Methods of treating, ameliorating and / or preventing fibrodysplasia ossificans progressiva and heterotopic ossification, and kits for the same

Abstract

Disclosed herein are methods for treating, ameliorating and / or preventing fibrodysplasia ossificans progressiva (FOP) in a subject in need thereof, and methods for treating, ameliorating and / or preventing non-FOP heterotopic ossification (HO) in a subject in need thereof. The methods include downregulating matrix metalloproteinase 9 (MMP-9) level and / or activity in the subject. Also disclosed are kits for carrying out the methods disclosed herein.

Description

Attorney Docket No.046483-7440WO1(03745) METHODS OF TREATING, AMELIORATING AND / OR PREVENTING FIBRODYSPLASIA OSSIFICANS PROGRESSIVA AND HETEROTOPIC OSSIFICATION, AND KITS FOR THE SAME CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No.63 / 587,880, filed October 4, 2023, which is incorporated herein by reference in its entirety. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The XML file named "046483-7440WO1_Seq Listing.xml" created on September 27, 2024, comprising 61,462 bytes, is hereby incorporated by reference in its entirety. BACKGROUND

[0003] Fibrodysplasia ossificans progressiva (FOP; MIM#135100) is the most catastrophic 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 (ossified), forming bone outside the skeleton (extra-skeletal or heterotopic bone) that constrains movement.

[0004] Individuals with FOP appear normal at birth except for characteristic malformations of the great toes that are present in all classically affected individuals. During the first decade of life, episodic soft tissue swellings (or flare-ups) arise in the neck and back and undergo pathological metamorphosis into mature heterotopic bone through an endochondral pathway. Minor trauma such as intramuscular immunizations, mandibular blocks for dental work, muscle over-exertion, blunt muscle trauma, bumps, bruises, falls, or influenza-like viral illnesses can trigger flare-ups of FOP that lead to progressive heterotopic ossification (HO). Most patients are immobilized by the age 30, and require lifelong assistance with activities of daily living. The median estimated lifespan is 56 years; death often results from complications of thoracic insufficiency syndrome.

[0005] Currently, palovarotene is approved for the treatment of FOP. The efficacy of this compound, however; is not satisfactory and the current standard-of-care medical management is largely supportive in nature. There is a need for methods and kits that can be used for treating, ameliorating, and / or preventing FOP. The present invention addresses this need.

[0006] Apart from genetic predisposition in the case of FOP, heterotopic ossification can 1 53121668.1Attorney Docket No.046483-7440WO1(03745) also be caused by trauma or injury in the bone, severe burns, stroke, polio, tetanus, syringomyelia, myelodysplastic syndromes, multiple sclerosis (MS), carbon monoxide poisoning, tumors in the spine, among others. Thus, there is a need for methods and kits that can be used for treating, ameliorating, and / or preventing non-FOP HO. The present invention addresses this need, as well. SUMMARY

[0007] In some aspects, the present invention is directed to the following non-limiting embodiments: Method of treating, ameliorating, and / or preventing fibrodysplasia ossificans progressiva (FOP)

[0008] In some aspects, the present invention is directed to a method of treating, ameliorating, and / or preventing fibrodysplasia ossificans progressiva (FOP) in a subject in need thereof.

[0009] In some embodiments, the method comprising down-regulating matrix metalloproteinase 9 (MMP-9) level and / or activity in the subject.

[0010] In some embodiments, down-regulating the MMP-9 level and / or activity in the subject comprises administering to the subject an effective amount of a small molecule MMP-9 inhibitor.

[0011] In some embodiments, down-regulating the MMP-9 level and / or activity in the subject comprises administering to the subject an effective amount of a protein MMP-9 inhibitor.

[0012] In some embodiments, down-regulating the MMP-9 level and / or activity in the subject comprises administering to the subject an effective amount of a nucleic acid (and / or an expression vector expressing the nucleic acid) that downregulates MMP-9 by RNA interference.

[0013] In some embodiments, down-regulating the MMP-9 level and / or activity in the subject comprises administering to the subject an effective amount of a ribozyme (and / or a vector expressing the ribozyme) that downregulates MMP-9.

[0014] In some embodiments, down-regulating the MMP-9 level and / or activity in the subject comprises administering to the subject an effective amount of an expression vector comprising an expression cassette, wherein the expression cassette expresses CRISPR components that downregulate MMP-9 by CRISPR knockout and / or CRISPR knockdown.

[0015] In some embodiments, down-regulating the MMP-9 level and / or activity in the 2 53121668.1Attorney Docket No.046483-7440WO1(03745) subject comprises administering to the subject an effective amount of a trans-dominant negative mutant protein of MMP-9, and / or an expression vector that expresses the trans- dominant negative mutant protein of MMP-9.

[0016] In some embodiments, the subject has a mutant ACVR1 gene.

[0017] In some embodiments, the mutant ACVR1 gene encodes a constitutively active ACVR1 polypeptide.

[0018] In some embodiments, the ACVR1 polypeptide comprises at least one mutation selected from the group consisting of L196P, P197-F198 del ins L, R202I, R206H, Q207E, F246Y, R258S, R258G, G325A, G328E, G328W, G328R, G356D, R375P, and K400E.

[0019] In some embodiments, the small molecule MMP-9 inhibitor is selected from the group consisting of metformin, doxycycline, incyclinide, and minocycline, or a salt or solvate thereof.

[0020] In some embodiments, the protein MMP-9 inhibitor is an anti-MMP-9 antibody or an antigen binding fragment thereof.

[0021] In some embodiments, the anti-MMP-9 antibody or the antigen binding fragment thereof comprises a heavy chain complementarity-determining region 1 (CDR1) set forth in SEQ ID NO:3, a heavy chain CDR2 set forth in SEQ ID NO:4, a heavy chain CDR4 set forth in SEQ ID NO:5, a light chain CDR1 set forth in SEQ ID NO:6, a light chain CDR2 set forth in SEQ ID NO:7, and a light chain CDR3 set forth in SEQ ID NO:8.

[0022] In some embodiments, the anti-MMP-9 antibody or the antigen binding fragment thereof comprises a heavy chain CDR1 set forth in SEQ ID NO:15, a heavy chain CDR2 set forth in SEQ ID NO:16, a heavy chain CDR4 set forth in SEQ ID NO:17, a light chain CDR1 set forth in SEQ ID NO:18, a light chain CDR2 set forth in SEQ ID NO:19, and a light chain CDR3 set forth in SEQ ID NO:20.

