Methods of treating, ameliorating and / or preventing progressive ossification fiber tissue dysplasia and heterotopic ossification and kits thereof

Abstract

Disclosed herein are methods for treating, ameliorating, and / or preventing progressive ossified fibrous tissue dysplasia (FOP) in a subject in need thereof. The methods include downregulating the level and / or activity of matrix metalloproteinase 9 (MMP-9) in a subject. Kits for performing the methods disclosed herein are also disclosed.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 324,842, filed on March 29, 2022, the entire contents of which are incorporated herein by reference.

[0003] Reference Sequence Listing

[0004] The accompanying sequence listing is submitted in the form of an xml file named "046483-7360WO1.xml", created on March 28, 2023, with a size of 4,200 bytes, which is incorporated herein by reference. Background Art

[0005] Fibrodysplasia ossificans progressiva (FOP; MIM#135100) is the most severe form of extraskeletal bone formation in humans. In this disease, muscle tissue and connective tissue such as tendons and ligaments are gradually replaced by bone (ossification), forming bone outside the skeleton (exosseous or heterotopic bone), which restricts movement.

[0006] Individuals with FOP appear normal at birth, except for a characteristic great toe deformity that is present in all typically affected individuals. During the first decade of life, episodic soft tissue swellings (or flare-ups) occur in the neck and back, and pathological deformation into mature heterotopic bone occurs via the endochondral route. Minor trauma, such as intramuscular immunizations, jaw blocks for dental work, muscle overexertion, blunt muscle trauma, bruises, bruises, falls, or influenza-like viral illnesses, may trigger a FOP flare-up, which results in progressive heterotopic ossification (HO). Most patients are immobilized by age 30 and require lifelong assistance with activities of daily living. Estimated median life expectancy is 56 years; death is usually from complications of thoracic insufficiency syndrome.

[0007] There is currently no definitive treatment for FOP, and current standard medical management is supportive only. 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

[0008] In some aspects, the present invention relates to the following non-limiting embodiments:

[0009] In some aspects, the present invention provides a method of treating, ameliorating and / or preventing fibrodysplasia ossificans progressiva (FOP) in a subject in need thereof.

[0010] In some embodiments, the method comprises downregulating the level and / or activity of matrix metalloproteinase 9 (MMP-9) in the subject.

[0011] In some embodiments, downregulating the level and / or activity of MMP-9 in a subject comprises administering to the subject an effective amount of:

[0012] Small molecule MMP-9 inhibitors,

[0013] Protein MMP-9 inhibitors,

[0014] A nucleic acid (and / or an expression vector expressing the nucleic acid) that downregulates MMP-9 by RNA interference,

[0015] A ribozyme (and / or a vector expressing a ribozyme) that downregulates MMP-9,

[0016] An expression vector comprising an expression cassette, wherein the expression cassette expresses a CRISPR component that downregulates MMP-9 by CRISPR knockout and / or CRISPR knockdown, and

[0017] A trans-dominant negative mutant protein of MMP-9, and / or an expression vector expressing a trans-dominant negative mutant protein of MMP-9.

[0018] In some embodiments, the subject has a mutated ACVR1 gene.

[0019] In some embodiments, the mutated ACVR1 gene encodes a constitutively active ACVR1 polypeptide.

[0020] In some embodiments, the ACVR1 polypeptide comprises at least one mutation selected from the group consisting of L196P, P197-F198del ins L, R202I, R206H, Q207E, F246Y, R258S, R258G, G325A, G328E, G328W, G328R, G356D, R375P, and K400E.

[0021] 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.

[0022] In some embodiments, the protein MMP-9 inhibitor is an anti-MMP-9 antibody or an antigen-binding fragment thereof.

[0023] In some embodiments, the method further comprises surgically removing the ossified tissue from the subject.

[0024] In some embodiments, the surgical removal step is performed after the level or activity of MMP-9 in the subject is downregulated.

[0025] In some embodiments, the subject is a human.

[0026] 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.

[0027] In some embodiments, the kit includes: a compound for downregulating the level and / or activity of matrix metalloproteinase 9 (MMP-9) in a subject; and instructions for administering an effective amount of the compound to the subject.

[0028] In some embodiments, the compound includes at least one selected from the group consisting of:

[0029] Small molecule MMP-9 inhibitors,

[0030] Protein MMP-9 inhibitors,

[0031] A nucleic acid (and / or an expression vector expressing the nucleic acid) that downregulates MMP-9 by RNA interference,

[0032] A ribozyme (and / or a vector expressing a ribozyme) that downregulates MMP-9,

[0033] An expression vector comprising an expression cassette, wherein the expression cassette expresses a CRISPR component that downregulates MMP-9 by CRISPR knockout and / or CRISPR knockdown, and

[0034] A trans-dominant negative mutant protein of MMP-9, and / or an expression vector expressing a trans-dominant negative mutant protein of MMP-9.

[0035] In some embodiments, the subject has a mutated ACVR1 gene.

[0036] In some embodiments, the mutated ACVR1 gene encodes a constitutively active ACVR1 polypeptide.

[0037] In some embodiments, the ACVR1 polypeptide comprises at least one mutation selected from the group consisting of L196P, P197-F198del ins L, R202I, R206H, Q207E, F246Y, R258S, R258G, G325A, G328E, G328W, G328R, G356D, R375P, and K400E.

[0038] In some embodiments, the small molecule MMP-9 inhibitor is selected from the group consisting of doxycycline, cyclocycline, and minocycline.

[0039] In some embodiments, the protein MMP-9 inhibitor is an anti-MMP-9 antibody or an antigen-binding fragment thereof.

[0040] In some embodiments, the instructions further include instructions for surgically removing the ossified tissue from the subject.

[0041] In some embodiments, the instructions further include instructions for performing the surgical removal step after the level or activity of MMP-9 in the subject is downregulated.

[0042] In some embodiments, the subject is a human. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The following detailed description of exemplary embodiments will be better understood when read in conjunction with the accompanying drawings. For illustrative purposes, non-limiting embodiments are shown in the accompanying drawings. However, it should be understood that the present description is not limited to the precise arrangements and tools of the embodiments shown in the accompanying drawings.

[0044] Figure 1 The progression of FOP in a typical patient according to some embodiments is shown.

[0045] Figures 2A-2D Features of FOP in Patient-R according to some embodiments are presented. Figure 2A This is a photo of a deformed toe. Figure 2B It's a radiograph of the toes. Figure 2C are scout CT images (collage) demonstrating the presence of congenital features of FOP and the absence of HO in patient-R. Figure 2D is an electropherogram showing a typical ACVR1 mutation (R206H, 617G>A) in patient-R.

[0046] Figures 3A-3C Results of MMP-9 assays in Patient-R according to some embodiments are shown. Figure 3A Patient-R was shown to have lower plasma MMP-9 (Myriad RBM assay) levels than all other FOP patients and controls (**p<0.01, n=77). Figure 3B is the result of gelatin zymography analysis, which showed that the total amount of MMP-9 in PBMCs of patient-R was less. Figure 3C The MMP-9 enzyme activity of PBMCs of patient-R compared with control subjects and other FOP patients is depicted. The results showed that the MMP-9 reserve was reduced in PBMCs of patient-R compared with PBMCs of other FOP patients.

[0047] Figures 4A-4B Demonstrated is the assessment of MMP-9 polymorphisms in Patient-R according to some embodiments. Figure 4A : Schematic diagram of the MMP-9 protein, indicating the different anatomical locations of the A20V and D165N SNPs that result in changes in amino acid residues. Figure 4B : PCR amplicon sequences confirmed the presence of A20V and D165N SNPs in patient-R.

[0048] Figure 5 According to some embodiments, MMP-9 is shown to be expressed in early stage diseased tissues. Figure 5 showed that the FOP model mouse (Acvr1 R206H / + ;CreERT2 - / + ) Images of early lesion tissue at 1, 3, and 5 days after cardiotoxin injury, demonstrating the expression of MMP-9 in inflammatory cells.

[0049] Figure 6A-6N These results suggest that downregulation of MMP-9 by genetic, pharmacological, or biological means in a FOP mouse model results in a decrease in HO. Fig. 6A :After soft tissue injury, MMP9 - / + ;Acvr1 R206H / + ;CreERT2 - / + Mice and MMP9 - / - ;Acvr1 R206H / + ;CreERT2 - / + The HO formation in mice was significantly less than that in Acvr1 R206H / + ;CreERT2 - / + Mouse. Figure 6B Depicted Fig. 6A Quantification of HO bone volume in the experiment was analyzed by MicroCT (***p<0.001, n=6). Figure 6C :MMP9 - / + ;Acvr1 R206H / + ;CreERT2 - / + and MMP9 - / - ;Acvr1 R206H / + ;CreERT2 - / + Mice produce significantly less MMP-9 than Acvr1 R206H / +；CreERT2- / + Control mice. Fig.6D MMP9 + / + ;Acvr1 + / + 、MMP9 + / + ;Acvr1 R206H / + ;CreERT2 - / + 、MMP9 - / - ;Acvr1 + / + and MMP9 - / - ;Acvr1 R206H / +;CreERT2 - / + Histology (H&E staining) of mice 14 days after cardiotoxin injury. Fig. 6E : Acvr1 treated with minocycline after soft tissue injury R206H / + ;CreERT2 - / + Mice formed significantly less HO than Acvr1 R206H / + ;CreERT2 - / + Control mice. Fig. 6F Depicted is the microCT analysis of Fig. 6E Quantification of HO bone volume in the experiment (**p<0.01, n=6). Figure 6G :Acvr1 treated with minocycline Q207D / + Mice developed significantly less HO after soft tissue injury than untreated Acvr1 Q207D / + Mice (***p<0.001, n=6). Figure 6H Depicted by MicroCT analysis of Figure 6G Quantification of HO bone volume in the experiment. Fig.6I demonstrated a protective effect of minocycline against HO when administered before or during lesion activation, suggesting that MMP-9 plays a role in inducing HO during the early inflammatory stage. Figure 6J The effects of different concentrations of minocycline on Acvr1 were shown. Q207D / + Effects on HO in mice. Minocycline at doses of 5 to 100 mg / kg effectively reduced HO. Figure 6K Depicted is the effect of blocking mAb MMP-9 (Gilead) on Acvr1 R206H / + ;CreERT2 - / + Results of HO bone volume after soft tissue injury in FOP mice. Figure 6L The results of gelatin zymography analysis. Cell culture supernatant from polarized THP-1, THP-1 A20V and THP-1 D165N M1-like and M2-like macrophages 48 hours after M0 macrophages. Figure 6M Quantification of MMP-9 enzymatic activity in gelatin zymography is shown. MMP-9 in M1-like and M2-like macrophages WT and MMP-9 A20V In contrast, MMP-9 D165N The enzyme activity decreased. Figure 6N Demonstrated in THP-1, THP-1 A20V and THP-1 D165N ELISA of activin A in the cell culture supernatants of M1-like and M2-like macrophages 48 hours after polarization of M0 macrophages. WT In contrast, MMP-9 A20V and MMP-9D165N Significantly reduced activin A levels in the supernatant.

[0050] Figure 7 A proposed molecular mechanism for the involvement of MMP-9 in FOP according to some embodiments is shown.

[0051] Figure 8 The results of protein-protein interaction (PPI) mapping of MMP-9 connectivity according to some embodiments are shown. PPI mapping was performed using IBM's Watson for Drug Discovery (WDD) and Ingenuity software. DETAILED DESCRIPTION

[0052] The following disclosure provides many different embodiments, or examples, for implementing different features of the provided subject matter. Specific examples of components and arrangements are described below to simplify the present disclosure. Of course, these are merely examples and are not intended to be limiting. For example, in the following description, a first feature may be formed above or on a second feature, and an embodiment in which the first feature and the second feature are directly in contact may be included, and an embodiment in which an additional feature may be formed between the first feature and the second feature so that the first feature and the second feature may not be in direct contact may be included. In addition, the present disclosure may repeat reference numbers and / or letters in various examples. This repetition is for simplicity and clarity, and does not itself determine the relationship between the various embodiments and / or configurations discussed.

[0053] Heterozygous missense mutations in activin receptor type I A, a bone morphogenetic protein (BMP) type I receptor, are found in all patients with sporadic or familial fibrodysplasia ossificans progressiva (FOP). Mutations in ACVR1 result in loss of ACVR1 autoinhibitory function, rendering it susceptible to dysregulated BMP pathway signaling. Activin A, a member of the transforming growth factor-β (TGF-β) family of molecules, antagonizes BMP signaling in the wild-type (WT) ACVR1 background, but specifically enhances BMP pathway signaling in cells carrying the ACVR1R206H mutation and drives ectopic bone formation in FOP.

[0054] The study described herein (the “Study”) identified a unique patient with fibrodysplasia ossificans progressiva (FOP) who had the classic R206H mutation in the Acvr1 BMP receptor. Although this patient had congenital features of FOP, he showed few FOP features after birth. The Study found that this patient had significantly suppressed inflammatory biomarkers and had compound heterozygosity for the MMP-9 gene (one allele of the MMP-9 gene had a polymorphism that resulted in an A20V mutation in the expressed polypeptide and the other allele had a polymorphism that resulted in a D165N mutation). The Study used protein structural modeling to predict that the compound heterozygosity for MMP-9 found in the patient resulted in reduced levels and activity of MMP-9 in the patient. The Study found that MMP-9 is expressed in early lesions in patients with FOP. The present study utilized several mouse models of FOP and found that reducing the level and / or activity of MMP-9 by genetic deletion of MMP-9 (including partial deletion), administration of a non-limiting example of a monoclonal antibody against MMP-9, or administration of a non-limiting small molecule inhibitor of MMP-9, all resulted in reduced levels of heterotopic ossification (bone formation) in response to injury in mouse models of FOP.

