[0190] Acvr1 Q207D / + Mice were a gift from Dr. Yuji Mishina (University of Michigan). To induce HO following Q207D expression, AV-Cre (University of Pennsylvania Vector Core; 1 × 10 per mouse) was used. 11 1000 particles) together with 50 µL of 10 µM cardiotoxin in 3-4 week old Acvr1 mice. Q207D / + Mice were injected into the popliteal fossa.

[0191] The experimental end point of the in vivo study to analyze the appearance of heterotopic ossification was set at 2 weeks, based on the fact that by this time, control mice had developed HO after muscle injury caused by the cardiotoxin.

[0192] Methods and Materials Related to MMP-9 Monoclonal Antibodies Mice were treated with anti-MMP-9 antibody (GS-622703, Gilead Sciences, Foster City, California, USA) at 50 mg / kg on day −3, then 15 mg / kg on days 0 (induction of HO) and 3, or with appropriate controls.

[0193] Quantification of MMP9 Total MMP9 protein (92 kDa pro-form and 82 kDa active form) was confirmed by sandwich ELISA in heparinized platelet-poor plasma (Human MMP-9 Quantikine ELISA Kit, DMP900 and Mouse Total MMP-9 Quantikine ELISA Kit, MMPT90, R&D Systems, Minneapolis, MN, USA).

[0194] Zymography Gelatin zymography was performed using equal amounts (10ug) of cell supernatant and cells after lysis to measure MMP9 using Novex Zymogram Plus Gels according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA, ZY00100BOX).

[0195] The gels were scanned, the images were inverted, and densitometric levels were determined using ImageJ software (imagej dot nih dot gov / ij / ). Profiles were generated that yielded peaks corresponding to each band (pro-MMP-9 and active MMP-9).

[0196] Immune cells / histochemistry Tissue samples were fixed in 4% paraformaldehyde for 24 h, decalcified with 10% EDTA for 7 days, and either snap-frozen or paraffin-embedded and serially sectioned at 8 μm. For antigen retrieval, deparaffinized and frozen sections were treated with 10 mM sodium citrate buffer (pH 6.0) at 95°C for 20 min.

[0197] For immunofluorescence staining, sections were blocked with Background Buster (Innovex Biosciences, Richmond, CA, USA; NB306), incubated with primary antibodies overnight at 4°C, and then incubated with appropriate secondary antibodies conjugated with Alexa 594 and Dapi mounting medium. Images were acquired with a Nikon microscope and NIS software.

[0198] For immunohistochemistry, endogenous peroxidase activity was quenched: sections were blocked and incubated with primary antibodies overnight at 4°C, followed by incubation with the appropriate host horseradish peroxidase (HRP) secondary antibody, DAB detection (SuperPicture Polymer 879263; Thermo Fisher Scientific), and hematoxylin / eosin counterstain.

[0199] Results were compared to negative controls that were not treated with primary antibodies. Primary antibodies used were: MMP-9 (Millipore, Sigma, Burlington, MA, USA; AB19016; 1:100 dilution).

[0200] CRISPR gene editing was used to insert 59C>T and 493G>A in MMP-9 in the THP-1 human leukemia monocytic cell line following the protocol from ThermoFisher Scientific using TrueCut HiFi Cas9 protein.

[0201] Micro-computed tomography To determine the volume of ectopic bone and obtain two-dimensional images in the medial sagittal plane of each limb, microcomputed tomography (μCT) was performed on hind limbs from mice obtained 14 days after injection with cardiotoxin or adenovirus-Cre / cardiotoxin using a Scanco VivaCT 40 instrument (Bruettisellen, Switzerland). Scanning was performed using a source voltage of 55 kV, a source current of 142 μA, and an isotropic voxel size of 10.5 μm. Bone was differentiated from "non-bone" by an upper threshold of 1000 Hounsfield units and a lower threshold of 150 Hounsfield units.

[0202] Structural Modeling Using sequence-based prediction algorithms, molecular modeling, and molecular dynamics simulations, the potential of the Ala20Val and Asp165Asn variants to alter the structure and activity of MMP9 was examined.