[0023] In some embodiments, the anti-MMP-9 antibody or the antigen binding fragment thereof comprises a heavy chain CDR1 set forth in SEQ ID NO:35, a heavy chain CDR2 set forth in SEQ ID NO:36, a heavy chain CDR4 set forth in SEQ ID NO:37, a light chain CDR1 set forth in SEQ ID NO:38, a light chain CDR2 set forth in SEQ ID NO:39, and a light chain CDR3 set forth in SEQ ID NO:40.

[0024] In some embodiments, the anti-MMP-9 antibody or the antigen binding fragment thereof comprises a light chain CDR1 set forth in SEQ ID NO:45, a light chain CDR2 set forth in SEQ ID NO:46, and a light chain CDR3 set forth in SEQ ID NO:47.

[0025] In some embodiments, the anti-MMP-9 antibody or the antigen binding fragment thereof comprises a heavy chain variable region set forth in SEQ ID NO:9, and a light chain 3 53121668.1Attorney Docket No.046483-7440WO1(03745) variable region set forth in SEQ ID NO:10.

[0026] In some embodiments, the anti-MMP-9 antibody or the antigen binding fragment thereof comprises a heavy chain variable region selected from the group consisting of SEQ ID NOs:21, 23, 25, 27 and 29, and a light chain variable region selected from the group consisting of SEQ ID NOs:22, 24, 26, 28 and 30.

[0027] In some embodiments, the anti-MMP-9 antibody or the antigen binding fragment thereof comprises a heavy chain variable region set forth in SEQ ID NO:21 and a light chain variable region set forth in SEQ ID NO:22.

[0028] In some embodiments, the anti-MMP-9 antibody or the antigen binding fragment thereof comprises a heavy chain variable region set forth in SEQ ID NO:27 and a light chain variable region set forth in SEQ ID NO:30.

[0029] In some embodiments, the anti-MMP-9 antibody or the antigen binding fragment thereof comprises a heavy chain variable region set forth in SEQ ID NO:41 and a light chain variable region set forth in SEQ ID NO:42.

[0030] In some embodiments, the anti-MMP-9 antibody or the antigen binding fragment thereof comprises a light chain variable region set forth in SEQ ID NO:48.

[0031] In some embodiments, the method further comprises surgically removing an ossified tissue from the subject.

[0032] In some embodiments, the surgically removing step is performed after the MMP-9 level or activity in the subject is down-regulated.

[0033] In some embodiments, the subject is a human. Method of treating, ameliorating and / or preventing a non-FOP heterotopic ossification (HO)

[0034] In some embodiments, the present invention is directed to a method of treating, ameliorating and / or preventing a non-FOP heterotopic ossification (HO) in a subject in need thereof.

[0035] In some embodiments, the method comprising down-regulating matrix metalloproteinase 9 (MMP-9) level and / or activity in the subject.

[0036] In some embodiments, down-regulating the MMP-9 level and / or activity in the subject comprises administering to the subject an effective amount of a small molecule MMP-9 inhibitor.

[0037] In some embodiments, down-regulating the MMP-9 level and / or activity in the subject comprises administering to the subject an effective amount of a protein MMP-9 inhibitor. 4 53121668.1Attorney Docket No.046483-7440WO1(03745)

[0038] In some embodiments, down-regulating the MMP-9 level and / or activity in the subject comprises administering to the subject an effective amount of a nucleic acid (and / or an expression vector expressing the nucleic acid) that downregulates MMP-9 by RNA interference.

[0039] In some embodiments, down-regulating the MMP-9 level and / or activity in the subject comprises administering to the subject an effective amount of a ribozyme (and / or a vector expressing the ribozyme) that downregulates MMP-9.

[0040] In some embodiments, down-regulating the MMP-9 level and / or activity in the subject comprises administering to the subject an effective amount of an expression vector comprising an expression cassette, wherein the expression cassette expresses CRISPR components that downregulate MMP-9 by CRISPR knockout and / or CRISPR knockdown.

[0041] In some embodiments, down-regulating the MMP-9 level and / or activity in the subject comprises administering to the subject an effective amount of a trans-dominant negative mutant protein of MMP-9, and / or an expression vector that expresses the trans- dominant negative mutant protein of MMP-9.

[0042] In some embodiments, the small molecule MMP-9 inhibitor is selected from the group consisting of metformin, doxycycline, incyclinide, and minocycline, or a salt or solvate thereof.

[0043] In some embodiments, the protein MMP-9 inhibitor is an anti-MMP-9 antibody or an antigen binding fragment thereof.

[0044] In some embodiments, the anti-MMP-9 antibody or the antigen binding fragment thereof comprises a heavy chain complementarity-determining region 1 (CDR1) set forth in SEQ ID NO:3, a heavy chain CDR2 set forth in SEQ ID NO:4, a heavy chain CDR4 set forth in SEQ ID NO:5, a light chain CDR1 set forth in SEQ ID NO:6, a light chain CDR2 set forth in SEQ ID NO:7, and a light chain CDR3 set forth in SEQ ID NO:8.

[0045] In some embodiments, the anti-MMP-9 antibody or the antigen binding fragment thereof comprises a heavy chain CDR1 set forth in SEQ ID NO:15, a heavy chain CDR2 set forth in SEQ ID NO:16, a heavy chain CDR4 set forth in SEQ ID NO:17, a light chain CDR1 set forth in SEQ ID NO:18, a light chain CDR2 set forth in SEQ ID NO:19, and a light chain CDR3 set forth in SEQ ID NO:20.

[0046] In some embodiments, the anti-MMP-9 antibody or the antigen binding fragment thereof comprises a heavy chain CDR1 set forth in SEQ ID NO:35, a heavy chain CDR2 set forth in SEQ ID NO:36, a heavy chain CDR4 set forth in SEQ ID NO:37, a light chain CDR1 set forth in SEQ ID NO:38, a light chain CDR2 set forth in SEQ ID NO:39, and a light chain 5 53121668.1Attorney Docket No.046483-7440WO1(03745) CDR3 set forth in SEQ ID NO:40.

[0047] In some embodiments, the anti-MMP-9 antibody or the antigen binding fragment thereof comprises a light chain CDR1 set forth in SEQ ID NO:45, a light chain CDR2 set forth in SEQ ID NO:46, and a light chain CDR3 set forth in SEQ ID NO:47.