[0055] Thus, in some aspects, the present invention relates to a method of treating, ameliorating and / or preventing fibrodysplasia ossificans progressiva (FOP) in a subject in need thereof.

[0056] In some aspects, the present invention relates to a kit for treating, ameliorating and / or preventing FOP in a subject in need thereof.

[0057] definition

[0058] As used herein, each of the following terms 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 of ordinary skill in the art to which the present disclosure belongs. In general, the terms used herein and laboratory procedures in animal pharmacology, pharmaceutics, peptide chemistry, and organic chemistry are well known and commonly used in the art. It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present teachings are still able to operate. The use of any section headings is to assist in reading this document and should not be construed as limiting; information related to the section headings may appear within or outside that particular section. The entire contents of all publications, patents, and patent documents mentioned in this document are incorporated herein by reference as if they were individually incorporated herein by reference.

[0059] In this application, when an element or component is referred to as being included in and / or selected from a list of enumerated elements or components, it should be understood that the element or component can be any one of the enumerated elements or components and can be selected from a group consisting of two or more enumerated elements or components.

[0060] In the methods described herein, the actions may be performed in any order unless a time or order of operations is explicitly stated. In addition, specified actions may be performed simultaneously unless explicit claim language specifies that they are to be performed separately. For example, a claim operation X and a claim operation Y may be performed simultaneously in a single operation, and the resulting method would fall within the literal scope of the claimed method.

[0061] As used herein, the terms "a", "an" or "the" are used to mean one or more than one, unless the context clearly dictates otherwise. The term "or" is used to refer to a non-exclusive "or" unless otherwise indicated. The statements "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".

[0062] As used herein, "about" when referring to a measurable value such as an amount, a length of time, and the like, is meant to include variations of ±20% or ±10%, in some embodiments ±5%, in some embodiments ±1%, and in some embodiments ±0.1% from the specified value, as such variations are suitable for practicing the disclosed methods.

[0063] "Disease" is a health condition in an animal in which the animal cannot maintain homeostasis and the animal's health will continue to deteriorate if the disease is not corrected.

[0064] A "disorder" in an animal is a health state in which the animal is able to maintain homeostasis, but the animal is in worse health than it would be without the disorder. If left untreated, a disorder does not necessarily lead to further deterioration in the animal's health.

[0065] A disease or disorder is "alleviated" if the symptoms of the disease or disorder are reduced in severity, in the frequency with which a patient experiences such symptoms, or both.

[0066] In one aspect, the terms "co-administered" and "co-administration" with respect to a subject refer to the administration of a compound and / or composition of the present disclosure to a subject together with a compound and / or composition that can also treat or prevent a disease or disorder contemplated herein. In certain embodiments, the co-administered compounds and / or compositions are administered separately, or in any combination as part of a single treatment. The co-administered compounds and / or compositions can be formulated in any combination as a mixture of solids and liquids in various solid, gel and liquid formulations, or as solutions.

[0067] As used herein, the term "pharmaceutical composition" or "composition" refers to a mixture of at least one compound useful within the scope of the present disclosure and a pharmaceutically acceptable carrier. Pharmaceutical compositions facilitate administration of the compound to a patient. There are a variety of techniques for administering compounds in the art, including but not limited to subcutaneous, intravenous, oral, aerosol, inhalation, rectal, vaginal, transdermal, intranasal, buccal, sublingual, parenteral, intrathecal, intragastric, ophthalmic, pulmonary, and topical administration.

[0068] As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, that does not diminish the biological activity or properties of a compound and is relatively nontoxic, i.e., the material can be administered to a subject without causing adverse biological effects or interacting in a deleterious manner with any of the ingredients of the composition in which it is contained.

[0069] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener, solvent or encapsulating material, which is involved in carrying or transporting the useful compounds of the present disclosure to or on the patient's body to enable it to perform its intended function. Each carrier must be "acceptable", meaning that it is compatible with the other ingredients of the formulation, including the useful compounds of the present disclosure, and will not cause harm to the patient. Materials that can be used 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, absorption delaying agents, etc., which are compatible with the activity of the useful compounds of the present disclosure and are physiologically acceptable to the patient. "Pharmaceutically acceptable carriers" may further include pharmaceutically acceptable salts of useful compounds of 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.

[0070] As used herein, "pharmaceutically acceptable salts" refer to salts of the administered compound prepared from pharmaceutically acceptable non-toxic acids and bases, including inorganic acids, inorganic bases, organic acids, inorganic bases, solvates, hydrates and clathrates thereof.

[0071] As used herein, a "pharmaceutically effective amount," "therapeutically effective amount," or "effective amount" of a compound refers to an amount of the compound sufficient to provide a beneficial effect to a subject to which the compound is administered.

[0072] As used herein, the term "prevent" or "prevention" means that if the disorder or disease has not already occurred, the disorder or disease does not occur; if the disorder or disease has already occurred, the disorder or disease does not further occur. The ability to prevent some or all symptoms associated with a disorder or disease is also contemplated.

[0073] As used herein, the terms "subject," "individual," and "patient" are used interchangeably and may refer to a human or non-human mammal or bird. Non-human mammals include, for example, livestock and pets, such as sheep, cattle, pigs, dogs, cats, and murine mammals. In certain embodiments, the subject is a human.

[0074] As used herein, the term "treatment" or "treating" 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 agent), to a patient, or to an isolated tissue or cell line (e.g., for diagnostic or ex vivo applications) of a patient suffering from a disease or disorder and / or symptoms of a disease or disorder, for the purpose of treating, curing, alleviating, relieving, altering, remedying, improving, modifying, or affecting the disease or disorder and / or symptoms of a disease or disorder. Such treatments can be specifically tailored or modified based on knowledge gained from the field of pharmacogenomics.

[0075] As used herein, the term "fibrodysplasia ossificans progressiva" or "FOP" refers to a genetic disease of congenital skeletal malformations and progressive heterotopic ossification (HO). In all individuals with sporadic or familial FOP, heterozygous (i.e., the patient has one mutant Acvr1 gene copy and one wild-type Acvr1 gene copy) missense mutations in a bone morphogenetic protein (BMP) type I receptor, type I activin receptor A / activin-like kinase 2 (ACVR1 / ALK2) have been found. An activating R206H mutation was found on ACVR1 in most FOP patients. For a detailed description of this disease, see, for example, Kaplan et al. (Best Practice & Research Clinical Rheumatology, March 2008, Vol. 22, No. 1, pp. 191-205) and Kaplan et al. (Bone, November 2020, Vol. 140, 115539).

[0076] As used herein, the term "heterotopic ossification" or "HO" refers to the formation of extraskeletal bone in muscle and soft tissue. Heterotopic ossification is one of the postnatal characteristics of FOP and is also a common phenomenon after trauma in non-FOP patients. A detailed description of heterotopic ossification is provided, for example, by Meyers et al. (JBMR Plus 2019, 3: e10172) and Dey et al. (Transl Res. 2017 Aug; 186: 95-111).

[0077] The term "flare-up", when used herein in connection with FOP, refers to the inflammatory soft tissue swelling experienced by most FOP patients, which begins in childhood and develops throughout life. Flare-ups include unpredictable episodes of soft tissue swelling, pain, reduced movement, and / or stiffness. These flare-ups often result in additional bone formation, but not always. For a detailed description of FOP flare-ups, see, for example, Pignolo et al. (J Bone Miner Res. 2016 Mar; 31(3): 650-6).

[0078] Non-limiting abbreviations used herein: FOP: fibrodysplasia ossificans progressiva; HO: heterotopic ossification; PBMC: peripheral blood mononuclear cell; SNP: single nucleotide polymorphism.

[0079] Method for treating, ameliorating and / or preventing fibrodysplasia ossificans progressiva

[0080] This study identified a patient with fibrodysplasia ossificans progressiva (FOP) who had a typical R206H mutation in the Acvr1 BMP receptor. Although this patient had congenital features of FOP, few FOP features were present after birth. This study found that inflammatory biomarkers were significantly suppressed in this patient, and that this patient had compound heterozygosity for the MMP-9 gene (one allele of the MMP-9 gene had a polymorphism that resulted in A20V and the other allele had a polymorphism that resulted in D165N). This study used protein structural modeling to predict that the MMP-9 compound heterozygosity found in the patient resulted in reduced MMP-9 levels and activity in the patient. This study found that MMP-9 was expressed in early lesion tissues in FOP subjects. Using several mouse FOP models as non-limiting examples, this study found that reducing MMP-9 levels and / or activity by genetic deletion of MMP-9 (including partial deletion), administration of non-restrictive MMP-9 monoclonal antibodies, or administration of non-restrictive small molecule inhibitors of MMP-9 all resulted in reduced levels of heterotopic ossification (bone formation) in response to soft tissue injury.

[0081] Therefore, in some aspects, the present invention relates to a method of treating, ameliorating and / or preventing fibrodysplasia ossificans progressiva (FOP) in a subject. 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.

[0082] This study found that inhibition of MMP-9 can significantly reduce the level of heterotopic ossification in Acvr1 BMP receptor Q270D mutant model animals. Since the Q270D mutation does not cause spontaneous heterotopic ossification, but only causes HO in response to injury and produces very strong post-traumatic HO, the Q270D model is sometimes considered a model of HO. Therefore, in some aspects, the present invention relates to a method for treating, improving and / or preventing heterotopic ossification 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.

[0083] In some embodiments, the subject is treated with a bone marrow transplant that replaces some of the hematopoietic stem cells in the subject with hematopoietic stem cells having reduced MMP-9 levels and / or activity. It will be appreciated by those of ordinary skill in the art that MMP-9, although expressed in a variety of tissues, is strongly expressed in bone marrow and lymphoid tissues. As described elsewhere herein, this study found that partially reducing the expression of MMP-9 by deleting only one MMP-9 allele was sufficient to counteract heterotopic ossification in FOP model animals induced by soft tissue injury. Therefore, in some embodiments, only partially reducing and / or eliminating the expression and / or activity of MMP-9 in the bone marrow is sufficient to simulate the partial reduction in MMP-9 expression in FOP model mice. In some embodiments, the bone marrow stem cells used for transplantation are genetically engineered to reduce MMP-9 levels and / or activity. For example, Daniel-Moreno et al. (Bone Marrow Transplantation Vol. 54, pp. 1940-1950 (2019)) describe genetic engineering of hematopoietic stem cells in the bone marrow. In some embodiments, the bone marrow used for transplantation is from a donor having an MMP-9 polymorphism that reduces MMP-9 levels and / or activity, such as the MMP-9 polymorphism found in Patient-R described herein.

[0084] In some embodiments, the subject has a mutated ACVR1 gene. In some embodiments, the mutated ACVR1 gene encodes a constitutively active ACVR1 polypeptide. In other embodiments, the mutated 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 relative 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 relative to the wild-type polypeptide. In some embodiments, the ACVR1 polypeptide comprises one or more amino acid substitutions, deletions, and / or insertions relative to the wild-type ACVR1 polypeptide, wherein the wild-type ACVR1 polypeptide comprises the amino acid sequence described in SEQ ID NO:3. GenBank Accession No. NP_001104537 discloses an ACVR1 polypeptide, which is incorporated herein by reference in its entirety. 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 relative 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 L196P, P197-F198 del-ins L, R202I, R206H, Q207E, F246Y, R258S, R258G, G325A, G328E, G328W, G328R, G356D, R375P and K400E, wherein del represents a deletion and ins represents an insertion. In some embodiments, relative to a wild-type ACVR1 polypeptide comprising the sequence set forth in SEQ ID NO:3, the ACVR1 peptide 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, wherein del represents a deletion and ins represents an insertion.ACVR1 mutations implicated in FOP are also described in Haupt et al. (Bone April 2018, Vol. 109, pp. 232-240) and Mukaddam et al. (Genetics of Fibrodysplasia Ossificans Progressiva, eLS, Vol 2: 1-8, 2021), each of which is incorporated herein by reference in its entirety.

[0085] 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 is downregulated in the subject.

[0086] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0087] Small molecule inhibitors of MMP-9

[0088] In some embodiments, compounds that downregulate MMP-9 levels and / or activity include small molecule inhibitors of MMP-9.

[0089] As used herein, the term "small molecule" refers to a molecule having a molecular weight of about 2000 Daltons or less, such as 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, cipemastat, 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, 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. For a description of MMP-9 inhibitors, see, for example, Fields (Cells. 2019 Sep; 8 (9): 984.).

[0090] In some embodiments, the small molecule inhibitor of MMP-9 includes a selective MMP-9 small molecule inhibitor. As used herein, the term "selective MMP-9 inhibitor" refers to an IC 50 Equal to or higher than the IC for any other matrix metalloproteinases 50 Small molecule compounds, such as ICs of any other matrix metalloproteinases 50 0.1 or more, 0.2 or more, 0.3 or more, 0.5 or more, 0.8 or more, or 1.0 or more. Non-limiting examples of selective MMP-9 small molecule inhibitors include apigenin-7-glucuronide, FFAGLDD, FSL-1TFA, Ginkgolide C, isoliquiritin apioside, JNJ0966, luteolin 7-O-glucuronide, MMP-9-IN-1, (R)-ND-336, SM-7368, FFAGLDD TFA, and the like.