[0203] statistical analysis All experiments were performed with at least three technical and biological replicates. Sample sizes were indicated in the figure legends. GraphPad Prism 8.0 software (San Diego, CA, USA) was used for statistical analysis. Normality of data was examined using the D'Agostino & Pearson normality test. Parametric data were analyzed using the appropriate Student's t test when comparing two groups, or one-way (or two-way) analysis of variance when comparing more than two groups (or at least two independent factors), respectively, followed by post-hoc Tukey's test to compare two groups. Non-parametric data were analyzed using the Mann-Whitney U test when comparing two groups, or Kruskal-Wallis one-way analysis when comparing more than two groups. * P < 0.05, **P < 0.01, and *** P<0.001 was considered significant. Data are expressed as mean (+ / -) standard error of the mean (SEM).

[0204] Example 4: FOP Patient-R exhibited congenital features of FOP but lacked progressive heterotopic ossification after birth and had suppressed inflammatory biomarkers Patient-R was a 35-year-old male with the classic Acvr1R206H mutation. As shown in Figures 2A-2C, patient-R had classic developmental findings of FOP, including malformed toes and thumbs (Figures 2A-2B), ankylosis of several facet joints in the subaxial cervical spine, a short and wide femoral neck, hip dysplasia, proximal medial tibial osteochondroma, and a small amount of asymptomatic HO in the lower lumbar spine. The presence of the classic big toe malformation in FOP suggested that the mutant Acvr1 receptor in patient-R was active during early development.

[0205] However, patient-R demonstrated resilience to the postnatal features of FOP, e.g., the disabling flare-ups and severe progressive HO found in essentially all other FOP patients were absent in patient-R (Figure 2C).

[0206] To elucidate the mechanism behind the resilience to postnatal features of FOP in Patient-R, this study measured the levels of inflammatory biomarkers in the plasma of Patient-R, as well as in the plasma of other FOP patients and control subjects. 113 plasma proteins (Custom Human MAP panel), including chemokines and cytokines, in blood collected from FOP patients and control subjects were measured and analyzed by quantitative multiplexed immunoassay (Myriad RBM). The analysis showed that the postnatal inflammatory markers in Patient-R were reduced compared to other FOP patients. As shown in Table 1, the inflammatory biomarkers in Patient-R were significantly suppressed compared to quiescent FOP patients, as the statistical probability (calculated by p-value) of observing reduced observations for these biomarkers was extremely small. Notably, the p-value of plasma MMP-9 levels in Patient-R was 6.8x10 -5 was calculated to be smaller than

[0207] (Table 1) Quiescent FOP * Plasma analytes of inflammation in patients with RR were suppressed compared to patients with TIFF2025513951000001.tif110128 Plasma analytes of inflammation in patient R (two independent samples taken more than 1 year apart) were *The following plasma analytes of inflammation were not significantly different between patients-RR and those with quiescent FOP: CRP=C-reactive protein; FRTN=ferritin; IgA=immunoglobulin A; IL-1ra=interleukin-1 receptor antagonist; IL-1R1=interleukin-1 receptor type 1; IL-18=interleukin-18; KLK-7=Kallekrein-7; MIP-1β=macrophage inflammatory protein-1β; MIF=macrophage inhibitory factor; MMP-2=matrix metalloproteinase-2; MMP-9=matrix metalloproteinase-9; TN-C=tenascin-C; TIMP-1=tissue inhibitor of metalloproteinase-1; TNFR=tumor necrosis factor-2. Adiponectin; Compliment-3; Interleukin-1 receptor type 2; Matrix metalloproteinase-3; Prostasin; T cell specific RANTES; Thymus expressed chemokine; Tissue inhibitor of metalloproteinases-3. The following plasma analytes of inflammation were too low to be detected in all samples (Patient-R and quiescent groups): B lymphocyte chemoattractant; Granulocyte-macrophage colony stimulating factor; Interferon gamma; Interleukin-1 alpha; Interleukin-1 beta; Interleukin-2, 3, 4, 5, 6, 7, 8, 10, 15, 17, 23; Macrophage inflammatory protein-1 alpha; Matrix metalloproteinases 1, 7, 10; Monocyte chemotactic protein-1, 3; Transforming growth factor beta-3; Tumor necrosis factor alpha; Tumor necrosis factor beta. * The quiescent FOP group was defined as those whose last flare-up occurred at least 2 years prior to plasma sample collection.