[0048] In some embodiments, the anti-MMP-9 antibody or the antigen binding fragment thereof comprises a heavy chain variable region set forth in SEQ ID NO:9, and a light chain variable region set forth in SEQ ID NO:10.

[0049] In some embodiments, the anti-MMP-9 antibody or the antigen binding fragment thereof comprises a heavy chain variable region selected from the group consisting of SEQ ID NOs:21, 23, 25, 27 and 29, and a light chain variable region selected from the group consisting of SEQ ID NOs:22, 24, 26, 28 and 30.

[0050] In some embodiments, the anti-MMP-9 antibody or the antigen binding fragment thereof comprises a heavy chain variable region set forth in SEQ ID NO:21 and a light chain variable region set forth in SEQ ID NO:22.

[0051] In some embodiments, the anti-MMP-9 antibody or the antigen binding fragment thereof comprises a heavy chain variable region set forth in SEQ ID NO:27 and a light chain variable region set forth in SEQ ID NO:30.

[0052] In some embodiments, the anti-MMP-9 antibody or the antigen binding fragment thereof comprises a heavy chain variable region set forth in SEQ ID NO:41 and a light chain variable region set forth in SEQ ID NO:42.

[0053] In some embodiments, the anti-MMP-9 antibody or the antigen binding fragment thereof comprises a light chain variable region set forth in SEQ ID NO:48.

[0054] In some embodiments, the non-FOP HO is caused by a trauma or injury in the bone, a severe burn, a stroke, a head trauma, paralysis, an orthopaedic procedure, a hip replacement, a severe crush injury, an explosive wound, a war wound, a severe civilian trauma, polio, tetanus, a syringomyelia, a myelodysplastic syndrome, multiple sclerosis (MS), carbon monoxide poisoning, or a tumor in the spine.

[0055] In some embodiments, the subject is a human. Kit for treating, ameliorating and / or preventing fibrodysplasia ossificans progressiva (FOP) or a non-FOP heterotopic ossification (HO)

[0056] In some aspects, the present invention is directed to a kit for treating, ameliorating and / or preventing fibrodysplasia ossificans progressiva (FOP) or a non-FOP heterotopic ossification (HO) in a subject in need thereof. 6 53121668.1Attorney Docket No.046483-7440WO1(03745)

[0057] In some embodiments, the kit comprises a compound for down-regulating matrix metalloproteinase 9 (MMP-9) level and / or activity in the subject; and an instruction for administering an effective amount of the compound to the subject.

[0058] In some embodiments, the compound comprises a small molecule MMP-9 inhibitor.

[0059] In some embodiments, the compound comprises a protein MMP-9 inhibitor.

[0060] In some embodiments, the compound comprises a nucleic acid (and / or an expression vector expressing the nucleic acid) that downregulates MMP-9 by RNA interference.

[0061] In some embodiments, the compound comprises a ribozyme (and / or a vector expressing the ribozyme) that downregulates MMP-9.

[0062] In some embodiments, the compound comprises an expression vector comprising an expression cassette, wherein the expression cassette expresses CRISPR components that downregulate MMP-9 by CRISPR knockout and / or CRISPR knockdown.

[0063] In some embodiments, the compound comprises a trans-dominant negative mutant protein of MMP-9, and / or an expression vector that expresses the trans-dominant negative mutant protein of MMP-9.

[0064] In some embodiments, the kit is for treating, ameliorating and / or preventing FOP, and the subject has a mutant ACVR1 gene.

[0065] In some embodiments, the mutant ACVR1 gene encodes a constitutively active ACVR1 polypeptide.

[0066] In some embodiments, the ACVR1 polypeptide comprises at least one mutation selected from the group consisting of L196P, P197-F198 del ins L, R202I, R206H, Q207E, F246Y, R258S, R258G, G325A, G328E, G328W, G328R, G356D, R375P, and K400E.

[0067] In some embodiments, the kit is for treating, ameliorating and / or preventing non- FOP HO, and the HO is caused by a trauma or injury in the bone, a severe burn, a stroke, a head trauma, paralysis, an orthopaedic procedure, a hip replacement, a severe crush injury, an explosive wound, a war wound, a severe civilian trauma, polio, tetanus, a syringomyelia, a myelodysplastic syndrome, multiple sclerosis (MS), carbon monoxide poisoning, or a tumor in the spine.

[0068] In some embodiments, the small molecule MMP-9 inhibitor is selected from the group consisting of metformin, doxycycline, incyclinide, and minocycline.

[0069] In some embodiments, the protein MMP-9 inhibitor is an antibody against MMP-9 or an antigen binding fragment thereof.

[0070] In some embodiments, the anti-MMP-9 antibody or the antigen binding fragment 7 53121668.1Attorney Docket No.046483-7440WO1(03745) thereof comprises a heavy chain complementarity-determining region 1 (CDR1) set forth in SEQ ID NO:3, a heavy chain CDR2 set forth in SEQ ID NO:4, a heavy chain CDR4 set forth in SEQ ID NO:5, a light chain CDR1 set forth in SEQ ID NO:6, a light chain CDR2 set forth in SEQ ID NO:7, and a light chain CDR3 set forth in SEQ ID NO:8.

[0071] In some embodiments, the anti-MMP-9 antibody or the antigen binding fragment thereof comprises a heavy chain CDR1 set forth in SEQ ID NO:15, a heavy chain CDR2 set forth in SEQ ID NO:16, a heavy chain CDR4 set forth in SEQ ID NO:17, a light chain CDR1 set forth in SEQ ID NO:18, a light chain CDR2 set forth in SEQ ID NO:19, and a light chain CDR3 set forth in SEQ ID NO:20.

[0072] In some embodiments, the anti-MMP-9 antibody or the antigen binding fragment thereof comprises a heavy chain CDR1 set forth in SEQ ID NO:35, a heavy chain CDR2 set forth in SEQ ID NO:36, a heavy chain CDR4 set forth in SEQ ID NO:37, a light chain CDR1 set forth in SEQ ID NO:38, a light chain CDR2 set forth in SEQ ID NO:39, and a light chain CDR3 set forth in SEQ ID NO:40.

[0073] In some embodiments, the anti-MMP-9 antibody or the antigen binding fragment thereof comprises a light chain CDR1 set forth in SEQ ID NO:45, a light chain CDR2 set forth in SEQ ID NO:46, and a light chain CDR3 set forth in SEQ ID NO:47.