[0091] In some embodiments, small molecule inhibitors of MMP-9 include compounds that are used in the medical field for purposes other than inhibiting MMP-9 and are known to be generally safe. Non-limiting examples of such compounds include tetracyclines, such as doxycycline, cyclocycline, and minocycline. Examples of tetracyclines capable of inhibiting MMP-9 are described in Griffin et al. (Am J Physiol Cell Physiol: C539-C548.), which is incorporated herein by reference in its entirety.

[0092] Protein inhibitors of MMP-9

[0093] In some embodiments, compounds that downregulate MMP-9 levels and / or activity include protein inhibitors of MMP-9.

[0094] In some embodiments, the protein inhibitor of MMP-9 includes an anti-MMP-9 antibody or an antigen-binding fragment thereof. Examples of anti-MMP-9 antibodies 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, and the like.

[0095] 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 of the tissue inhibitor of metalloproteinases (TIMP) family, such as TIMP-1, TIMP-3, etc., FFAGLDD peptide, FFAGLDD TFA, cyclic CTTHWGFTLC, cyclic CTTHWGFTLC TFA, etc.

[0096] RNA interference nucleic acid and its expression vector for down-regulating MMP-9

[0097] In some embodiments, the compound that down-regulates the level and / or activity of MMP-9 includes a nucleic acid that down-regulates the level of MMP-9 by RNA interference, and / or an expression vector that expresses the nucleic acid.

[0098] In some embodiments, the nucleic acid that downregulates the level of MMP-9 by RNA interference includes an isolated nucleic acid. In other embodiments, the modulator is an RNAi molecule (such as but not limited to siRNA and / or shRNA and / or miRNA) or an antisense molecule that inhibits the expression and / or activity of MMP-9. In still other embodiments, the nucleic acid includes a promoter / regulatory sequence, such that the nucleic acid is preferably capable of directing the expression of the nucleic acid. Therefore, the present specification provides expression vectors and methods for introducing exogenous DNA into cells and simultaneously expressing the exogenous DNA in cells, such as described in 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 elsewhere herein.

[0099] In certain embodiments, siRNA is used to reduce the level of MMP-9. RNA interference (RNAi) is a phenomenon in which double-stranded RNA (dsRNA) is introduced into various organisms and cell types to cause degradation of complementary mRNA. In cells, long dsRNAs are cleaved into short small interfering RNAs or siRNAs of 21-25 nucleotides by a ribonuclease called Dicer. The siRNA is then assembled with protein components into an RNA-induced silencing complex (RISC) and loosened in the process. The activated RISC then binds to the complementary transcript through the base pairing interaction between the siRNA antisense strand and the mRNA. The bound mRNA is cleaved, and the sequence-specific degradation of the mRNA leads to gene silencing. See, for example, U.S. Pat. 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 a method for chemically modifying siRNA that facilitates systemic administration by intravenous injection. Optimization of siRNA requires consideration of overall G / C content, terminal C / T content, Tm, and 3' overhang nucleotide content. See, for example, Schwartz et al., 2003, Cell, 115: 199-208 and Khvorova et al., 2003, Cell 115: 209-216. Thus, the present description also includes methods of reducing MMP-9 levels using RNAi technology.

[0100] In certain embodiments, the present specification provides a vector comprising siRNA or antisense polynucleotides. In other embodiments, siRNA or antisense polynucleotides inhibit the expression of MMP-9. Adding the desired polynucleotides to the vector and the selection of the vector are well known in the art.

[0101] In certain embodiments, the expression vectors described herein encode short hairpin RNA (shRNA) inhibitors. shRNA inhibitors are well known in the art and are directed against the mRNA of the target, thereby reducing the expression of the target. In certain embodiments, the encoded shRNA is expressed by the cell and then processed into siRNA. For example, in some cases, the cell has a natural enzyme (e.g., a dicer) that can cleave the shRNA to form the siRNA.

[0102] siRNA, shRNA or antisense polynucleotides can be cloned into various types of vectors described in other parts of this article. In order to express siRNA or antisense polynucleotides, at least one module in each promoter plays a role in positioning the start site of RNA synthesis.

[0103] In order to evaluate the expression of siRNA, shRNA or antisense polynucleotide, the expression vector of the cell can also include a selectable marker gene or a reporter gene or both, so as to identify and select the expressing cell from the cell group that attempts to use viral vector transfection or infection. In some embodiments, the selectable marker can be carried on a separate DNA and used in the cotransfection process. The flanks of the selectable marker and the reporter gene can have appropriate regulatory sequences to express in the host cell. Useful selectable markers are known in the art and include, for example, antibiotic resistance genes, such as neomycin resistance, etc.

[0104] After generating siRNA polynucleotides, it will become apparent to the skilled artisan that the siRNA polynucleotides possess certain properties that can be modified to improve the siRNA as a therapeutic compound. Thus, in certain embodiments, siRNA polynucleotides are further designed to resist degradation by modifying the siRNA polynucleotide to include phosphorothioates, or other linkers, methylphosphonates, sulfones, sulfates, ketyls, phosphorodithioates, phosphoramides, phosphates, 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)).

[0105] Any polynucleotide can be further modified to increase its stability in vivo. Possible modifications include, but are not limited to, adding flanking sequences at the 5' and / or 3' ends; using phosphorothioate or 2'O-methyl instead of phosphodiester linkages in the backbone; and / or adding non-traditional bases such as inosine, quinoline, and whitinoside, etc., as well as acetyl, methyl, thio and other modified forms of adenine, cytosine, guanine, thymine and uracil.

[0106] In certain embodiments, the antisense nucleic acid sequence expressed by the plasmid vector is used to inhibit the expression of MMP-9 protein. The antisense expression vector is used to transfect mammalian cells or the mammal itself, thereby reducing the endogenous expression of MMP-9.

[0107] Antisense molecules and their use in inhibiting gene expression are well known in the art (see, e.g., Cohen, 1989, In: Oligodeoxyribonucleotides, Antisense Inhibitors of Gene Expression, CRC Press). Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule (Weintraub, 1990, Scientific American 262:40), as defined elsewhere herein. In cells, antisense nucleic acids hybridize with corresponding mRNA to form double-stranded molecules, thereby inhibiting the translation of genes.

[0108] The use of antisense methods to inhibit gene translation is known in the art, for example, as described in Marcus-Sakura (1988, Anal. Biochem. 172: 289). Such antisense molecules can be provided to cells by gene expression using DNA encoding the antisense molecules, as taught by Inoue, 1993, U.S. Pat. No. 5,190,931.

[0109] Alternatively, the antisense molecules in this specification can also be synthesized and then provided to the cell. Since antisense oligomers are easy to synthesize and import into target cells, antisense oligomers of about 10 to about 30 nucleotides, more preferably antisense oligomers of about 15 nucleotides can be used. Synthetic antisense molecules considered in this specification include oligonucleotide derivatives known in the art, which have better biological activity (see U.S. Patent No. 5,023,243) compared with unmodified oligonucleotides.

[0110] Ribozyme for downregulating MMP-9 and vector for expressing ribozyme

[0111] In some embodiments, the compound that down-regulates the level and / or activity of MMP-9 comprises a ribozyme that down-regulates MMP-9, and / or a vector that expresses the ribozyme.

[0112] Ribozymes are used to inhibit MMP-9 protein expression. Ribozymes useful for inhibiting the expression of target molecules can be designed by adding a target sequence complementary to, for example, an mRNA sequence encoding MMP-9 to the basic ribozyme structure. Ribozymes are antisense RNAs whose catalytic sites are capable of specifically cleaving complementary RNAs. Therefore, ribozymes with sequences complementary to the MMP-9 mRNA sequence can downregulate the expression of MMP-9 by reducing the level of MMP-9 mRNA. Ribozymes targeting MMP-9 can be synthesized using commercially available reagents (Applied Biosystems, Inc., Foster City, CA), or they can be genetically expressed from DNA encoding them. In some embodiments, DNA encoding ribozymes is incorporated into vectors described elsewhere herein.

[0113] Compounds that downregulate MMP-9 via CRISPR knockout / knockdown and other knockout / knockdown technologies

[0114] In some embodiments, compounds that downregulate MMP-9 levels and / or activity include compounds that downregulate MMP-9 by CRISPR knockout / knockdown and other knockout / knockdown techniques. In some embodiments, the compound includes an expression vector that includes an expression cassette, wherein the expression cassette expresses a CRISPR component that downregulates MMP-9 by CRISPR knockout or CRISPR knockdown.

[0115] In some embodiments, the compound that downregulates the activity or level of MMP-9 comprises a CRISPR / Cas9 system for knocking out MMP-9.

[0116] The CRISPR / Cas9 system is a simple and efficient system for inducing targeted genetic changes. Target recognition by the Cas9 protein requires a "seed" sequence in the guide RNA (gRNA) and a conserved dinucleotide containing a protospacer adjacent motif (PAM) upstream of the gRNA binding region. Therefore, by redesigning the gRNA in cell lines (such as 293T cells), primary cells, and CAR T cells, the CRISPR / Cas9 system can be designed to cut almost any DNA sequence. The CRISPR / Cas9 system can simultaneously target multiple genomic sites by co-expressing a single Cas9 protein and two or more gRNAs, making this system particularly suitable for multi-gene editing or coordinated activation of target genes.

[0117] The Cas9 protein and guide RNA form a complex that recognizes and cuts the target sequence. Cas9 consists of six domains: REC I, REC II, Bridge Helix, PAM interaction, 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 designed to be complementary to the target DNA sequence at its 5' end. Once the guide RNA binds to the Cas9 protein, a conformational change occurs, thereby activating the protein. Once activated, Cas9 will search for target DNA by binding to a sequence that matches its protospacer adjacent motif (PAM) sequence. PAM is a two or three nucleotide base sequence within one nucleotide downstream of the complementary region of the guide RNA. In a non-limiting example, the PAM sequence is 5'-NGG-3'. When the Cas9 protein finds its target sequence with an appropriate PAM, it melts the base upstream of the PAM and pairs it with the complementary region on the guide RNA. The RuvC and HNH nuclease domains then cleave the target DNA after the third nucleotide base upstream of the PAM.

[0118] A non-limiting example of a CRISPR / Cas system for inhibiting gene expression, CRISPRi, is described in U.S. Patent Application Publication No. US2014 / 0068797. CRISPRi induces permanent gene damage, which uses RNA-guided Cas9 endonucleases to introduce DNA double-strand breaks, which triggers error-prone repair pathways to cause frameshift mutations. Catalytically dead Cas9 lacks endonuclease activity. When co-expressed with guide RNA, a DNA recognition complex is generated that specifically interferes with transcriptional elongation, RNA polymerase binding, or transcription factor binding. This CRISPRi system effectively inhibits the expression of targeted genes.

[0119] CRISPR / Cas gene disruption occurs when a guide nucleic acid sequence and a Cas endonuclease specific for a targeted gene are introduced into a cell and form a complex that enables the Cas endonuclease to introduce a double-strand break on the targeted gene. In certain embodiments, the CRISPR / Cas system includes an expression vector, such as, but not limited to, a pAd5F35-CRISPR vector. In other embodiments, the Cas expression vector induces expression of the 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.

[0120] In certain embodiments, inducing the Cas expression vector includes exposing the cell to a preparation that activates an inducible promoter in the Cas expression vector. In such an embodiment, the Cas expression vector includes an inducible promoter, such as a promoter that can be induced by exposure to an antibiotic (e.g., by tetracycline or a derivative of tetracycline, such as doxycycline). However, it should be understood that other inducible promoters can be used. The inducer can be a selective condition that causes the inducible promoter to be induced (e.g., exposure to a preparation, such as an antibiotic). This results in expression of the Cas expression vector.

[0121] In certain embodiments, guide RNA(s) and Cas9 can be delivered to cells as a ribonucleoprotein (RNP) complex. RNP consists of a purified Cas9 protein complexed with a gRNA, and is well known in the art for being effectively delivered to a variety of cell types, including but not limited to neurons, stem cells, and immune cells (Addgene, Cambridge, MA, Mirus Bio LLC, Madison, WI).

[0122] The guide RNA is specific for a genomic region of interest and targets the region for Cas endonuclease-induced double-strand breaks. The target sequence of the guide RNA sequence can be located within a gene locus or within a non-coding region of the genome. In certain embodiments, the 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.

[0123] Guide RNA (gRNA), also known as "short guide RNA" or "sgRNA", provides targeting specificity and scaffolding / binding capacity for the Cas9 nuclease. The gRNA can be a synthetic RNA composed of a target sequence and a scaffolding sequence, the scaffolding sequence being derived from endogenous bacterial crRNA and tracrRNA. The gRNA is used to target Cas9 to a specific genomic site in genome engineering experiments. The guide RNA can be designed using standard tools known in the art.

[0124] In the context of CRISPR complex formation, "target sequence" refers to a sequence to which a guide sequence is designed to have some complementarity, and hybridization between the target sequence and the guide sequence promotes the formation of the CRISPR complex. Complete complementarity is not necessarily required, as long as there is sufficient complementarity to cause hybridization and promote the formation of the CRISPR complex. The target sequence may include any polynucleotide, such as a DNA or RNA polynucleotide. In certain embodiments, the target sequence is located in the nucleus or cytoplasm of a cell. In other embodiments, the target sequence may be located in an organelle of a eukaryotic cell, such as a mitochondria or a nucleus. Typically, in the context of an endogenous CRISPR system, the formation of a CRISPR complex (including a guide sequence that hybridizes to a target sequence and complexes with one or more Cas proteins) results in the cleavage of one or both strands in 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). As with the target sequence, it is believed that complete complementarity is not required, as long as the complementarity is sufficient to function.