[0208] Example 5: Patient-R had low plasma MMP-9 levels and was a compound heterozygote for MMP-9 polymorphic variants As shown in Figures 3A-3C, the plasma MMP-9 level in Patient-R is low compared to other FOP patients. As shown in Figures 3A-3C, in this study, a commercial multiplex set of 113 plasma soluble analytes in Patient-R was compared with the classical ACVR1 R206H The same analytes were compared in 40 FOP patients with mutations and 40 age- and sex-matched controls. Total MMP-9 in patient R was higher than that in the classical ACVR1 R206H The incidence was found to be significantly lower (p<0.0002) compared to other patients with the mutation and age- and sex-matched controls (Pignolo et al., J Bone Miner Res, 2021).

[0209] In patient R and 19 additional FOP patients with the classical phenotype, whole-exome sequencing was performed using DNA isolated from blood or saliva according to the UCSF Biospecimens and Skeletal Tissues for Rare and Orphan Genetics (BSTROnG) and University of Pennsylvania cohorts and sequenced as described in the Methods section. All 20 FOP patients were found to have ACVR1 by Ingenuity Variant Analysis (IVA, Qiagen). R206H The results confirmed that all subjects were heterozygous for ACVR1 and no additional ACVR1 mutations were identified in any of the subjects. For each FOP patient, approximately 80,000 total variants were identified compared to the human reference genome, UCSC Browser GrCh38.

[0210] Patient-R had several unique variants compared to 19 classic FOP patients. All unique variants were further classified according to pathogenicity using the American College of Medical Genetics (ACMG) Standards and Guidelines. Based on gene sequencing of MMP-9 from the parents of patient-R, this study identified two MMP-9 polymorphisms inherited from two different alleles (Figure 4A-4B). Specifically, one allele of the MMP-9 gene in patient-R harbors a polymorphism resulting in the A20V protein sequence change (population frequency of Rs1805088-A20V-T = 0.02201 [https: / / www.ncbi.nlm.nih.gov / snp / rs1805088#frequency_tab]). In contrast, the other allele of the MMP-9 gene in patient R harbors a polymorphism resulting in the D165N protein sequence change (population frequency of Rs8125581-D165N-A = 0.000360 [https: / / www.ncbi.nlm.nih.gov / snp / rs8125581#frequency_tab]).

[0211] Example 6: Protein structure modeling of the MMP-9 polymorphism in patient-R suggested lower functionality than the normal allele Our in silico studies suggest that the A20V variant of MMP-9 likely disrupts signal peptide cleavage, interferes with translation on the rough endoplasmic reticulum (ER), perturbs folding within the ER, or disrupts the tethering of MMP-9 to membranes of the secretory pathway.

[0212] The D165N polymorphism is located within the MMP-9 catalytic domain and was predicted by multiple computational approaches to be highly detrimental to MMP9 structure and function, resulting in a non-functional variant (Bhatnager et al., Computational Biology and Chemistry, Volume 77, December 2018, Pages 97-108). The D165N variant of MMP-9 corresponds to the metal-binding region of the MMP9 catalytic domain, and the stability of MMP9 depends on the interaction of the peptide with calcium ions (Bhatnager, 2018). Therefore, the D165N variant is likely to be ineffective in inhibiting enzyme activity, as it cannot form the necessary ion pair with His118 as in wild-type MMP-9. Importantly, the D165N variant of MMP-9 is predicted to be highly unstable, which would reduce its folding efficiency in the endoplasmic reticulum, a quality control site that regulates the transport of correctly folded secreted proteins. Because the ion pair stabilizes the loop only a few residues C-terminal to the MMP-9 cleavage and activation site, the loop in the mutant D165N MMP-9 protein may not adopt an optimal conformation for proteolysis by the activated enzyme plasminogen (Gong et al., J Clin Invest 118:3012-3024, 2008).

[0213] Whereas the D165N single nucleotide polymorphism (SNP) found in patient-R is rare and non-pathogenic in the general population, this variant was predicted to be protective in patients with aortic aneurysms. Furthermore, the specific enzymatic activity (total activity / amount of secreted protein) of the D165N MMP-9 variant was studied and found to be significantly lower than that of wild-type MMP-9, suggesting that this amino acid substitution has a significant effect on MMP-9 enzymatic activity in addition to reducing secreted protein. The MMP-9 variants (A20V and D165N) detected in patient-R provide a structural basis for the loss of function of secreted MMP-9, with implications for HO inhibition in FOP.