[0074] In some embodiments, the anti-MMP-9 antibody or the antigen binding fragment thereof comprises a heavy chain variable region set forth in SEQ ID NO:9, and a light chain variable region set forth in SEQ ID NO:10.

[0075] In some embodiments, the anti-MMP-9 antibody or the antigen binding fragment thereof comprises a heavy chain variable region selected from the group consisting of SEQ ID NOs:21, 23, 25, 27 and 29, and a light chain variable region selected from the group consisting of SEQ ID NOs:22, 24, 26, 28 and 30.

[0076] In some embodiments, the anti-MMP-9 antibody or the antigen binding fragment thereof comprises a heavy chain variable region set forth in SEQ ID NO:21 and a light chain variable region set forth in SEQ ID NO:22.

[0077] In some embodiments, the anti-MMP-9 antibody or the antigen binding fragment thereof comprises a heavy chain variable region set forth in SEQ ID NO:27 and a light chain variable region set forth in SEQ ID NO:30.

[0078] In some embodiments, the anti-MMP-9 antibody or the antigen binding fragment thereof comprises a heavy chain variable region set forth in SEQ ID NO:41 and a light chain variable region set forth in SEQ ID NO:42.

[0079] In some embodiments, the anti-MMP-9 antibody or the antigen binding fragment 8 53121668.1Attorney Docket No.046483-7440WO1(03745) thereof comprises a light chain variable region set forth in SEQ ID NO:48.

[0080] In some embodiments, the instruction further instructions for surgically removing an ossified tissue from the subject.

[0081] In some embodiments, the instruction further comprises instructions to perform the surgically removing after the level or the activity of MMP-9 in the subject is down-regulated.

[0082] In some embodiments, the subject is a human. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] The following detailed description of exemplary embodiments will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating, non-limiting embodiments are shown in the drawings. It should be understood, however, that the instant specification is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0084] Figs.1A-1D: characterization of FOP in patient-R, in accordance with some embodiments. Fig.1A: Photo of malformations in toes (arrows). Fig.1B: Radiographs of feet. Arrows indicate malformed great toes with absence of the interphalangeal joints. Fig. 1C: Scout image of a whole-body low dose CT scan showing a paucity of HO typical of someone with FOP. Arrow indicates a small area of HO associated with the lumbar spine. Fig.1D: Electropherogram showing the classic ACVR1 mutation (617G>A) in patient-R. The sequences shown in Fig.1C are CAGTGGCTCRCCAGATTACACT (SEQ ID NO:1, R=G and A) and CAGTGGCTCGCCAGATTACACT (SEQ ID NO:2)

[0085] Figs.2A-2D illustrate some results of the MMP-9 assays in controls, FOP patients, and patient-R, in accordance with some embodiments. Fig.2A: Box and Whisker plot of total MMP-9 in unaffected controls (n=39) and FOP patients (n=38) (Myriad RBM assay). Fig. 2B: Box and Whisker plot of FOP patients (n=38) and Patient-R (n=2). Fig.2C: Gelatin zymography assay reveal less active MMP-9 in cell culture supernatant of PBMCs (cultured for 72 h with TNF-D^ of patient-R versus controls and FOP patients (n=3). Fig.2D: Quantification of gelatin zymography in G. Figs.2A-2B: Mann-Whitney test was used. Fig. 2D: Tukey’s multiple comparisons test was used. **p<0.01, *p<0.05, ns - no significant difference.

[0086] Fig.3A-3B illustrate the MMP-9 variants in Patient-R, in accordance with some embodiments. Fig.3A: Cartoon of MMP-9 protein denoting the locations of the A20V and D165N SNPs resulting in amino acid residue changes. Fig.3B: Sequence of PCR amplicons 9 53121668.1Attorney Docket No.046483-7440WO1(03745) confirms the presence of A20V and D165N SNPs in Patient-R.

[0087] Figs.4A-4C illustrate the structural basis for loss-of-function of secreted MMP-9 zymogen, in accordance with some embodiments. Fig.4A: Overlay of five high resolution structures from the PDB shows that a short (1.8 A) ion pair / H-bond with optimal geometry is invariant, i.e., not an artifact of crystallization. Fig.4B: The inhibitory a-helical prodomain, which blocks the active site including a bound catalytic Zn2+ion via cysteine ligation, requires cleavage adjacent the ion pair. Fig.4C: Four sidechains extend out from the folded core of the protein C-terminal to the site of the zymogen cleavage, indicative of a potential role in substrate binding by plasminogen, the activating protease.

[0088] Fig.5 demonstrates that the treatment with minocycline decreases HO in FOP mouse model at 14 days after injury with cardiotoxin, in accordance with some embodiments. Micro CT images of Acvr1Q207D / +mice not treated (left panel) and treated with minocycline (right panel). Heterotopic bone (arrows) was quantified (n=6). Unpaired t-test was used.***p<0.001.

[0089] Fig.6 demonstrates that minocycline decreases HO at doses down to 5 mg / kg in Acvr1Q207D / +mice, in accordance with some embodiments. Quantification of heterotopic bone in Acvr1Q207D / +mice not treated (controls) and treated with minocycline at varying concentrations (100 mg / kg to 1 mg / kg). Minocycline at a dose of 100 mg / kg to 5 mg / kg is effective in decreasing HO in Acvr1Q207D / +mice. Data shown: mean ± SEM; one-way Anova was used;***p<0.001, **p<0.01,

[0090] Figs.7A-7B demonstrate that the treatment with a monoclonal antibody against MMP-9 decreases HO in FOP mouse models at 14 days after injury with cardiotoxin, in accordance with some embodiments. Fig.7A: Micro CT images of Acvr1ARC-R206H / +;CreERT2- / +mice not treated (n=18) (left panel) and treated with monoclonal antibody against MMP-9 (n=16) (right panel). Heterotopic bone (arrows) was quantified. Fig.7B: Micro CT images of Acvr1Q207D / +mice not treated (left panel) and treated with antibodies against MMP-9 (leftbone (arrows) was quantified (n=8). Unpaired t-test was used.****p<0.0001, **p<0.01.