[0125] In certain embodiments, one or more vectors driving the expression of one or more elements of the CRISPR system are introduced into a host cell so that the expression of the elements of the CRISPR system is guided to form a CRISPR complex at one or more target sites. For example, the Cas enzyme, the guide sequence connected to the tracr-mate sequence, and the tracr sequence can be connected to different regulatory elements on different vectors, respectively. Alternatively, two or more elements expressed by the same or different regulatory elements can be combined in a single vector, and any components of the CRISPR system not included in the first vector are provided by one or more additional vectors. The CRISPR system elements combined in a single vector can be arranged in any suitable direction, such as one element is located 5' ("upstream") or 3' ("downstream") relative to the second element. The coding sequence of an element can be located on the same or opposite chain of the coding sequence of the second element and positioned in the same or opposite direction. In certain embodiments, a single promoter drives expression of a transcript encoding a CRISPR enzyme and one or more guide sequences, a tracr mate sequence (optionally operably linked to the guide sequence), and a tracr sequence embedded in 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).

[0126] 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 in addition to the CRISPR enzyme). The CRISPR enzyme fusion protein may include any additional protein sequences, and optionally a linker sequence between any two domains. Examples of protein domains that may be fused to the CRISPR enzyme include, but are not limited to, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Additional domains that may constitute a portion of a fusion protein that includes a CRISPR enzyme are described in U.S. Patent Application Publication No. US20110059502, which is incorporated herein by reference. In certain embodiments, a labeled CRISPR enzyme is used to determine the location of a target sequence.

[0127] Traditional viral and non-viral gene transfer methods can be used to introduce nucleic acids in mammalian and non-mammalian cells or target tissues. These methods can be used to introduce nucleic acids encoding CRISPR system components into cells in culture or into host organisms. Non-viral vector delivery systems include DNA plasmids, RNA (transcripts of vectors described herein), naked nucleic acids, and nucleic acids compounded with liposome-like delivery vectors. Viral vector delivery systems include DNA and RNA viruses, which have exogenous or integrated genomes after delivery to cells (Anderson, 1992, Science 256: 808-813; and Yu, et al., 1994, Gene Therapy 1: 13-26).

[0128] In some embodiments, 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 can be from Streptococcus pyogenes, Streptococcus thermophilus, or other species.

[0129] In general, the Cas protein includes at least one RNA recognition and / or RNA binding domain. The RNA recognition and / or RNA binding domain interacts with the guide RNA. The Cas protein may also include a nuclease domain (i.e., a DNase or RNase domain), a DNA binding domain, a helicase domain, an RNAse domain, a protein-protein interaction domain, a dimerization domain, and other domains. The Cas protein may be modified to increase nucleic acid binding affinity and / or specificity, change enzyme activity, and / or change another property 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, Cas may be derived from a modified Cas9 protein. For example, the amino acid sequence of the Cas9 protein may be modified to change one or more properties of the protein (e.g., nuclease activity, affinity, stability, etc.). Alternatively, the domains of the Cas9 protein that are not involved in RNA-guided cutting may also be eliminated from the protein, so that the modified Cas9 protein is smaller than the wild-type Cas9 protein. In general, the Cas9 protein includes at least two nuclease (i.e., DNase) domains. For example, the Cas9 protein may include a RuvC-like nuclease domain and a HNH-like nuclease domain. RuvC and HNH domains jointly cut single strands to break double strands of DNA. (Jinek, et al., 2012, Science, 337: 816-821). In some embodiments, the Cas9-derived protein may be modified to contain only one functional nuclease domain (RuvC-like or HNH-like nuclease domain). For example, the Cas9-derived protein may be modified to delete or mutate one of the nuclease domains so that it is no longer functional (i.e., it has no nuclease activity). In some embodiments in which one of the nuclease domains is inactive, the Cas9-derived protein can introduce a nick into a double-stranded nucleic acid (such a protein is called a "nickase"), but cannot cleave double-stranded DNA. In any of the above embodiments, well-known methods such as directed mutagenesis, PCR-mediated mutagenesis, and full gene synthesis, as well as other methods known in the art, may be used to inactivate any or all of the nuclease domains by one or more deletion mutations, insertion mutations, and / or substitution mutations.

[0130] In a non-limiting embodiment, the vector drives the expression of the CRISPR system. There is no lack of suitable vectors useful for this specification in the art. The vector to be used is suitable for replication and optionally integrated into a eukaryotic cell. A typical vector comprises a transcription and translation terminator, a start sequence, and a promoter for regulating the expression of the desired nucleic acid sequence. The vector in this specification can also be used for nucleic acid standard gene delivery schemes. Gene delivery methods are known in the art (U.S. Patents 5,399,346, 5,580,859, and 5,589,466, the entire contents of which are incorporated herein by reference).

[0131] In addition, the vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art, such as described in Sambrook et al. (4th edition, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 2012) and other virology and molecular biology manuals. Viruses that can be used as vectors include but are not limited to retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, Sindbis viruses, gamma retroviruses and slow viruses. In general, suitable vectors include a replication origin, a promoter sequence, a convenient restriction endonuclease site, and one or more selectable markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193) that work in at least one organism.

[0132] In some embodiments, the compound that downregulates the activity or expression level of MMP-9 includes a nucleic acid that downregulates the expression level of MMP-9 by CRISPR knockdown. CRISPR knockdown includes but is not limited to CRISPRCas13 knockdown. (For example, see 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. The entire contents of which are incorporated herein by reference).

[0133] In some embodiments, the present invention includes any other method for achieving gene knockdown and / or editing that can achieve deletion and / or inactivation of MMP-9, such as, but not limited to, the method described in WO 2018 / 236840 (the entire contents of which are incorporated herein by reference).

[0134] Compounds that downregulate MMP-9 by inactivation and / or chelation

[0135] In some embodiments, the compound that downregulates the activity or expression level of MMP-9 includes a protein that downregulates the activity of MMP-9 by inactivating and / or chelating MMP-9. In some embodiments, the compound includes a nucleic acid that expresses a protein that downregulates the activity of MMP-9 by inactivating and / or chelating MMP-9. In some embodiments, the compound includes an expression vector that expresses a protein that downregulates the activity of MMP-9 by inactivating and / or chelating MMP-9 (see the "Vector" section for a non-limiting description of the vector).

[0136] In some embodiments, the compound that downregulates the expression level of MMP-9 is a trans dominant negative mutant of MMP-9, and / or a nucleic acid or vector that expresses a trans dominant negative mutant of MMP-9.

[0137] Kit for treating, ameliorating and / or preventing fibrodysplasia ossificans progressiva

[0138] 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.

[0139] In some aspects, the present invention relates to a kit for treating, ameliorating and / or preventing heterotopic ossification in a subject in need thereof.

[0140] In some embodiments, the kit includes a compound that downregulates the level and / or activity of MMP-9 in a subject; and a manual for instructing the administration of an effective amount of the compound to the subject. In some embodiments, the compound that downregulates the level and / or activity of MMP-9 in a subject is the same or similar to the compound described elsewhere herein, such as in the section "Methods for treating, ameliorating and / or preventing fibrodysplasia ossificans progressiva".

[0141] Carrier

[0142] Vectors can improve the stability of a nucleic acid, make delivery easier, or allow expression of the nucleic acid or its protein product in a cell.

[0143] Therefore, in some embodiments, a protein inhibitor or a nucleic acid that downregulates MMP-9 activity or expression level is incorporated into the vector.

[0144] In some embodiments, this specification relates to a vector comprising the nucleotide sequence of this specification or the construct of this specification. The selection of the vector depends on the host cell to be subsequently introduced. In certain embodiments, the vector of this specification is an expression vector. Suitable host cells include various prokaryotic and eukaryotic host cells. In certain embodiments, the expression vector is selected from the group consisting of viral vectors, bacterial vectors and mammalian cell vectors. Systems based on prokaryotic and / or eukaryotic vectors can be used in this specification to produce polynucleotides or their homologous polypeptides. Many such systems can be widely available on the market.

[0145] In some embodiments, the vector is a viral vector. Viral vector technology is well known in the art, for example, described in virology and molecular biology handbooks. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and slow viruses. In general, suitable vectors include a replication origin, a promoter sequence, a convenient restriction endonuclease site, and one or more selectable markers that work in at least one organism. (See, for example, WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0146] In some embodiments, the viral vector is a suitable adeno-associated virus (AAV), such as the adeno-associated virus AAV1-AAV8 family. In some embodiments, the viral vector is a viral vector that can infect humans. The desired nucleic acid sequence, such as the nucleic acid for downregulating MMP-9 described above, can be inserted between the terminal inverted repeats (ITRs) of 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 enables the desired nucleic acid to be expressed in bone marrow, lymphoid tissue, connective tissue, kidney, bladder, and other tissues expressing MMP-9. In some embodiments, the promoter is a neuron-selective promoter or a neuron-specific promoter. AAV can be a recombinant AAV, wherein the capsid is from an 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 capsids. In various embodiments, the ITR in AAV is at least one ITR selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and AAV8 ITRs. In various embodiments, the present specification contemplates an AAV8 viral vector (recombinant or non-recombinant) containing a desired nucleic acid expression sequence and at least one promoter sequence, which, when administered to a subject, causes increased systemic expression of the desired nucleic acid. In some embodiments, the viral vector is a recombinant or non-recombinant AAV2 or AAV5 containing any desired nucleic acid expression sequence described herein.

[0147] In some embodiments, the vector of the introduced nucleic acid sequence is a plasmid, which may or may not be integrated into the genome of the host cell when it is introduced into the host cell. Illustrative, non-limiting examples of vectors into which the nucleotide sequence of this specification or the gene construct of this specification can be inserted include tet-on inducible vectors for expression in eukaryotic cells.

[0148] The vector can be obtained by conventional methods known to those skilled in the art (Sambrook et al., 2012). In certain embodiments, the vector is a vector useful for transforming animal cells.

[0149] In certain embodiments, the recombinant expression vector may further comprise a nucleic acid molecule encoding a peptide or peptidomimetic inhibitor of the present specification as described elsewhere herein.

[0150] A promoter may be one naturally associated with a gene or polynucleotide sequence, which may be obtained by isolating 5' non-coding sequences upstream of the coding segment and / or exons. Such promoters may be referred to as "endogenous" promoters. Similarly, an enhancer may be one naturally associated with a polynucleotide sequence, either downstream or upstream of the sequence. Alternatively, certain advantages may be obtained by placing the coding polynucleotide fragment under the control of a recombinant or heterologous promoter. A recombinant or heterologous promoter refers to a promoter that is not normally associated with a polynucleotide sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer that is not normally associated with a polynucleotide sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, promoters or enhancers isolated from any other prokaryotic cell, virus or eukaryotic cell, and promoters or enhancers that are not "naturally occurring", i.e., promoters or enhancers that contain different elements of different transcriptional regulatory regions and / or mutations that alter expression. In addition to synthetically producing nucleic acid sequences for promoters and enhancers, recombinant cloning and / or nucleic acid amplification techniques, including PCR, may be used. TM , in combination with the compositions disclosed herein, production sequences (U.S. Pat. No. 4,683,202, U.S. Pat. No. 5,928,906). In addition, it is contemplated that control sequences directing transcription and / or expression of sequences in non-nuclear organelles such as mitochondria, chloroplasts, etc., may also be used.

[0151] It is important to use promoters and / or enhancers that effectively direct the expression of the DNA fragment in the selected cell type, organelle, and organism. Those of ordinary skill in the field of molecular biology generally know how to use promoters, enhancers, and cell type combinations to express proteins. The promoter used can be constitutive, tissue-specific, inducible, and / or can be used to direct high-level expression of the introduced DNA fragment under appropriate conditions, such as having advantages in large-scale production of recombinant proteins and / or peptides. The promoter can be heterologous or endogenous.

[0152] The recombinant expression vector may also contain a selectable marker gene, which facilitates the selection of transformed or transfected host cells. Suitable selectable marker genes are proteins encoding genes, such as G418 and hygromycin, beta-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or immunoglobulins or portions thereof, such as the Fc portion of an immunoglobulin (preferably IgG) that confer resistance to certain drugs. Selectable markers may be introduced into different vectors from target nucleic acids.

[0153] Combination therapy

[0154] In some embodiments, methods of treating, ameliorating, and / or preventing the diseases and / or disorders contemplated herein comprise administering to a subject an effective amount of at least one compound and / or composition contemplated within the present disclosure.

[0155] In some embodiments, at least one additional agent for treating, improving and / or preventing the diseases and / or disorders contemplated herein is further administered to the subject. In other embodiments, the compound and at least one additional agent are co-administered to the subject. In still other embodiments, the compound and at least one additional agent are co-formulated.

[0156] The compounds contemplated in the present disclosure are intended to be used in combination with one or more additional compounds. These additional compounds may include a compound of the present disclosure and / or at least one additional agent for treating a neurodegenerative disorder, and / or at least one additional agent for treating one or more diseases or disorders contemplated herein.

[0157] For example, the synergy effect can be calculated using a suitable method, such as, for example, Sigmoid-E max Equation (Holford & Scheiner, 1981, Clin. Pharmacokinet. 6: 429-453), Loewe addition equation (equation of Loewe additivity) (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 114: 313-326) and median-effect equation (median-effect equation) (Chou & Talalay, 1984, Adv. Enzyme Regul. 22: 27-55). Each of the above equations can be applied to experimental data to generate corresponding charts to help evaluate the effect of drug combinations. The corresponding charts related to the above equations are concentration-effect curves, isobologram curves and combination index curves.