[0214] Example 7: MMP-9 is expressed in early disease tissues of subjects with FOP In certain embodiments, biopsies of early diseased tissue from classically affected FOP human patients (i.e., human FOP patients who have the Acvr1 R206H mutation and who exhibit postnatal symptoms of FOP) show positive staining for MMP-9 in inflammatory cells.

[0215] Similar results were found in FOP model mice. Figure 5 shows that FOP model mice (Acvr1 R206H / + ;CreERT2 - / + ) images of lesion tissue show early expression of MMP-9 in inflammatory cells in the lesions. Furthermore, MMP-9 expression in lesion tissue gradually decreases from day 1 to day 5.

[0216] Example 8: Downregulation of MMP-9 levels or activity reduces heterotopic bone formation in a mouse FOP model As shown in Figures 6A-6C, Acvr1 R206H / + ;CreERT2 - / + Mice (FOP model mice with the classical R206H mutation in one Acvr1 allele), Acvr1 R206H / + ;CreERT2 - / + MMP-9 - / + Mice (FOP model mice with only one allele of the MMP-9 gene) or Acvr1 R206H / + ;CreERT2 - / + MMP-9 - / - Soft tissue injury was induced by cardiotoxin in mice (FOP model mice with both alleles of the MMP-9 gene deleted), and the level of heterotopic ossification (HO) in response to injury was detected and quantified by micro-CT.

[0217] As shown in Figure 6C, MMP9 - / + ;Acvr1 R206H / + ;CreERT2 - / + and MMP9 - / - ;Acvr1 R206H / + ;CreERT2 - / +Mice are Acvr1 R206H / + ;CreERT2 - / + They produce significantly less MMP-9 than control mice.

[0218] As visualized in FIG. 6A and quantified in FIG. 6B, Acvr1 R206H / + ;CreERT2 - / + Mice showed high levels of HO, whereas Acvr1 R206H / + ;CreERT2 - / + MMP-9 - / - Mice showed significantly lower levels of HO. Interestingly, Acvr1 R206H / + ;CreERT2 - / + MMP-9 - / + Mice are Acvr1 R206H / + ;CreERT2 - / + mice had significantly lower levels of HO and Acvr1 R206H / + ;CreERT2 - / + MMP-9 - / - The mice exhibited levels of HO comparable to those observed in control mice, indicating that partial reduction in MMP-9 expression levels and activity was sufficient to reduce injury-induced HO in response to soft tissue injury in the FOP model mice.

[0219] Small molecule compounds that inhibit MMP-9 activity are available, so in this study we tested the effects of one such MMP-9 inhibitor, minocycline, in our FOP mouse model.

[0220] Minocycline is a broad-spectrum tetracycline antibiotic used to treat urinary tract infections, respiratory infections, skin infections, severe acne, gonorrhea, Rocky Mountain spotted fever, chlamydia infections, and more.

[0221] Looking at Figures 7D-7F, Acvr1 R206H / + ;CreERT2 - / + FOP mouse model and another mouse FOP mouse model, Acvr1 Q207D / +(Fukuda et al, Genesis. 2006 Apr;44(4):159-67) was used to study the effect of minocycline on heterotopic ossification caused by cardiotoxin-induced soft tissue injury.

[0222] As shown in Figures 6D-6F, minocycline inhibited Acvr1 R206H / + ;CreERT2 - / + Minocycline almost completely abolished soft tissue injury-induced HO in FOP mice. Q207D / + It also significantly reduced the levels of soft tissue injury-induced HO in FOP mice.

[0223] See Figure 6I, when minocycline is administered before or at the time of lesion activation, the compound prevents HO. The data indicate that MMP-9 acts to induce HO during the early inflammatory phase.

[0224] Next, this study attempted to downregulate MMP-9 using anti-MMP-9 monoclonal antibody (Marshall et al. PLoS One. 2015 May 11;10(5):e0127063). As shown in Figure 6K, Acvr1 R206H / + ;CreERT2 - / + In FOP mice, antibody administration significantly reduced heterotopic bone formation in the FOP mouse model.