[0091] Figs.8A-8C illustrates some results of the analysis of MMP-9 and Activin A in THP-1 cells (Wild Type, A20V, and D165N), in accordance with some embodiments. Cell culture supernatants were from M1-like and M2a-like macrophages at 48 hours after polarization of Wild Type, A20V, and D165N M0 macrophages. Fig.8A: Gelatin zymography of active MMP-9. Fig.8B: Quantification of gelatin zymography in A. Two-way Anova was used. Fig.8C: ELISA for Activin A in cell culture supernatants from M1-like and 10 53121668.1Attorney Docket No.046483-7440WO1(03745) M2a-like macrophages. The level of Activin A in the supernatant is dramatically reduced by MMP-9A20Vand MMP-9D165Ncompared to MMP-9WT. Data shown: mean ± SEM; ****p<0.0001, **p<0.01, *p<0.05.

[0092] Fig.9 shows a hypothetical schema of MMP-9 activity in heterotopic ossification in FOP, in accordance with some embodiments. Chevrons = Major categories; Arrows = Activation; Arrowhead = Activation Complex; Blunt-end red line = Inhibition; MMP-9 (Green) = Target; FAPs = Fibro-adipogenic progenitor cells; HOFOP= Heterotopic ossification in FOP

[0093] Fig.10 demonstrates that minocycline reduces Activin A, in accordance with some embodiments. ELISA for Activin A in cell culture supernatants from THP-1 derived M1-like macrophages. THP-1 M0 derived macrophages were daily treated with minocycline during of polarization into M1-like macrophages. Activin A was measured by ELISA in cell culture supernatants at 48 hours. One-way Anova was used. Data shown are mean - / +SEM; ****p<0.0001, ***p<0.01, **p<0.01, *p<0.05.

[0094] Fig.11: Amino acid and DNA sequences of the heavy chain and the light chain of a non-limiting mouse MMP9 antibody according to some embodiments. Fig.11: variable domain sequences of a non-limiting mouse IgG1 chain (SEQ ID NO:57), as well as the DNA molecule encoding the variable domain (SEQ ID NO:58), in accordance with some embodiments. MGWSSIILFLVATATGVHSQVQLQQPGSVLVRPGASVKLSCTASGYTFTSYWMNWV Y T G T T C C

[0095] Fig.12: Variable domain sequences of a non-limiting mouse kappa chain (SEQ ID NO:59), as well as the DNA molecule encoding the variable domain (SEQ ID NO:60), in 11 53121668.1Attorney Docket No.046483-7440WO1(03745) accordance with some embodiments. MSSAQFLGLLLLCFQGTRCDIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQK PDGTFKLLIYYTSILHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYGWLPRTF G A C A C G

[0096] Fig.13 depicts the amino acid sequences of the heavy chain and the light chain of a non-limiting humanized MMP9 antibody according to some embodiments (The sequences shown in the figure are SEQ ID NO:56 for the Light Chain and SEQ ID NO:55). DETAILED DESCRIPTION

[0097] 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. These are, of course, merely examples and are not intended to be limiting. For example, the formation of a first feature over or on a second feature in the description that follows may include embodiments in which the first and second features are formed in direct contact, and may also include embodiments in which additional features may be formed between the first and second features, such that the first and second features may not be in direct contact. In addition, the present disclosure may repeat reference numerals and / or letters in the various examples. This repetition is for the purpose of simplicity and clarity and does not in itself dictate a relationship between the various embodiments and / or configurations discussed.

[0098] Heterozygous missense mutations in activin receptor A type I, a bone morphogenetic protein (BMP) type I receptor, have been identified in all individuals with sporadic or familial fibrodysplasia ossificans progressiva (FOP). ACVR1 mutations cause 12 53121668.1Attorney Docket No.046483-7440WO1(03745) loss of autoinhibition of ACVR1 and render it susceptible to dysregulated BMP pathway signaling. Activin A, a member of the transforming growth factor-ȕ^^7*)-ȕ^^IDPLO\^RI^ molecules that antagonizes BMP signaling in a wild-type (WT) ACVR1 background, specifically enhances BMP pathway signaling within cells harboring the ACVR1R206H mutation and drives heterotopic bone formation in FOP.