[0158] Administration / dosage / formulation

[0159] The administration regimen may affect what constitutes an effective amount. The therapeutic formulations contemplated in the present disclosure may be administered to a subject before or after the onset of the disease and / or disorder contemplated herein. In addition, several separate doses may be administered daily or sequentially, as well as staggered doses, or may be infused continuously, or may be bolus injections. In addition, the dosage of the therapeutic formulations contemplated in the present disclosure may be increased or decreased in proportion to the exigencies of the therapeutic or preventive situation.

[0160] The compositions contemplated in the present disclosure are administered to a patient, preferably a mammal, more preferably a human, using known procedures, at a dosage and 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 disease or disorder state of the patient; the age, sex and weight of the patient; and the ability of the therapeutic compound contemplated by the present disclosure to treat the disease and / or disorder contemplated herein in the patient. The dosage regimen may be adjusted to provide the optimal therapeutic response. For example, several divided doses may be administered daily, or the dose may be reduced proportionally according to the exigencies of the therapeutic situation. A non-limiting example of an effective dosage range for a therapeutic compound contemplated in the present disclosure is from about 1 to 5,000 mg / kg body weight / day. A person of ordinary skill in the art will be able to study the relevant factors and determine the effective amount of the therapeutic compound without conducting unnecessary experiments.

[0161] Actual dosage levels of the active ingredients in the pharmaceutical compositions contemplated by the present disclosure may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without causing toxicity to the patient.

[0162] In particular, the selected dosage level will depend upon a variety of factors, including the activity of the particular compound employed, the time of administration, the excretion rate of the compound, the duration of the 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.

[0163] A physician, such as a physician or veterinarian, having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, a physician or veterinarian can start the pharmaceutical composition with a dose of a compound contemplated by the present disclosure that is lower than that required to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved.

[0164] In certain embodiments, it is particularly advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as unit dosages for patients to be treated; each unit contains a predetermined amount of the therapeutic compound calculated to produce the desired therapeutic effect in combination with the desired pharmaceutical carrier. The dosage unit forms contemplated in the present disclosure are determined by and directly dependent upon: (a) the unique characteristics of the therapeutic compound and the specific therapeutic effect to be achieved; and (b) the inherent limitations of the art of compounding / formulation of such therapeutic compounds contemplated herein for use in treating diseases and / or disorders.

[0165] In certain embodiments, the compositions of the present disclosure are formulated using one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the pharmaceutical compositions of the present disclosure include a therapeutically effective amount of a compound of the present disclosure and a pharmaceutically acceptable carrier.

[0166] The carrier can be a solvent or dispersion medium, which contains, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.), suitable mixtures thereof and vegetable oils. Suitable fluidity can be maintained by, for example, using coatings such as lecithin, maintaining the desired particle size under dispersion conditions, and using surfactants. The effects of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferred to add isotonic agents, such as sugars, sodium chloride or polyols such as mannitol and sorbitol, to the composition. The absorption of the injectable composition can be extended by adding a preparation that delays absorption, such as aluminum monostearate or gelatin to the composition.

[0167] In certain embodiments, the compositions of the present disclosure are administered to patients at a dosage of 1 to 5 times or more per day. In another embodiment, the compositions of the present disclosure are administered to patients at a dosage range including, but not limited to, once a day, once every two days, once every three days to once a week, once every two weeks. It will be readily apparent to those of ordinary skill in the art that the frequency of administration of the various compositions of the present disclosure varies from person to person, depending on many factors, including, but not limited to, age, disease or disorder to be treated, sex, general health, and other factors. Therefore, the present disclosure should not be considered to be limited to any specific dosage regimen, and the precise dosage and composition administered to any patient is determined by the attending physician after considering all other factors of the patient.

[0168] The compounds of the present disclosure may be administered in an amount ranging from 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, 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 whole or partial increments therebetween.

[0169] In some embodiments, the dosage of the disclosed compounds is from about 1 mg to about 2500 mg. In some embodiments, the dosage of the disclosed compounds 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. Likewise, 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 whole or partial increments thereof.

[0170] In certain embodiments, the disclosure is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the disclosure, alone or in combination with a second agent; and instructions for using the compound to treat, prevent, or alleviate one or more symptoms of a neurodegenerative disorder in a patient.

[0171] The preparation can be mixed with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances, which are suitable for intracranial, intrathecal, oral, parenteral, nasal, intravenous, subcutaneous, enteral or any other suitable administration known in the art. The pharmaceutical preparation can be sterilized and, if necessary, can also be mixed with adjuvants, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure, buffers, colorants, flavorings and / or aromatic substances, etc. It can also be used in combination with other active agents, such as other analgesics, as needed.

[0172] The administration route of any composition of the present disclosure includes oral, nasal, rectal, vaginal, parenteral, oral, sublingual or topical. The compound used in the present disclosure can be formulated to be administered by any suitable route, such as oral or parenteral, such as transdermal, transmucosal (e.g., sublingual, lingual, (through) oral, (through) urethral, ​​vaginal (e.g., vaginal and perivaginal), nasal (intra), (through) rectal), intravesical, intrapulmonary, intraduodenal, intragastric, intrathecal, subcutaneous, intramuscular, intradermal, intraarterial, intravenous, intrabronchial, inhalation and topical administration.

[0173] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, lozenges, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, slurries, lozenges, creams, ointments, patches, lotions, disks, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosol 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.

[0174] Oral administration

[0175] For oral use, tablets, dragees, liquids, drops, suppositories or capsules, caplets and gelatin caps are particularly suitable. Compositions for oral administration may be prepared according to any method known in the art, and such compositions may contain one or more selected from inert, non-toxic, pharmaceutically acceptable excipients suitable for the manufacture of tablets. These 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 coated by known techniques to maintain elegance or to delay the release of the active ingredient. Oral preparations may also be presented as hard gelatin capsules in which the active ingredient is mixed with an inert diluent.

[0176] For oral administration, the compounds of the present disclosure can be in the form of tablets or capsules prepared by conventional methods with pharmaceutically acceptable excipients, such as binders (e.g., polyvinyl pyrrolidone, hydroxypropyl cellulose or hydroxypropyl methylcellulose); fillers (e.g., corn starch, lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g., magnesium stearate, talc or silicon dioxide); disintegrants (e.g., sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). If desired, the tablets can be coated using suitable methods and coating materials, such as OPADRY available from Colorcon, West Point, Pennsylvania, USA. TM Film coating systems (eg OPADRY TM OY type, OYC type, organic enteric-coated OY-P type, aqueous enteric-coated OY-A type, OY-PM type and OPADRY TM White, 32K18400). Liquid preparations for oral administration may be in the form of solutions, syrups or suspensions. Liquid preparations may be prepared by conventional methods with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methylcellulose or hydrogenated edible fats); emulsifiers (e.g., lecithin or gum arabic); non-aqueous carriers (e.g., almond oil, oily esters or ethanol); and preservatives (e.g., methyl or propyl parabens or sorbic acid).

[0177] The present disclosure also includes a multilayer tablet comprising a layer providing delayed release of one or more compounds of the present disclosure and an additional layer providing immediate release of another drug.Using wax / pH sensitive polymer mixtures, gastro-insoluble compositions can be obtained in which the active ingredient is entrapped, ensuring its delayed release.

[0178] Parenteral administration

[0179] For parenteral administration, the compounds of the present disclosure may be formulated for injection or infusion, e.g., intravenous, intramuscular or subcutaneous injection or infusion, or administered by bolus administration and / or continuous infusion. Suspensions, solutions or emulsions in oily or aqueous vehicles may be used, optionally containing other formulating agents, such as suspending agents, stabilizers and / or dispersing agents.

[0180] Other forms of administration

[0181] Other dosage forms disclosed herein include those described in U.S. Pat. Nos. 6,340,475, 6,488,962, 6,451,808, 5,972,389, 5,582,837 and 5,007,790. Other dosage forms disclosed herein also include those described in U.S. Patent Application Nos. 20030147952, 20030104062, 20030104053, 20030044466, 20030039688 and 2002005182. Other dosage forms disclosed herein include those described in PCT Application Nos. WO 03 / 35041, WO 03 / 35040, WO 03 / 35029, WO 03 / 35177, WO 03 / 35039, WO 02 / 96404, WO 02 / 32416, WO 01 / 97783, WO 01 / 56544, WO 01 / 32217, WO 98 / 55107, WO 98 / 11879, WO 97 / 47285, WO 93 / 18755, and WO 90 / 11757.

[0182] Controlled release formulations and drug delivery systems

[0183] In certain embodiments, the formulations of the present disclosure may be, but are not limited to, short-term, rapid-release, and controlled, such as sustained-release, delayed-release, and pulsatile-release formulations.

[0184] The term sustained release is traditionally used to refer to a drug formulation that provides a gradual release of a drug over a long period of time, which can, but does not necessarily, keep the blood drug level essentially constant over a long period of time. This period of time can be as long as a month or more, and the release time should be longer than the same amount of drug administered in a bolus form.

[0185] For sustained release, the compound may be formulated with a suitable polymer or hydrophobic material which provides the compound with sustained release characteristics.Thus, the compounds used in the disclosed methods may be administered in microparticulate form, for example by injection or by implantation in the form of a wafer or disk.

[0186] In certain embodiments of the present disclosure, a compound of the present disclosure, alone or in combination with another pharmaceutical agent, is administered to a patient using a sustained release formulation.

[0187] The term delayed release is used herein in its conventional sense to refer to a drug formulation that provides an initial release of the drug after a delay following drug administration, which may, but does not necessarily, include a delay of from about 10 minutes to about 12 hours.

[0188] The term pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides for release of the drug in a manner that produces a pulsatile plasma profile of the drug following administration.

[0189] The term immediate release is used in its traditional sense to refer to a drug formulation that releases the drug immediately after administration.

[0190] As used herein, short-term refers to any time period after drug administration 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 after drug administration.

[0191] As used herein, rapid-offset refers to any time period after drug administration 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 whole or partial increments thereof.

[0192] dose

[0193] The therapeutically effective amount or dosage of the compounds disclosed herein depends on the age, sex and weight of the patient, the patient's current physical condition and the progression of the neurodegenerative disorder of the patient being treated. Those skilled in the art can determine the appropriate dosage based on these and other factors.

[0194] Suitable doses of compounds of the present disclosure may be in the range of about 0.01 mg to about 5,000 mg per day, such as about 0.1 mg to about 1,000 mg, for example, about 1 mg to about 500 mg, such as about 5 mg to about 250 mg per day. The dose may be a single dose or multiple doses, for example 1 to 4 times or more per day. When multiple doses are used, the amount of each dose may be the same or different. For example, a dose of 1 mg per day may be administered in two portions, 0.5 mg each time, with an interval of about 12 hours between each dose.

[0195] It is understood that, in non-limiting examples, the amount of compound administered per day may be administered once every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, in the case of administration every other day, a 5 mg dose may be started on Monday, with the first subsequent 5 mg dose administered on Wednesday, the second subsequent 5 mg dose administered on Friday, and so on.

[0196] In the case where the patient's condition does improve, the modulator of the present disclosure is optionally administered continuously, according to the doctor's discretion; optionally, the medication is temporarily reduced or suspended for a period of time (i.e., a "drug holiday"). The length of the drug holiday is optionally between 2 days and 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 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

[0197] Once the patient's condition improves, a maintenance dose may be administered if necessary. Subsequently, the dosage or frequency of administration, or both, is reduced to maintain the level of improved disease, depending on the patient's condition. In certain embodiments, the patient requires long-term intermittent treatment when any symptoms and / or infection recur.

[0198] The compounds used in the disclosed methods can be formulated into unit dosage forms. The term "unit dosage form" refers to physically discrete units, suitable for use as unit dosages for patients receiving treatment, each unit containing a predetermined amount of active substance, calculated to produce the desired therapeutic effect, optionally used with a suitable pharmaceutical carrier. The unit dosage form can be used for a single daily dose or multiple daily doses (e.g., about 1 to 4 times or more per day). When multiple doses are used per day, the unit dosage form for each dose can be the same or different.

[0199] The toxicity and efficacy of such treatment regimens are optionally determined in cell cultures or experimental animals, including but not limited to LD 50 (a dose lethal to 50% of the population) and ED 50 The ratio of the dose between the toxic effect and the therapeutic effect is the therapeutic index, expressed as LD 50 and ED 50 Capsid assembly modulators with high therapeutic indices are preferred. Data obtained from cell culture assays and animal studies can be used to formulate a dosage range for use in humans. The dosage of such capsid assembly modulators is preferably within a circulating concentration range that includes the ED with minimal toxicity. 50 The dosage optionally varies within this range depending upon the dosage form employed and the route of administration utilized.

[0200] Those of ordinary skill in the art recognize or can determine, using no more than routine experimentation, many 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 claims appended hereto. For example, it should be understood that modifications to detection and / or reaction conditions using art-recognized alternatives and methods that do not exceed routine experimentation are within the scope of this application.

[0201] It should be understood that, wherever numerical values ​​and ranges are provided herein, all numerical values ​​and ranges included in these numerical values ​​and ranges are included in the scope of the present disclosure. In addition, all numerical values ​​within these ranges and the upper or lower limits of the numerical ranges are also within the consideration scope of the application.

[0202] Example

[0203] This specification is further described in detail with reference to the following experimental examples. These examples are for illustrative purposes only and are not intended to be limiting unless otherwise stated. Therefore, this specification should not be construed as being limited to the following examples, but should be construed as including any and all variations that are apparent due to the teachings provided herein.