[0225] Example 9: Selected Observations This study indicates that MMP-9 plays a key role in the pathogenesis of HO in FOP. In patient-R, the presence of classic developmental findings of FOP (osteochondroma in the proximal tibia, congenital cervical fusion, hallux valgus deformity, and shortened thumb) and very few postnatal flare-ups and HO suggested that FOP was active during embryogenesis but relatively inactive postnatally, possibly due to the absence of inflammatory triggers of HO. The findings of significantly reduced MMP-9 storage and activity in patient-R and the putative protective polymorphism in the MMP-9 gene drew attention to factors that were further validated as contributing to the HO-inhibited phenotype by in vivo studies in FOP mice. Collectively, these results support that MMP-9 mediates HO by regulating the inflammatory response to tissue degeneration.

[0226] MMP-9 (or gelatinase B) is conserved throughout the animal kingdom and belongs to a multigene family of over 20 matrix metalloproteinases that process or degrade numerous pericellular substrates. MMP-9 plays an essential and multifaceted role as an inflammatory modulator in remodeling the extracellular matrix; activating, deactivating, and / or modifying proinflammatory cytokines and other signaling molecules; and orchestrating stem and tissue progenitor cell migration in a wide range of physiological and pathological processes, including embryonic development, skeletal morphogenesis, fracture repair, inflammation, wound healing, angiogenesis, heart disease, arthritis, and cancer. Specifically, MMP-9 induces the degradation and remodeling of the extracellular matrix, the release and activation of growth factors, the remodeling of the stem cell niche, and the recruitment and migration of inflammatory and stem cells.

[0227] MMP-9 is expressed in neutrophils, macrophages, and mast cells, is regulated by inflammatory cytokines, and is a modulator of inflammation and innate immunity, integrating multiple immunoregulatory pathways and promoting the breakdown of biochemical and physical barriers to T-cell trafficking. MMP-9 is also expressed at high levels in ischemic skeletal muscle present in early FOP flare-ups.

[0228] MMP-9 is also a signal inducer of endochondral ossification. It regulates the differentiation of chondrogenic and osteogenic cells during the early stages of fracture repair and is expressed throughout the entire process of fracture repair. - / - Mice exhibit abnormal fracture healing and nonunion.

[0229] The role of MMP-9, if any, in the pathogenesis of FOP has not been investigated. Our clinical, biochemical, and genetic findings in patient R suggest that even partial reduction in MMP-9 levels may be sufficient to prevent HO in FOP mice, as haploinsufficiency of MMP-9 prevents HO in FOP mice. R206H The presence of the mutation appears to offer protection against disabling flare-ups and HO.

[0230] In vivo studies in a genetically precise mouse model of trauma-induced FOP support this hypothesis, demonstrating that MMP-9 plays a central role in the development of HO and that genetic, pharmacological, and biological reduction of MMP-9 levels prevents the induction of HO in FOP mouse models. Collectively, these data indicate that HO can be prevented by pharmacological reduction of tissue levels of MMP-9 in FOP patients.

[0231] This study describes the tetracycline class of drugs. Tetracyclines have been used therapeutically as antibiotics against Gram-positive and Gram-negative bacteria for almost 40 years. However, in addition to their antibiotic properties, the second-generation semisynthetic tetracyclines minocycline and doxycycline are potent MMP-9 inhibitors at subantibacterial doses.

[0232] In summary, this study identifies MMP-9 as a key modulator of HO in FOP and demonstrates that the systematic study of a single resilient individual can reveal unexpected disease mechanisms that may lead to novel therapeutic strategies.