[0099] The study described herein (“the present study”) discovered a special fibrodysplasia ossificans progressiva (FOP) patient having the classical R206H mutation in Acvr1 BMP receptor. Although this patient had the congenital features of FOP, he developed very few post-natal FOP features. The present study discovered that this patient had significantly suppressed biomarkers of inflammation, and that the patient had a compound heterozygosity for MMP-9 gene (one allele of the MMP-9 gene having a polymorphism resulting in an A20V mutation in the expressed polypeptide and the other allele having a polymorphism resulting in a D165N mutation). Using protein structure modeling, the present study predicted that the compound heterozygosity of MMP-9 found in the patient resulted in reduced MMP-9 level and activity in the patient. The present study discovered that MMP-9 is expressed in early lesional tissues of FOP subjects. Using several mouse FOP models, the present study discovered that reducing MMP-9 level and / or activity by genetic deletion of MMP-9 (including partial deletion), administration of non-limiting examples of monoclonal antibodies against MMP-9, or administration of non-limiting small molecule inhibitors of MMP-9 all resulted in decreased level of heterotopic ossification (bone formation) in response to injury in the mouse FOP models. [000100] Accordingly, in some aspects, the instant invention is directed to a method of treating, ameliorating, and / or preventing fibrodysplasia ossificans progressiva (FOP) in a subject in need thereof. [000101] In some aspects, the instant invention is directed to a kit for treating, ameliorating, and / or preventing FOP in a subject in need thereof. [000102] It should be noted that the present invention is not limited to the treatment and prevention of heterotopic ossification (HO) in the case of FOP. Transgenic mice with R206H or Q207D mutation in the Acvr1 gene are widely accepted in the art as animal models for the study of HO in general (see e.g., Meyers et al., JBMR Plus.2019 Apr; 3(4): e10172). The fact that the methods and kits herein can be used to prevent or reduce HO in these animal models show that these methods and kits are applicable in treating HO in general, such as HO caused by trauma or injury in the bone, severe burns, stroke, head trauma, paralysis, orthopaedic procedures, hip replacements, severe crush injury, explosive wounds (such as those from 13 53121668.1Attorney Docket No.046483-7440WO1(03745) improvised explosive), war wounds, severe civilian trauma, polio, tetanus, syringomyelia, myelodysplastic syndromes, multiple sclerosis (MS), carbon monoxide poisoning, or tumors in the spine. [000103] Accordingly, in some aspects, the present invention is directed to a method of treating, ameliorating, and / or preventing heterotopic ossification (HO) in a subject in need thereof. [000104] In some aspects, the present invention is directed to a kit for treating, ameliorating, and / or preventing heterotopic ossification (HO) in a subject in need thereof. Definitions [000105] As used herein, each of the following terms has the meaning associated with it in this section. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Generally, the nomenclature used herein and the laboratory procedures in animal pharmacology, pharmaceutical science, peptide chemistry, and organic chemistry are those well-known and commonly employed in the art. It should be understood that the order of steps or order for performing certain actions is immaterial, so long as the present teachings remain operable. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference. [000106] In the application, where an element or component is said to be included in and / or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components and can be selected from a group consisting of two or more of the recited elements or components. [000107] In the methods described herein, the acts can be carried out in any order, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process. [000108] In this document, the terms "a," "an," or "the" are used to include one or more than one unless the context clearly dictates otherwise. The term "or" is used to refer to a 14 53121668.1Attorney Docket No.046483-7440WO1(03745) nonexclusive "or" unless otherwise indicated. The statement "at least one of A and B" or "at least one of A or B" has the same meaning as "A, B, or A and B." [000109] "About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, in certain embodiments ±5%, in certain embodiments ±1%, in certain embodiments ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. [000110] As used herein, the language "pharmaceutically acceptable salt" refers to a salt 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. [000111] As used herein, a "pharmaceutically effective amount," "therapeutically effective amount," or "effective amount" of a compound is that amount of compound that is sufficient to provide a beneficial effect to the subject to which the compound is administered. [000112] As used herein, the term "prevent" or "prevention" means no disorder or disease development if none had occurred, or no further disorder or disease development if there had already been development of the disorder or disease. Also considered is the ability of one to prevent some or all of the symptoms associated with the disorder or disease. [000113] Non-limiting abbreviations used herein: FOP: fibrodysplasia ossificans progressiva. HO: heterotopic ossification. PBMC: peripheral blood mononuclear cell. SNP: single nucleotide polymorphism. Method of Treating, Ameliorating and / or Preventing Fibrodysplasia Ossificans Progressiva [000114] The present study discovered a fibrodysplasia ossificans progressiva (FOP) patient having the classical R206H mutation in Acvr1 BMP receptor. Although this patient had the congenital features of FOP, developed very few post-natal FOP features. The present study discovered that this patient had significantly suppressed biomarkers of inflammation, and that the patient had a compound heterozygosity for MMP-9 gene (one allele of the MMP-9 gene having a polymorphism resulting in A20V and the other allele having a polymorphism resulting in D165N). Using protein structure modeling, the present study predicted that the compound heterozygosity of MMP-9 found in the patient resulted in reduced MMP-9 level and activity in the patient. The present study discovered that MMP-9 is expressed in early lesional tissues of FOP subjects. Using several mouse FOP models as non-limiting examples, the present study discovered that reducing MMP-9 level and / or activity by genetic deletion of 15 53121668.1Attorney Docket No.046483-7440WO1(03745) MMP-9 (including partial deletion), administration of a non-limiting MMP-9 monoclonal antibody, or administration of a non-limiting small molecule inhibitor of MMP-9 all resulted in decreased level of heterotopic ossification (bone formation) in response to soft tissue injury. [000115] Accordingly, in some aspects, the present invention is directed to a method of treating, ameliorating and / or preventing fibrodysplasia ossificans progressiva (FOP) in a subject. In some embodiments, the method includes administering to the subject a compound that down-regulates MMP-9 level and / or activity in the subject. [000116] The present study discovered that the inhibition of MMP-9 is able to significantly reduce the level of heterotopic ossification in model animals with Q270D mutation in Acvr1 BMP receptor. Since Q270D mutation does not cause spontaneous heterotopic ossification but rather results in HO in response to injury only and produces very robust post-traumatic HO, the Q270D model is considered a model for HO. Accordingly, In some aspects, the present invention is directed to a method of treating, ameliorating and / or preventing heterotopic ossification in a subject in need thereof. In some embodiments, the method includes administering to the subject a compound that down-regulates MMP-9 level and / or activity in