[0204] Example 1: Related Information and Overview

[0205] The progressive transformation of muscle, tendon, ligament, aponeurosis, and fascia into extraskeletal bone tissue is the defining feature of fibrodysplasia ossificans progressiva (FOP; MIM#135100), the most severe form of extraskeletal bone formation in humans. Individuals with FOP appear normal at birth, but have a characteristic deformity of the great toe that is present in all typical patients. During the first decade of life, sporadic soft tissue swellings (or flares) occur in the neck and back, with pathological metamorphosis into mature heterotopic bone via the endochondral route. FOP flares may be triggered by minor trauma such as intramuscular immunization, jaw block for dental work, muscle overexertion, blunt muscle trauma, blows, contusions, falls, or viral illnesses such as influenza, resulting in progressive heterotopic ossification (HO).

[0206] Most patients are immobile by age 30 and require lifelong assistance with activities of daily living. The estimated median life expectancy is 56 years, and death is usually from complications of thoracic hypoplasia syndrome. There is currently no definitive cure for FOP, and there is an unmet need for effective therapies. The current standard of medical management is supportive.

[0207] Heterozygous missense mutations in a bone morphogenetic protein (BMP) type I receptor, activin receptor A type I / activin-like kinase 2 (ACVR1 / ALK2), are found in all individuals with sporadic or familial FOP. ACVR1 mutations result in loss of ACVR1 autoinhibitory function, 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 antagonizes BMP signaling in the wild-type (WT) ACVR1 background, is expressed in individuals with ACVR1. R206H BMP pathway signaling is specifically enhanced in mutant cells and drives ectopic bone formation in FOP.

[0208] Loss of autoinhibition by the mutant receptor is necessary for multiple developmental features of FOP but appears to be insufficient to induce the sporadic acute attacks that lead to disabling postnatal HO. Acute attacks of FOP are closely associated with underlying inflammatory triggers.

[0209] In this study, we found that patient-R had typical developmental features of FOP and a typical ACVR1 (R206H) mutation at age 22 years, but was remarkably resilient to postnatal features of FOP at age 35 years, including the absence of disabling acute attacks and severe, progressive HO. The paradoxical presence of typical developmental abnormalities of FOP and the minimal postnatal features of FOP led to the hypothesis that patient-R lacked an inflammatory trigger that triggered the acute attacks and subsequent heterotopic ossification. Biomarker analysis and genetic studies of patient-R revealed reduced basal activity of matrix metalloproteinase-9 (MMP-9) and compound heterozygosity for MMP-9. These observations prompted studies in FOP mice, which showed that even partial inhibition of MMP-9 activity by genetic, pharmacological, or biological means effectively suppressed HO and revealed unexpected molecular targets for FOP and perhaps more common forms of HO.

[0210] Example 2: Case Report

[0211] Asymptomatic patient R presented to the FOP clinic at the age of 22 years. He was born by term pregnancy and caesarean section. Short malformations of toes and thumbs with absent interphalangeal joints were noted at birth. He received routine immunizations during childhood and had no acute episodes. At the age of three years, he was bitten by a venomous spider on his left thigh. There was no skin necrosis, but a one-centimeter bony nodule was present in his left quadriceps muscle, which did not affect mobility. He had a very active childhood, often participating in contact sports, and had no acute episodes or loss of mobility. There was no family history of skeletal deformities or HO.

[0212] At the age of 21, he presented with multiple episodes of vomiting due to suspected viral gastroenteritis, after which a firm nodule developed on the left side of his neck. Excisional biopsy of the nodule revealed a fibroproliferative lesion. A two-centimeter nonrestrictive band of HO was present at the surgical site. Skeletal examination revealed typical developmental deformities of FOP, including malformed toes and thumbs, multiple ankyloses of the subaxial cervical spine, short and wide femoral necks, hip dysplasia, proximal medial tibial osteochondroma, and a small asymptomatic HO in the lower lumbar spine ( Figure 2A ). Genetic testing confirmed a typical FOP mutation (ACVR1 c.617G>A; R206H). There was no evidence of mosaicism.

[0213] Physical examination revealed that the patient was a 22-year-old healthy male with missing interphalangeal joints of the thumbs and big toes, bilateral proximal medial tibial osteochondromas, and normal range of motion of all other joints of the axial and accessory skeleton except for slightly reduced range of motion of the cervical spine and hip joints. His cumulative analog joint involvement scale (CAJIS) score was 2 / 30 (age-related range: 12-22; median: 18), which is a valid tool for assessing the disease burden of FOP. Patient-R participated in a sponsor-sponsored natural history study of classic FOP patients, in which the patient underwent a baseline whole-body computed tomography (WBCT) to assess the systemic burden of HO. Patient-R's WBCT HO volume was 47,000mm 3 (Average of 13 subjects aged 25-35: 434,000mm 3 ).

[0214] Patient-R had the mildest typical FOP reported to date in a patient of his age, with only one mild acute episode of FOP after trauma, and an approximately 90% reduction in ectopic bone compared with age-matched controls. The presence of typical developmental features of FOP ( Figure 2A ) suggests that the mutant receptor is active during embryonic development. However, the rarity of postnatal inflammatory flares and subsequent HO in FOP and the fact that adults with FOP remain remarkably mobile suggest that FOP patients may lack the inflammatory triggers for flares and HO.

[0215] Researchers have been working to identify factors that lead to acute postnatal onset and HO in this unique, now 35-year-old FOP patient (Patient-R) and to determine if these factors are associated with HO in an accurate FOP mouse model. Identification of such factors may reveal previously unknown protective mechanisms in FOP and herald new therapeutic strategies to prevent and treat the relentless progression of this debilitating disease.

[0216] Example 3: Methods and Materials

[0217] Patient Study Approval

[0218] Patient-R and unaffected control samples were obtained with informed consent. The study protocol was approved by the Research Review Board of the Perelman School of Medicine of the University of Pennsylvania. All human biopsies were obtained before the diagnosis of FOP, as tissue trauma in FOP frequently induces heterotopic ossification.

[0219] Animal research approval

[0220] All animal experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania.

[0221] Venous blood collection

[0222] Venous blood samples were collected from FOP patients and their family members as part of routine clinical care in the Department of Orthopaedics at the Perelman School of Medicine at the University of Pennsylvania. Blood was collected in 10 ml K2 EDTA tubes (BD cat#366643) from adults and children older than two years, and in 4 ml K2 EDTA tubes (BD cat#367861) from children younger than two years. Samples were stored at room temperature until processed 2–24 hours later.

[0223] Peripheral blood mononuclear cells (PBMC) were obtained as follows: the buffy coat was diluted 1:2 with phosphate buffered saline (PBS), then slowly layered on an equal volume of Ficoll-Paque and centrifuged at 400g for 30 minutes to separate PBMC from red blood cells. The mononuclear cell layer was carefully collected and washed twice with PBS. PBMC were stored at -80°C.

[0224] Preparation of venous blood samples

[0225] Plasma was separated using one of two methods, depending on whether PBMCs were isolated from the sample for other reasons. In one or two 15 ml conical tubes, undiluted blood was layered over Ficoll-Paque (GE Healthcare Cat#17-1440-02) and centrifuged at 800 x g, no brake, for 20 min at room temperature. Plasma was collected from the top layer (leaving PBMCs, erythrocytes, and Ficoll) and then transferred to a new 15 ml tube. Plasma was centrifuged at 1400 x g for 10 min to remove residual cells, and the supernatant was aliquoted into 1.5 ml cryogenic vials. Samples were stored below -80°C. Alternatively, undiluted blood was centrifuged at 1400 x g for 20 minutes at room temperature in the original collection tube. The plasma was transferred to a new 15 ml tube and centrifuged at 1400 x g for 10 min to remove residual cells. The resulting supernatant was aliquoted into 1.5 ml cryogenic vials and stored below -80°C.

[0226] One cryovial of each selected sample was thawed on ice, the tube was flicked to mix briefly, and then 500 μl aliquots were dispensed into pre-labeled and frozen 1.5 ml screw-cap microcentrifuge tubes. The tubes were flash frozen in a dry ice / ethanol bath and then placed in a freezer. The tubes were stored at -80°C and shipped overnight on dry ice to Myriad Rules Based Medicine (RBM; Salt Lake City, UT) for multiplex analysis.

[0227] Luminex Multiplex

[0228] Luminex multiplex analysis was performed by Myriad RBM (Pignolo et al., J Bone Miner Res., 2021). All samples were stored at below -70°C prior to testing. Samples were thawed at room temperature, vortexed, spun at 3700 x g for 5 min to clarify the samples, and transferred to the master microplate. Using automated pipetting, aliquots of each sample were added to a single microsphere complex of the selected Multi Analyte Profile and blocker. The mixture was mixed thoroughly and incubated at room temperature for 1 hour. A multiplex mixture of biotinylated reporter antibodies was automatically added, mixed thoroughly, and incubated for another 1 hour at room temperature. The multifactorial reaction was 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 3 times. After the last wash, buffer was added to increase the volume, analyzed using the Luminex instrument, and the resulting data were interpreted using proprietary software developed by Myriad RBM.

[0229] For each multiplex reaction, calibrators and controls were included on each microplate. Eight-point calibrators were run in the first and last columns of each plate to form standard curves, and controls were run in duplicate at three concentration levels. Standard curve, control, and sample QC were performed to ensure proper analytical performance. Study sample values ​​for each analyte were determined using 4- and 5-parameter logistics analysis using weighted and non-weighted curve fitting algorithms in the data analysis software package.

[0230] Cell culture

[0231] PBMCs were thawed and suspended in AIM-V medium at 2.5 × 10 6Cells were cultured in 24-well plates at a density of 10 cells / ml and cultured at 37°C in a 5% CO2 environment. PBMCs were co-cultured with 20 ng / ml TNF-α for 72 hours. Cells were lysed with RIPA lysis and extraction buffer (ThermoFisher Scientific, Grand Island, NY, USA; #89901) containing 1x protease inhibitor cocktail (ThermoFisher Scientific, Grand Island, NY, USA; #89901), and cell culture supernatants were collected for analysis.

[0232] Clinical determination of acute exacerbation

[0233] Forty patient samples, as well as age- and sex-matched controls as closely as possible, were stratified according to the acute exacerbation status at the time of sample collection (Pignolo et al., J Bone Miner Res, 2021). Levels of 113 analytes were measured in plasma samples from four groups of subjects: unaffected individuals and individuals with FOP divided by acute exacerbation status (active, remote, quiescent). Acute exacerbation status was arbitrarily defined at the time of sample collection based on the clinically determined time of onset of symptoms and signs consistent with the last acute exacerbation. Acute exacerbation status was defined as follows: (1) an active acute exacerbation was a clinical acute exacerbation that was present / ongoing at the time of sample collection; (2) an acute exacerbation was remote when it occurred within 1-2 years of sample collection; and (3) a quiescent state occurred when the last acute exacerbation occurred more than 2 years before sample collection. Protein-Protein Interaction (PPI) Maps

[0234] PPI mapping was performed by IBM Watson for Drug Discovery (WDD) and Ingenuity software to generate a relationship network from biomarker predictions. The biological relationship network was extracted for proteins significantly associated with FOP genotype and acute exacerbation status according to previously described methods for analyzing pathway interactions between differentially expressed proteins. Confidence was set at >95% and captured links were supported by at least two published articles.

[0235] FOP mice (cardiotoxin skeletal muscle injury-minocycline and doxycycline treatment)

[0236] Conditional knock-in mouse model Acvr1 [R206H]FlEx (Acvr1 R206H / + ), which generates tamoxifen-induced expression of the full R206H mutant allele after recombination by Cre recombinase (Hatsell et al., Sci Transl Med. 2015 Sep 2; 7(303): 303ra137). R206H / + and MMP-9- / - Acvr1 was generated by crossing CreERT2 mice (The Jackson Laboratory, Bar Harbor, ME, USA; Stock#007084) with CreERT2 mice (The Jackson Laboratory, Bar Harbor, ME, USA; Stock#008463). R206H / + ;CreERT2 - / + 、Acvr1 R206H / + ;CreERT2 - / + ; MMP-9 - / + and Acvr1 R206H / + ;CreERT2 - / + ; MMP-9 - / - Mouse.

[0237] Mice were injected intraperitoneally with tamoxifen (100 mg / kg body weight, 10 mg / ml stock solution, Sigma, St. Louis, MO, USA; #T5648) in corn oil to induce inversion of the R206H mutant allele for a total of 5 injections over 2 weeks (starting at 4 weeks of age).

[0238] The quadriceps muscles of mice (7 weeks old) were injured by injecting 50 μL of 10 μM cardiotoxin from Naja mossambica (Sigma-Aldrich, St. Louis, MO, USA; #C9759).

[0239] Mice were injected intraperitoneally daily with 100 mg / kg minocycline (Cayman Chemical, Ann Arbor, Michigan, USA; #14454) or appropriate controls in pharmaceutical grade saline 3 days before and 14 days after HO induction.

[0240] Acvr1 Q207D / + Mice were a gift from Dr. Yuji Mishina (University of Michigan). To induce the expression of Q207D and subsequently HO, AV-Cre (Vector Center, University of Pennsylvania; 1 × 10 per mouse) was transfected into mice at 3–4 weeks of age. 11 granules) were injected into Acvr1 Q207D / + in the knee fossa of mice.

[0241] The experimental endpoint for in vivo studies analyzing the occurrence of heterotopic ossification was determined to be 2 weeks, based on the fact that control mice already developed HO at this time after muscle injury with cardiotoxin.

[0242] MMP-9 monoclonal antibody related methods and materials

[0243] Mice were treated with MMP-9 antibody (GS-622703, Gilead Sciences, Foster City, California, USA) at 50 mg / kg on day -3, then 15 mg / kg on day 0 (induction of HO) and day 3, or with appropriate controls.