[0233] Enumerated aspects: In some aspects, the invention relates to the following non-limiting embodiments. Embodiment 1 : A method of treating, ameliorating, and / or preventing fibrodysplasia ossificans progressiva (FOP) in a subject in need thereof, comprising downregulating the level and / or activity of matrix metalloproteinase 9 (MMP-9) in said subject. Embodiment 2: The step of downregulating MMP-9 level and / or activity in the subject comprises administering to the subject an effective amount of Small molecule MMP-9 inhibitors, Protein MMP-9 inhibitors, A nucleic acid that downregulates MMP-9 by RNA interference (and / or an expression vector that expresses said nucleic acid), A ribozyme that downregulates MMP-9 (and / or a vector expressing said ribozyme), An expression vector comprising an expression cassette expressing a CRISPR component that downregulates MMP-9 by CRISPR knockout and / or CRISPR knockdown; and Transdominant-negative mutant protein of MMP-9 and / or expression vector expressing said transdominant-negative mutant protein of MMP-9 The method of embodiment 1, comprising administering to said subject Embodiment 3: The method of any one of embodiments 1 to 2, wherein the subject has a mutant ACVR1 gene. Embodiment 4: The method of embodiment 3, wherein the mutant ACVR1 gene encodes a constitutively active ACVR1 polypeptide. Embodiment 5: The method of embodiment 4, wherein said ACVR1 polypeptide comprises at least one mutation selected from the group consisting of L196P, P197-F198 del ins L, R202I, R206H, Q207E, F246Y, R258S, R258G, G325A, G328E, G328W, G328R, G356D, R375P, and K400E. Embodiment 6: The method of any one of embodiments 2-5, wherein said small molecule MMP-9 inhibitor is selected from the group consisting of doxycycline, incyclinide, and minocycline, or a salt or solvate thereof. Embodiment 7: The method of any one of embodiments 2 to 5, wherein said proteinaceous MMP-9 inhibitor is an anti-MMP-9 antibody or an antigen-binding fragment thereof. Embodiment 8: The method of any one of embodiments 1 to 7, further comprising the step of surgically removing ossified tissue from said subject. Embodiment 9: The method of embodiment 8, wherein the surgical removal step is performed after the level or activity of MMP-9 in said subject is downregulated. Embodiment 10: The method of any one of embodiments 1 to 9, wherein the subject is a human. Embodiment 11: A kit for treating, ameliorating, and / or preventing fibrodysplasia ossificans progressiva (FOP) in a subject in need thereof, the method comprising downregulating a level and / or activity of matrix metalloproteinase 9 (MMP-9) in the subject, the kit comprising: a compound for downregulating the level and / or activity of matrix metalloproteinase 9 (MMP-9) in said subject; instructions for administering to said subject an effective amount of said compound; The kit comprising: Aspect 12: The compound is Small molecule MMP-9 inhibitors, Protein MMP-9 inhibitors, A nucleic acid that downregulates MMP-9 by RNA interference (and / or an expression vector that expresses said nucleic acid), A ribozyme that downregulates MMP-9 (and / or a vector expressing said ribozyme), An expression vector comprising an expression cassette expressing a CRISPR component that downregulates MMP-9 by CRISPR knockout and / or CRISPR knockdown; and Transdominant-negative mutant protein of MMP-9 and / or expression vector expressing said transdominant-negative mutant protein of MMP-9 The kit of embodiment 11, comprising at least one selected from the group consisting of: Embodiment 13: The kit of any one of embodiments 11 to 12, wherein the subject has a mutant ACVR1 gene. Embodiment 14: The kit of embodiment 13, wherein the mutant ACVR1 gene encodes a constitutively active ACVR1 polypeptide. Embodiment 15: The kit of embodiment 14, wherein said ACVR1 polypeptide comprises at least one mutation selected from the group consisting of L196P, P197-F198 del ins L, R202I, R206H, Q207E, F246Y, R258S, R258G, G325A, G328E, G328W, G328R, G356D, R375P, and K400E. Embodiment 16: The kit of any one of embodiments 11 to 15, wherein said small molecule MMP-9 inhibitor is selected from the group consisting of doxycycline, incyclinide, and minocycline. Embodiment 17: The kit according to any one of embodiments 11 to 15, wherein said proteinaceous MMP-9 inhibitor is an antibody to MMP-9 or an antigen-binding fragment thereof. Embodiment 18: The kit of any one of embodiments 11 to 17, wherein the manual further comprises instructions for surgically removing ossified tissue from said subject. Embodiment 19: The kit of embodiment 18, wherein said instructions further comprise instructions for performing surgical removal after said level or said activity of MMP-9 in said subject is downregulated. Embodiment 20: The kit according to any one of embodiments 11 to 19, wherein the subject is a human.

[0234] The above outlines the features of some embodiments so that those skilled in the art may better understand the aspects of the present disclosure.Those skilled in the art should recognize that they can easily use this disclosure as a basis for designing or improving other processes and structures to achieve the same purpose and / or realize the same advantages of the embodiments introduced herein.Those skilled in the art should also understand that such equivalent structures do not depart from the spirit and scope of the present disclosure, and that various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the present disclosure.