the subject. [000117] In some embodiments, the subject is treated with a bone marrow transplantation which replaces some hematopoietic stem cells in the subject with those that having reduced level and / or activity of MMP-9. One of ordinary skill in the art would understand that MMP- 9, although expressed in multiple tissues, is strongly expressed in bone marrow and lymphoid tissues. As described elsewhere herein, the present study discovered that partial reduction of MMP-9 expression by deleting only one MMP-9 allele is sufficient to counter the soft tissue injury induced heterotopic ossification in FOP model animals. As such, in certain embodiments, partial reduction and / or elimination of MMP-9 expression and / or activity only in the bone marrow is sufficient to mimic the partial reduction of MMP-9 expression in FOP model mice. In some embodiments, the bone marrow stem cells used for the transplantation are genetically engineered to reduce MMP-9 level and / or activity. Genetic engineering of the hematopoietic stem cells in the bone marrow is described in, for example, Daniel-Moreno et al. (Bone Marrow Transplantation volume 54, pages 1940–1950 (2019)). In some embodiments, the bone marrow used for the transplantation is from a donor having MMP-9 polymorphisms that reduce the MMP-9 level and / or activity, such as the MMP-9 polymorphisms found in Patient-R described herein. [000118] In some embodiments, the subject has a mutant ACVR1 gene. In some 16 53121668.1Attorney Docket No.046483-7440WO1(03745) embodiments, the mutant ACVR1 gene encodes a constitutively active ACVR1 polypeptide. In other embodiments, the mutant ACVR1 gene comprises, relative to a wild-type gene, 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. 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 a wild-type polypeptide. In some embodiments, the ACVR1 polypeptide comprises one or more amino acid substitutions, deletions, and / or insertions relative to a wild-type ACVR1 polypeptide, wherein the wild-type ACVR1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:54. Genbank Accession No. NP_001104537, which is herein incorporated by reference in its entirety, discloses an ACVR1 polypeptide. In some embodiments, the ACVR1 polypeptide comprises one or more mutations on 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 on amino acid residues L196, P197, F198, R202, R206, Q207, F246, R258, G325, G328, G356, R375 or K400 relative to the wild-type ACVR1 polypeptide comprising the sequence set forth in SEQ ID NO:54. In some embodiments, the ACVR1 polypeptide comprises one or more mutations selected from the group consisting of L196P, P197-F198 del ins L, R202I, R206H, Q207E, F246Y, R258S, R258G, G325A, G328E, G328W, G328R, G356D, R375P, and K400E, wherein del represents deletion and ins represents insertion. In some embodiments, the ACVR1 polypeptide comprises one or more mutations selected from the group consisting of L196P, P197-F198 del ins L, R202I, R206H, Q207E, F246Y, R258S, R258G, G325A, G328E, G328W, G328R, G356D, R375P, and K400E relative to the wild- type ACVR1 polypeptide comprising the sequence set forth in SEQ ID NO:54, wherein del represents deletion and ins represents insertion. ACVR1 mutations involved in FOP are also described in Haupt et al. (Bone Volume 109, April 2018, Pages 232-240) and Mukaddam et al., (Genetics of Fibrodysplasia Ossificans Progressiva, eLS, Vol 2: 1–8, 2021), each of which is herein incorporated by reference in its entirety. Human ACVR1, NP_001104537 L S I17 53121668.1Attorney Docket No.046483-7440WO1(03745) QGENVAVKIFSSRDEKSWFRETELYNTVMLRHENILGFIASDMTSRHSSTQLWLITHY HEMGSLYDYLQLTTLDTVSCLRIVLSIASGLAHLHIEIFGTQGKPAIAHRDLKSKNILV Rossified tissue from the subject. In some embodiments, the surgical removal of ossified tissue is performed after the level or the activity of MMP-9 in the subject has been down-regulated. [000120] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. Small Molecule Inhibitor of MMP-9 [000121] In some embodiments, the compound that down-regulates MMP-9 level and / or activity includes a small molecule inhibitor of MMP-9. [000122] As used herein, the term “small molecule” means molecules have 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 referred to as GM6001), JNJ0966, luteolin 7-O-glucuronide, marimastat, MMP- 2 / MMP-9 Inhibitor I, MMP-2 / MMP-9 Inhibitor II, MMP3 inhibitor 1, MMP-9-IN-1, MMP-9 Inhibitor I, MMP-9 Inhibitor II, MMP-9 / MMP-13 inhibitor I, MMP Inhibitor II, MMP13-IN- 3, MMPI-1154, morroniside, ND-336, NNGH, (R)-ND-336, PF-00356231 hydrochloride, PD-166793, prinomastat, prinomastat hydrochloride, salvianolic acid A, S 3304, SB-3CT, SM-7368, tanomastat, tetracycline derivatives such as doxycycline, incyclinide, and minocycline, UK 356618, UK-370106, XL-784, and the like. MMP-9 inhibitors are described in, for example, Fields (Cells.2019 Sep; 8(9): 984.) [000123] 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 small molecule compounds that having IC50toward MMP-9 equal to or higher than 18 53121668.1Attorney Docket No.046483-7440WO1(03745) the IC50 toward any other matrix metalloproteinases, such as higher by 0.1 or more, higher by 0.2 or more, higher by 0.3 or more, higher by 0.5 or more, higher by 0.8 or more, or higher by 1.0 or more than the IC50 toward any other matrix metalloproteinases. Non-limiting examples of selective MMP-9 small molecule inhibitors include apigenin-7-glucuronide, FFAGLDD, FSL-1 TFA, ginkgolide C, isoliquiritin apioside, JNJ0966, luteolin 7-O- glucuronide, MMP-9-IN-1, (R)-ND-336, SM-7368, FFAGLDD TFA, and the like. [000124] In some embodiments, the small molecule inhibitor of MMP-9 includes a compound that are in use in the medical field for purposes other than inhibiting MMP-9 and is known to be generally safe. Non-limiting examples of such compounds include tetracyclines such as doxycycline, incyclinide, and minocycline. Examples of tetracyclines that are able to inhibit MMP-9 are described in, e.g., Griffin et al. (Am J Physiol Cell Physiol.2010 Sep; 299(3): C539–C548.), the entirety of the reference is hereby incorporated herein by reference. [000125] In some embodiments, the small molecule inhibitor of MMP-9 includes metformin. Metformin has been shown to be an indirect and a direct inhibitor of MMP-9 (see e.g., Hwang et al., Br J Pharmacol.2010 Jul; 160(5): 1195–1211 and Chen et al., J Cardiovasc Dev Dis.2023 Jan 30;10(2):54. doi: 10.3390 / jcdd10020054). Protein Inhibitor of MMP-9 [000126] In some embodiments, the compound that down-regulates MMP-9 level and / or activity includes a protein inhibitor of MMP-9. [000127] In some embodiments, the protein inhibitor of MMP-9 includes an antibody against MMP-9, or an antigen binding fragment thereof. Examples of antibody against MMP-9 include anti-Ac-MMP-9 antibody (4A3), anti-MMP-9 antibody (E-11), anti-MMP-9 antibody (2C3), and anti-MMP-9 antibody (6-6B) by Santa Cruz Biotechnology, MMP9 monoclonal antibody (5G3), MMP9 Recombinant Rabbit Monoclonal Antibody (JA80-73), and MMP9 Monoclonal Antibody (5C3) by Invitrogen, and the like. [000128] MMP9 antibodies are disclosed in, for example, WO 2013 / 130078 A1, WO 2013 / 130905 A1, and U.S. Patent No.9,732,156, each of which is incorporated herein by reference in its entirety. [000129] In some embodiments, the MMP9 antibody comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3 comprising about 80% or more identity, about 85% or more identity, about 90% or more identity, about 92% or more identity, about 95% or more identity, about 98% or more identity, about 99% or more identity, or 100% identity to the heavy chain CDR1, the heavy 19 53121668.1Attorney Docket No.046483-7440WO1(03745) chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, and the light chain CDR3 set forth in Table 1, respectively: Table 1 Heavy Chain CDR1 GYTFTSYWMN (SEQ ID NO:3) [000130] In some embo mens, e an o y compr ses a eavy chain variable region and a light chain variable region comprising about 80% or more identity, about 85% or more identity, about 90% or more identity, about 