[0244] MMP9 quantification

[0245] Total MMP9 protein (92 kDa prototype and 82 kDa active form) in heparin-treated platelet-poor plasma was measured by sandwich ELISA (human MMP-9 quantitative ELISA kit, DMP900 and mouse total MMP-9 quantitative ELISA kit, MMPT90, R&D Systems, Minneapolis, MN, USA).

[0246] Zymogram analysis

[0247] Equal amounts of cell supernatant and lysed cells (10 ug) were used for gelatin zymography to measure MMP9 using Novex Zymogram Plus gels according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA, ZY00100BOX).

[0248] The gel was scanned, the image inverted, and the densitometry levels determined using ImageJ software (imagej dot nih dot gov / ij / ). The resulting profile yielded peaks corresponding to each band (pro-MMP-9 and active-MMP-9).

[0249] Immunocytology / Histochemistry

[0250] Tissue samples were fixed in 4% paraformaldehyde for 24 hours, decalcified with 10% EDTA for 7 days, snap-frozen or embedded in paraffin, and serially sectioned at 8 μm thickness. Deparaffinized and frozen sections were treated with 10 mM sodium citrate buffer (pH 6.0) at 95°C for 20 min for antigen retrieval.

[0251] For immunofluorescence staining, sections were blocked using Background Buster (Innovex Biosciences, Richmond, CA, USA; NB306), incubated with primary antibodies overnight at 4°C, and then incubated with appropriate Alexa 594-conjugated secondary antibodies and Dapi mounting medium. Images were acquired on a Nikon microscope using NIS software.

[0252] Endogenous peroxidase activity was quenched for immunohistochemistry. Sections were blocked and incubated with primary antibodies overnight at 4°C, followed by appropriate host horseradish peroxidase (HRP) secondary antibodies, DAB detection (SuperPicture Polymer 879263; Thermo Fisher Scientific), and hematoxylin / eosin counterstaining.

[0253] The results were compared with the negative control without primary antibody treatment. The primary antibodies used were as follows: MMP-9 (Millipore, Sigma, Burlington, MA, USA; AB19016; 1:100 dilution).

[0254] CRISPR gene editing of MMP-9 in the THP-1 human leukemic monocytic cell line was performed using TrueCut HiFi Cas9 protein according to the ThermoFisher Scientific protocol to insert 59C>T and 493G>A.

[0255] Micro-computed tomography

[0256] Microcomputed tomography (μCT) of mouse hindlimbs obtained 14 days after injection of cardiotoxin or adenovirus-Cre / cardiotoxin was performed using a Scanco VivaCT 40 device (Bruettisellen, Switzerland) to determine the volume of ectopic bone and obtain two-dimensional images of the medial sagittal plane of each limb. The source voltage used for scanning was 55 kV, the source current was 142 μA, and the isotropic voxel size was 10.5 μm. The upper threshold for distinguishing bone from "non-bone" was 1000 Hounsfield units, and the lower threshold was 150 Hounsfield units.

[0257] Structural Modeling

[0258] Sequence-based prediction algorithms, molecular modeling, and molecular dynamics simulations were used to test the potential of the Ala20Val and Asp165Asn variants to alter MMP9 structure and activity.

[0259] Statistical analysis

[0260] All experiments were performed with at least three technical and biological replicates, and the sample sizes are indicated in the figure legends. Statistical analyses were performed using GraphPad Prism 8.0 software (San Diego, CA, USA). The normality of the data was checked using the D'Agostino & Pearson normality test. Parametric data were analyzed using the appropriate Student's t test when comparing two groups of data; one-way (or two-way) ANOVA followed by post hoc Tukey's test was used when comparing more than two groups of data (or at least two independent factors). Nonparametric data were analyzed using the Mann-Whitney U test when comparing two groups of data; Kruskal-Wallis one-way analysis of variance was used when comparing more than two groups of data. *P < 0.05, **P < 0.01, ***P < 0.001 were considered significant. Data are presented as mean (+ / -) standard error of the mean (SEM).

[0261] Example 4: FOP patient-R has congenital features of FOP but does not have progressive heterotopic ossification after birth and has suppressed inflammatory biomarkers

[0262] Patient-R is a 35-year-old male with a typical Acvr1R206H mutation. Figure 2A-2C Patient-R had typical developmental lesions of FOP, including malformed toes and thumbs ( Figure 2A-2B ), multiple ankyloses of the subaxial cervical spine, a short and wide femoral neck, hip dysplasia, medial proximal tibial osteochondroma, and a small amount of asymptomatic HO in the lower lumbar spine. The presence of the typical big toe deformity in FOP suggests that the mutated Acvr1 receptor in patient-R is active during early development.

[0263] However, Patient-R demonstrated resilience to the postnatal features of FOP. For example, the disabling acute onset and severe progressive HO that characterizes essentially all other FOP patients were absent in Patient-R ( Figure 2C ).

[0264] To elucidate the mechanisms behind patient-R’s resilience to postnatal features of FOP, the present study measured levels of inflammatory biomarkers in patient-R plasma as well as in the plasma of other FOP patients and control subjects. A panel of 113 plasma proteins, including chemokines and cytokines (Custom Human MAP panel), was measured by a quantitative multiplex immunoassay (Myriad RBM) and analyzed in blood collected from FOP patients and control subjects. The results of the analysis revealed that patient-R had fewer postnatal inflammatory markers compared to other FOP patients. As shown in Table 1, inflammatory biomarkers were significantly suppressed in patient-R compared to quiescent FOP patients, as the statistical probability (calculated as p-value) of observing low levels of these biomarkers was very small. Notably, the p-value for plasma MMP-9 levels in patient-R was calculated to be less than 6.8 x 10 -5 .

[0265] Biomarkers p-value(<) CRP 0.03 FRTN 0.03 IgA 0.05 IL-1ra 0.02 IL-1RI 0.05 IL-18 0.02 KLK-7 0.01 MIP-1β 0.001 MIF 0.01 MMP-2 0.04 MMP-9 <![CDATA[6.8x 10 -5 <!-- 26 -->]]> TN-C 0.02 TIMP-1 <![CDATA[3.0x 10 -4 ]]> TNFR 0.02

[0266] Table 1. Plasma inflammatory analytes were suppressed in patient-RR compared with patients with quiescent FOP*. Plasma inflammatory analytes were significantly suppressed in patient-R (two independent blood draws separated by more than one year) compared with patients with quiescent FOP* (N = 11; one sample per patient). P values ​​for each sample are indicated. 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 = angio-regulatory hormone-7; MIP-1β = macrophage inflammatory protein-1beta; MIF = macrophage inhibitory factor; MMP-2 = matrix metalloproteinase-2; MMP-9 = matrix metalloproteinase-9; TN-C = cytokine-C; TIMP-1 = tissue inhibitor of metalloproteinase-1; TNFR = tumor necrosis factor-2. The following plasma inflammatory analytes did not differ significantly between patients-RR and patients with quiescent FOP: adiponectin; complement-3; interleukin-1 receptor type 2; matrix metalloproteinase-3; prostaglandins; T-cell-specific RANTES; thymus-expressed chemokine; tissue inhibitor of metalloproteinase-3. The following plasma inflammatory analytes were too low to be detected in all samples (patient-R and quiescent groups): B-lymphocyte chemoattractant factor; granulocyte-macrophage colony-stimulating factor; gamma interferon; interleukin-1α; interleukin-1β; interleukins-2, 3, 4, 5, 6, 7, 8, 10, 15, 17, 23; matrix metalloproteinases 1, 7, 10; monocyte chemoattractant proteins-1, 3; transforming growth factor beta-3; tumor necrosis factor α; tumor necrosis factor β. *Quiet FOP group defined as: the last acute attack occurred at least 2 years before the plasma sample collection.

[0267] Example 5: Patient-R has low plasma MMP-9 levels and is a compound heterozygote for MMP-9 polymorphic variants

[0268] like Figures 3A-3C As shown, patient-R had lower plasma MMP-9 levels compared with other FOP patients. Figures 3A-3C In this study, a commercially available multiplex panel of 113 plasma soluble analytes from patient-R was compared with 40 typical ACVR1 R206H The same analytes were compared in FOP patients with the ACVR1 mutation and 40 age- and sex-matched controls. R206H Total MMP-9 was significantly reduced in patient-R compared with patients with the mutation and age- and sex-matched controls (p<0.0002) (Pignolo et al., J Bone Miner Res, 2021).

[0269] Whole exome sequencing was performed on patient-R and 19 additional FOP patients with typical phenotypes using DNA isolated from blood or saliva as requested by the UCSF Biosamples for Rare and Orphan Genetics and Bone Tissue (BSTROnG) and University of Pennsylvania cohorts and sequenced as described in the Methods section. Ingenuity variant analysis (IVA, Qiagen) confirmed that all 20 FOP patients had ACVR1 R206H No additional ACVR1 variants were found in any of the subjects who were heterozygous for FOP. A total of approximately 80,000 variants were identified in each FOP patient when compared to the human reference genome UCSC Browser GrCh38.

[0270] Patient-R had several unique variants compared with 19 typical FOP patients. All unique variants were further classified according to pathogenicity using the criteria and guidelines of the American College of Medical Genetics (ACMG). Based on sequencing of the MMP-9 gene in the parents of patient-R, this study identified two polymorphisms in MMP-9 that were transmitted by two different alleles ( Figures 4A-4B Specifically, one allele of the MMP-9 gene in patient-R has a polymorphism that causes a change in the A20V protein sequence (Rs1805088-A20V-T = 0.02201 population frequency [https: / / www.ncbi.nlm.nih.gov / snp / rs1805088#frequency_tab]), while the other allele of the MMP-9 gene in patient-R has a polymorphism that causes a change in the D165N protein sequence (Rs8125581-D165N-A = 0.000360 population frequency [https: / / www.ncbi.nlm.nih.gov / snp / rs8125581#frequency_tab]).

[0271] Example 6: Protein structure modeling of the MMP-9 polymorphism in patient-R suggests that it is less functional than the normal allele

[0272] Our in silico studies suggest that the A20V variant of MMP-9 may impair signal peptide cleavage, interfere with translation on the rough endoplasmic reticulum (ER), affect folding within the ER, or disrupt MMP-9 binding to secretory pathway membranes.

[0273] The D165N polymorphism is located within the catalytic domain of MMP-9 and is predicted by multiple computational methods to be highly detrimental to the structure and function of MMP9 and to result in a non-functional variant (Bhatnager et al., Computational Biology and Chemistry, Vol. 77, December 2018, pp. 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 may be ineffective in inhibiting enzyme activity because it cannot form the necessary ion pair with His118 like wild-type MMP-9. Importantly, the D165N variant of MMP-9 is predicted to be extremely unstable, which will reduce folding efficiency in the endoplasmic reticulum, which is a quality control site that regulates the transport of properly folded secretory proteins. Since ion pairs stabilize the loop of several residues C-terminal to the MMP-9 cleavage and activation site, the loop of the variant D165N MMP-9 protein may not adopt a morphological structure that is optimal for proteolysis of the activating enzyme plasminogen (Gong et al., J Clin Invest 118:3012-3024, 2008).

[0274] Although the D165N single nucleotide polymorphism (SNP) found in patient-R is rare and non-pathogenic in the general population, this variant is expected to have a protective effect in patients with aortic aneurysms. In addition, 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, indicating that in addition to the reduction in the amount of secreted protein, the amino acid substitution also has a great impact on the enzymatic activity of MMP-9. The MMP-9 variants detected in patient-R (A20V and D165N) provide a structural basis for the loss of function of secreted MMP-9 and have an impact on the inhibition of HO in FOP.

[0275] Example 7: MMP-9 is expressed in early lesion tissues of FOP patients

[0276] In certain embodiments, early lesional tissue biopsies from typically affected human patients with FOP (ie, human FOP patients with the Acvr1 R206H mutation and exhibiting postnatal symptoms of FOP) show positive staining for MMP-9 in inflammatory cells.

[0277] Similar results were found in FOP model mice. Figure 5 , FOP model mice (Acvr1 R206H / + ;CreERT2 - / +) Images of lesion tissue after cardiotoxin injury showed that MMP-9 was expressed in inflammatory cells in the lesion area early on. In addition, the expression of MMP-9 in lesion tissue gradually decreased from day 1 to day 5.

[0278] Example 8: Downregulation of MMP-9 levels or activity reduces heterotopic ossification in a mouse FOP model

[0279] refer to Figures 6A-6C , cardiotoxin-induced soft tissue injury in the following models: Acvr1 R206H / + ;CreERT2 - / + mice (FOP model mice with the classic R206H mutation in one Acvr1 allele), Acvr1 R206H / + ;CreERT2 - / + ; MMP-9 - / + mice (FOP model mice with only one MMP-9 gene allele) or Acvr1 R206H / + ;CreERT2 - / + ; MMP-9 - / - Mice (FOP model mice in which both pairs of MMP-9 gene alleles were deleted) were used, and the level of injury-induced heterotopic ossification (HO) was detected and quantified by microCT.

[0280] like Figure 6C As shown, MMP9 - / + ;Acvr1 R206H / + ;CreERT2 - / + and MMP9 - / - ;Acvr1 R206H / + ;CreERT2 - / + Mice produce significantly less MMP-9 than Acvr1 R206H / + ;CreERT2 - / + Control mice.