Claims

1. A composition for treating, relieving, and / or preventing fibrodysplasia ossificans progressive (FOP) in a subject where such treatment is necessary, wherein the composition comprises a compound for downregulating the level and / or activity of matrix metalloproteinase 9 (MMP-9) in the subject.

2. The compound is Small molecule MMP-9 inhibitors, Protein MMP-9 inhibitors, A nucleic acid (and / or an expression vector expressing said nucleic acid) that downregulates MMP-9 by RNA interference, A ribozyme that downregulates MMP-9 (and / or a vector expressing said ribozyme), An expression vector comprising an expression cassette expressing a CRISPR component that downregulates MMP-9 by CRISPR knockout and / or CRISPR knockdown, and / or A transdominant-negative mutant protein of MMP-9, and / or an expression vector expressing the transdominant-negative mutant protein of MMP-9. The composition according to claim 1, comprising:

3. The composition according to any one of claims 1 to 2, wherein the subject has a mutant ACVR1 gene.

4. The composition according to claim 3, wherein the mutant ACVR1 gene encodes a constitutively active ACVR1 polypeptide.

5. The composition according to claim 4, wherein the ACVR1 polypeptide comprises at least one mutation selected from the group consisting of L196P, P197-F198 del ins L, R202I, R206H, Q207E, F246Y, R258S, R258G, G325A, G328E, G328W, G328R, G356D, R375P, and K400E.

6. The composition according to claim 2, wherein the low molecular weight MMP-9 inhibitor is selected from the group consisting of doxycycline, incyclinide, and minocycline, or salts or solvates thereof.

7. The composition according to claim 2, wherein the protein MMP-9 inhibitor is an anti-MMP-9 antibody or its antigen-binding fragment.

8. The composition according to claim 1, wherein ossified tissue is surgically removed from the subject.

9. The composition according to claim 8, wherein the surgical removal step is performed after the level or activity of MMP-9 in the subject has been downregulated.

10. The composition according to claim 1, wherein the subject is a human.

11. A kit for treating, relieving, and / or preventing fibrodysplasia ossificans progressive (FOP) in subjects who require it, A compound for downregulating the level and / or activity of matrix metalloproteinase 9 (MMP-9) in the subject, Instructions for administering an effective amount of the compound to the subject. The kit includes the above.

12. The aforementioned compound, Small molecule MMP-9 inhibitors, Protein MMP-9 inhibitors, A nucleic acid (and / or an expression vector expressing said nucleic acid) that downregulates MMP-9 by RNA interference, A ribozyme that downregulates MMP-9 (and / or a vector expressing said ribozyme), An expression vector comprising an expression cassette expressing a CRISPR component that downregulates MMP-9 by CRISPR knockout and / or CRISPR knockdown, and A transdominant-negative mutant protein of MMP-9, and / or an expression vector expressing the transdominant-negative mutant protein of MMP-9. The kit according to claim 11, comprising at least one selected from the group consisting of the following.

13. The kit according to any one of claims 11 to 12, wherein the subject has a mutant ACVR1 gene.

14. The kit according to claim 13, wherein the mutant ACVR1 gene encodes a constitutively active ACVR1 polypeptide.

15. The kit according to claim 14, wherein the ACVR1 polypeptide comprises at least one mutation selected from the group consisting of L196P, P197-F198 del ins L, R202I, R206H, Q207E, F246Y, R258S, R258G, G325A, G328E, G328W, G328R, G356D, R375P, and K400E.

16. The kit according to claim 12, wherein the small molecule MMP-9 inhibitor is selected from the group consisting of doxycycline, incyclinide, and minocycline.

17. The kit according to claim 12, wherein the protein MMP-9 inhibitor is an antibody against MMP-9 or an antigen-binding fragment thereof.

18. The kit according to claim 11, wherein the instructions further include instructions for surgically removing ossified tissue from the subject.

19. The kit according to claim 18, wherein the instructions further include instructions for performing surgical removal after the level or activity of MMP-9 in the subject has been downregulated.

20. The kit according to claim 11, wherein the subject is a human.

Images