[0281] like Fig. 6A Shown and in Figure 6B Medium quantification, Acvr1 R206H / + ;CreERT2 - / + Mice displayed high levels of HO, while Acvr1 R206H / + ;CreERT2 - / + ; MMP-9 - / - Mice displayed significantly lower levels of HO. Interestingly, Acvr1 R206H / + ;CreERT2 - / + ; MMP-9 - / + Mice displayed significantly lower HO levels than Acvr1 R206H / + ;CreERT2 - / + Mouse, with Acvr1R206H / + ;CreERT2 - / + ; MMP-9 - / - These results suggest that even partial reduction of MMP-9 expression and activity is sufficient to reduce injury-induced HO in FOP model mice.

[0282] Since small molecule compounds that inhibit MMP-9 activity are available, this study tested the effects of minocycline, a MMP-9 inhibitor, on FOP model mice.

[0283] Minocycline is a broad-spectrum tetracycline antibiotic used to treat a variety of bacterial infections such as urinary tract infections, respiratory tract infections, skin infections, severe acne, gonorrhea, Rocky Mountain spotted fever, chlamydia, and many more.

[0284] refer to Figure 7 D-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.

[0285] refer to Figure 6D-6F , minocycline almost completely eliminated Acvr1 R206H / + ;CreERT2 - / + HO induced by soft tissue injury in FOP mice. Figure 6G-6H Minocycline also significantly reduced Acvr1 Q207D / + HO levels induced by soft tissue injury in FOP mice.

[0286] refer to Fig.6I , minocycline administration before or during lesion activation prevented HO. These data suggest that MMP-9 plays a role in inducing HO during the early inflammatory stage.

[0287] This study subsequently attempted to downregulate MMP-9 using anti-MMP-9 monoclonal antibodies (Marshall et al., PLoS One. 2015 May 11; 10(5): e0127063). Figure 6K , in Acvr1 R206H / + ;CreERT2 - / + In FOP mice, administration of this antibody significantly reduced heterotopic ossification in the FOP mouse model.

[0288] Example 9: Selective Discussion

[0289] This study demonstrates that MMP-9 plays a key role in the pathogenesis of HO in FOP. The presence of typical developmental findings of FOP (proximal tibial osteochondroma, congenital cervical fusion, hallux valgus deformity, and shortened thumbs) and the rare acute episodes and HO in patient-R after birth suggest that FOP is active during embryonic development but relatively inactive after birth, which may be due to the lack of inflammatory factors that trigger HO. The findings of significantly reduced MMP-9 reserves and activity in patient-R and the presence of putative protective polymorphisms in the MMP-9 gene have drawn attention to this factor, and studies in FOP mice have further verified the role of this factor in the HO-suppressed phenotype. Together, these results support that MMP-9 mediates HO by regulating the inflammatory response to tissue abnormalities.

[0290] MMP-9 (or gelatinase B) belongs to a multigene family of more than 20 matrix metalloproteinases that are conserved throughout the animal kingdom and that process or degrade many pericellular matrices. In a range of physiological and pathological processes including embryonic development, skeletal morphogenesis, fracture repair, inflammation, wound healing, angiogenesis, heart disease, arthritis, and cancer, MMP-9 plays an important multifaceted role as an inflammatory regulator in remodeling the extracellular matrix, activating, deactivating, and / or altering proinflammatory cytokines and other signaling molecules, and coordinating the migration of stem cells and tissue progenitor cells. Specifically, MMP-9 triggers degradation and remodeling of the extracellular matrix, release and activation of growth factors, remodeling of the stem cell niche, and recruitment and migration of inflammatory cells and stem cells.

[0291] MMP-9 is expressed in neutrophils, macrophages, and mast cells, is regulated by inflammatory cytokines, is a regulator of inflammation and innate immunity, integrates multiple immunomodulatory pathways, and promotes the destruction of biochemical and physical barriers to T cell trafficking. MMP-9 expression levels are also increased in ischemic skeletal muscle, which is present in early acute attacks of FOP.

[0292] MMP-9 is also a signaling inducer of endochondral ossification. In the early stages of fracture repair, MMP-9 regulates the differentiation of chondrocytes and osteoblasts and is expressed throughout the entire process of fracture repair. - / - Fracture healing and nonunion were abnormal in mice.

[0293] The role of MMP-9 in the pathogenesis of FOP has not been investigated. The clinical, biochemical, and genetic findings of patient-R in this study suggest that even partial reduction of MMP-9 levels can prevent typical ACVR1 R206H Disabling acute attacks and HO in the presence of mutations, as haploinsufficiency of MMP-9 is protective against HO in FOP mice.

[0294] In vivo studies in a genetically accurate trauma-induced FOP mouse model confirmed this hypothesis and demonstrated that MMP-9 plays a key role in the development of HO and that genetic, pharmacological, and biological reduction of MMP-9 levels protected the FOP mouse model from HO. Taken together, these data suggest that pharmacological reduction of MMP-9 levels in FOP patient tissues can prevent HO.

[0295] This study focused on tetracyclines. Tetracyclines have been used as antibiotics for nearly 40 years to treat both gram-positive and gram-negative bacteria. However, as second-generation semisynthetic tetracyclines, minocycline and doxycycline, in addition to their antibiotic properties, are potent inhibitors of MMP-9 at sub-antimicrobial doses.

[0296] In summary, this study identifies MMP-9 as an important regulator of HO in FOP and demonstrates that systematic investigation of a single resilient individual can reveal unexpected disease mechanisms, leading to new therapeutic strategies.

[0297] Examples of implementation methods

[0298] In some aspects, the present invention relates to the following non-limiting embodiments:

[0299] Embodiment 1: A method for treating, ameliorating and / or preventing fibrodysplasia ossificans progressiva (FOP) in a subject in need thereof, the method comprising downregulating the level and / or activity of matrix metalloproteinase-9 (MMP-9) in the subject.

[0300] Embodiment 2: The method according to embodiment 1, wherein downregulating the level and / or activity of MMP-9 in the subject comprises administering to the subject an effective amount of:

[0301] Small molecule MMP-9 inhibitors,

[0302] Protein MMP-9 inhibitors,

[0303] A nucleic acid (and / or an expression vector expressing the nucleic acid) that downregulates MMP-9 by RNA interference,

[0304] A ribozyme (and / or a vector expressing a ribozyme) that downregulates MMP-9,

[0305] An expression vector comprising an expression cassette, wherein the expression cassette expresses a CRISPR component that downregulates MMP-9 by CRISPR knockout and / or CRISPR knockdown, and

[0306] A trans-dominant negative mutant protein of MMP-9, and / or an expression vector expressing a trans-dominant negative mutant protein of MMP-9.

[0307] Embodiment 3: The method according to any one of embodiments 1-2, wherein the subject has a mutated ACVR1 gene.

[0308] Embodiment 4: The method according to embodiment 3, wherein the mutated ACVR1 gene encodes a constitutively active ACVR1 polypeptide.

[0309] Embodiment 5: The method according to embodiment 4, wherein the ACVR1 polypeptide comprises at least one mutation selected from the group consisting of L196P, P197-F198del ins L, R202I, R206H, Q207E, F246Y, R258S, R258G, G325A, G328E, G328W, G328R, G356D, R375P and K400E.

[0310] Embodiment 6: The method according to any one of embodiments 2-5, wherein the small molecule MMP-9 inhibitor is selected from the group consisting of doxycycline, cyclocycline and minocycline or a salt or solvate thereof.

[0311] Embodiment 7: The method according to any one of embodiments 2-5, wherein the protein MMP-9 inhibitor is an anti-MMP-9 antibody or an antigen-binding fragment thereof.

[0312] Embodiment 8: The method according to any one of embodiments 1-7, further comprising surgically removing ossified tissue from the subject.

[0313] Embodiment 9: The method according to embodiment 8, wherein the surgical removal step is performed after the level or activity of MMP-9 in the subject is downregulated.

[0314] Embodiment 10: The method according to any one of embodiments 1-9, wherein the subject is a human.

[0315] Embodiment 11: A kit for treating, ameliorating and / or preventing fibrodysplasia ossificans progressiva (FOP) in a subject in need thereof, the method comprising downregulating the level and / or activity of matrix metalloproteinase 9 (MMP-9) in the subject, comprising:

[0316] Compounds for downregulating the level and / or activity of matrix metalloproteinase 9 (MMP-9) in a subject; and

[0317] Instructions for administering to a subject an effective amount of the compound.

[0318] Embodiment 12: The kit according to embodiment 11, wherein the compound comprises at least one selected from the group consisting of:

[0319] Small molecule MMP-9 inhibitors,

[0320] Protein MMP-9 inhibitors,

[0321] A nucleic acid (and / or an expression vector expressing the nucleic acid) that downregulates MMP-9 by RNA interference,

[0322] A ribozyme (and / or a vector expressing a ribozyme) that downregulates MMP-9,

[0323] An expression vector comprising an expression cassette, wherein the expression cassette expresses a CRISPR component that downregulates MMP-9 by CRISPR knockout and / or CRISPR knockdown, and

[0324] A trans-dominant negative mutant protein of MMP-9, and / or an expression vector expressing a trans-dominant negative mutant protein of MMP-9.

[0325] Embodiment 13: The kit according to any one of embodiments 11-12, wherein the subject has a mutated ACVR1 gene.

[0326] Embodiment 14: The kit according to embodiment 13, wherein the mutated ACVR1 gene encodes a constitutively active ACVR1 polypeptide.

[0327] Embodiment 15: The kit according to embodiment 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.

[0328] Embodiment 16: The kit according to any one of embodiments 11-15, wherein the small molecule MMP-9 inhibitor is selected from the group consisting of doxycycline, cyclocycline and minocycline.

[0329] Embodiment 17: The kit according to any one of embodiments 11 to 15, wherein the protein MMP-9 inhibitor is an anti-MMP-9 antibody or an antigen-binding fragment thereof.

[0330] Embodiment 18: The kit according to any one of embodiments 11-17, wherein the manual further comprises instructions for surgically removing ossified tissue from the subject.

[0331] Embodiment 19: The kit according to embodiment 18, wherein the instructions further include instructions for performing the surgical removal after the level or activity of MMP-9 in the subject is downregulated.

[0332] Embodiment 20: The kit according to any one of embodiments 11-19, wherein the subject is a human.

[0333] The features of several embodiments are summarized above so that those of ordinary skill in the art can better understand the various aspects of the present disclosure. Those of ordinary skill in the art should recognize that they can easily use the present disclosure as a basis for designing or modifying other processes and structures for implementing the same purpose and / or achieving the same advantages of the embodiments described herein. Those of ordinary skill in the art should also recognize that such equivalent structures do not depart from the spirit and scope of the present disclosure, and that they can make various changes, substitutions and modifications to the present disclosure without departing from the spirit and scope of the present disclosure.

Claims

1. A method for treating, ameliorating and / or preventing fibrodysplasia ossificans progressiva (FOP) in a subject in need thereof, the method comprising downregulating the level and / or activity of matrix metalloproteinase-9 (MMP-9) in the subject.

2. The method of claim 1, wherein downregulating the level and / or activity of MMP-9 in the subject comprises administering to the subject an effective amount of: Small molecule MMP-9 inhibitors, Protein MMP-9 inhibitors, A nucleic acid (and / or an expression vector expressing said nucleic acid) which downregulates MMP-9 by RNA interference, A ribozyme (and / or a vector expressing said ribozyme) which downregulates MMP-9, An expression vector comprising an expression cassette, wherein the expression cassette expresses a CRISPR component that downregulates MMP-9 by CRISPR knockout and / or CRISPR knockdown, and A trans dominant negative mutant protein of MMP-9, and / or an expression vector expressing the trans dominant negative mutant protein of MMP-9.

3. The method of any one of claims 1-2, wherein the subject has a mutated ACVR1 gene.

4. The method of claim 3, wherein the mutated ACVR1 gene encodes a constitutively active ACVR1 polypeptide.

5. The method of 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 method according to any one of claims 2-5, wherein the small molecule MMP-9 inhibitor is selected from the group consisting of doxycycline, cyclocycline and minocycline, or a salt or solvate thereof.

7. The method according to any one of claims 2 to 5, wherein the protein MMP-9 inhibitor is an anti-MMP-9 antibody or an antigen-binding fragment thereof.

8. The method of any one of claims 1-7, further comprising surgically removing the ossified tissue from the subject.

9. The method of claim 8, wherein the surgical removal step is performed after the level or activity of MMP-9 in the subject is downregulated.

10. The method of any one of claims 1-9, wherein the subject is a human.

11. A kit for treating, ameliorating and / or preventing fibrodysplasia ossificans progressiva (FOP) in a subject in need thereof, the kit comprising: Compounds for downregulating the level and / or activity of matrix metalloproteinase 9 (MMP-9) in a subject; and Instructions for administering to a subject an effective amount of the compound.

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

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

14. The kit of claim 13, wherein the mutated ACVR1 gene encodes a constitutively active ACVR1 polypeptide.

15. The kit of claim 14, wherein the ACVR1 polypeptide comprises at least one mutation selected from the group consisting of L196P, P197-F198del ins L, R202I, R206H, Q207E, F246Y, R258S, R258G, G325A, G328E, G328W, G328R, G356D, R375P, and K400E.

16. The kit according to any one of claims 12-15, wherein the small molecule MMP-9 inhibitor is selected from the group consisting of doxycycline, cyclocycline and minocycline.

17. The kit according to any one of claims 12 to 15, wherein the protein MMP-9 inhibitor is an anti-MMP-9 antibody or an antigen-binding fragment thereof.

18. The kit of any one of claims 11-17, wherein the instructions further provide for surgically removing ossified tissue from the subject.

19. The kit of claim 18, wherein the instructions further comprise instructions for performing the surgical removal after the level or activity of MMP-9 in the subject is downregulated.

20. The kit according to any one of claims 11-19, wherein the subject is a human.

Images