92% or more identity, about 95% or more identity, about 98% or more identity, about 99% or more identity, or 100% identity to the heavy chain variable region and the light chain variable region set forth in Table 2, respectively: Table 2 Heavy chain variable region Y Y[000131] In some embodiments, the MMP 9 antibody comprises a heavy chain and a light 20 53121668.1Attorney Docket No.046483-7440WO1(03745) chain comprising about 80% or more identity, about 85% or more identity, about 90% or more identity, about 92% or more identity, about 95% or more identity, about 98% or more identity, about 99% or more identity, or 100% identity to the heavy chain and the light chain set forth in Table 3, respectively: Table 3 Heavy chain MGWSSIILFLVATATGVHSQVQLQQPGSVLVRPGASVKLSCTASGYTFTSYWMNWV E IV V P G K V[000132] In other embodiments, the MMP 9 antibody comprises an amino acid sequence as set forth in Table 3, with the proviso that the antibody does not comprise the signal peptide. [000133] In another embodiment, the MMP9 antibody comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, set forth in Table 1, wherein the antibody comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one 21 53121668.1Attorney Docket No.046483-7440WO1(03745) of the sequences as set forth in Tables 2 and 3. [000134] In another embodiment, the MMP9 antibody comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, set forth in Table 1, wherein the antibody comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences as set forth in Table 3, with the proviso that the antibody does not comprise the signal peptide. [000135] In some embodiments, the MMP 9 antibody comprises a heavy chain by a DNA sequence and a light chain encoded by a DNA sequence comprising about 80% or more identity, about 85% or more identity, about 90% or more identity, about 92% or more identity, about 95% or more identity, about 98% or more identity, about 99% or more identity, or 100% identity to the heavy chain DNA sequence and the light chain DNA sequence set forth in Table 4, respectively: Table 4 Heavy Chain DNA sequence t t t t tt t tt t t t t t t t tCAGGTGCAGCTGCAGCAGCC G C T G C C C C G G G G22 53121668.1Attorney Docket No.046483-7440WO1(03745) TGAACTCCGCCGCCTTCCCAGCCCCCATCGAAAAGACCATCAGCAAGACCAAGG GCAGACCCAAGGCCCCCCAGGTGTACACAATCCCGCCACCCAAAGAACAGATGG T A G C C A T G G G C T[000136] In some embodiments, the MMP9 antibody comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3 comprising about 80% or more identity, about 85% or more identity, about 90% or more identity, about 92% or more identity, about 95% or more identity, about 98% or more identity, about 99% or more identity, or 100% identical to the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, and the light chain CDR3 set forth in Table 5, respectively: Table 5 Heavy Chain CDR123 53121668.1Attorney Docket No.046483-7440WO1(03745) Heavy Chain CDR2 VIWTGGTTNYNSALMS (SEQ ID NO:16) [000137] In some emb, avy chain variable region and a light chain variable region comprising about 80% or more identity, about 85% or more identity, about 90% or more identity, about 92% or more identity, about 95% or more identity, about 98% or more identity, about 99% or more identity, or 100% identity to the heavy chain variable region and the light chain variable region set forth in Table 6, respectively: Table 6 Heavy chain variable region I F I F24 53121668.1Attorney Docket No.046483-7440WO1(03745) GGGTKVEIK (SEQ ID NO:24) Humanized V2 heavy chain variable region A F A F F[000138] In some embodiments, the MMP 9 antibody comprises a heavy chain and a light chain set forth in Table 7: Table 7 Heavy Chain V25 53121668.1Attorney Docket No.046483-7440WO1(03745) TVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPS GPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPD I T Y P Q[000139] In other embodiments, the MMP 9 antibody comprises an amino acid sequence as set forth in Table 7, with the proviso that the antibody does not comprise the signal peptide. [000140] In another embodiment, the MMP9 antibody comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, set forth in Table 5, wherein the antibody comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences as set forth in Tables 6 and 7. [000141] In another embodiment, the MMP9 antibody comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, set forth in Table 5, wherein the antibody comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences as set forth in Table 7, with the proviso that the antibody does not comprise the signal peptide. [000142] In some embodiments, the MMP9 antibody comprises a heavy chain variable region 26 53121668.1Attorney Docket No.046483-7440WO1(03745) and a light chain variable region set forth in Table 8: Table 8 Heavy chain variable region QVQLQESGPGLVKPSETLSLTCTVSGFSLLSYGVHWVRQPPGKGLEWL A F [000, light chain comprising about 80% or more identity, about 85% or more identity, about 90% or more identity, about 92% or more identity, about 95% or more identity, about 98% or more identity, about 99% or more identity, or 100% identity to the heavy chain and the light chain set forth in Table 9, respectively: Table 9 Heavy Chain R T S G D Y T L27 53121668.1Attorney Docket No.046483-7440WO1(03745) QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:34) V P P T P S G V T[000144] In other embodiments, the MMP 9 antibody comprises an amino acid sequence as set forth in Table 9, with the proviso that the antibody does not comprise the signal peptide. [000145] In another embodiment, the MMP9 antibody comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences as set forth in Tables 8 and 9. [000146] In another embodiment, the MMP9 antibody comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences as set forth in Table 9, with the proviso that the antibody does not comprise the signal peptide. [000147] In some embodiments, the MMP9 antibody comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3 comprising about 80% or more identity, about 85% or more identity, about 90% or 28 53121668.1Attorney Docket No.046483-7440WO1(03745) more identity, about 92% or more identity, about 95% or more identity, about 98% or more identity, about 99% or more identity, or 100% identity to the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, and the light chain CDR3 set forth in Table 10, respectively: Table 10 Heavy Chain CDR1 GFSLLSYGVH (SEQ ID NO:35) [000148] In some embodiments, the MMP9 antibody comprises a heavy chain variable region and a light chain variable region comprising about 80% or more identity, about 85% or more identity, about 90% or more identity, about 92% or more identity, about 95% or more identity, about 98% or more identity, about 99% or more identity, or 100% identity to the heavy chain variable region and the light chain variable region set forth in Table 11, respectively: Table 11 Heavy chain variable region I F29 53121668.1Attorney Docket No.046483-7440WO1(03745) [000149] In some embodiments, the MMP 9 antibody comprises a heavy chain and a light chain comprising about 80% or more identity, about 85% or more identity, about 90% or more identity, about 92% or more identity, about 95% or more identity, about 98% or more identity, about 99% or more identity, or 100% identity to the heavy chain and the light chain set forth in Table 12, respectively: Table 12 Heavy Chain MAVLVLFLCLVAFPSCVLSQVQLKESGPGLVAPSQSLSITCTVSGFSLLSYGVHWVR Y V S V A Y Rset forth in Table 12, with the proviso that the antibody does not comprise the signal peptide. [000151] In another embodiment, the MMP9 antibody comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a 30 53121668.1Attorney Docket No.046483-7440WO1(03745) light chain CDR3, set forth in Table 10, wherein the antibody comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences as set forth in Tables 11 and 12. [000152] In another embodiment, the MMP9 antibody comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, set forth in Table 10, wherein the antibody comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences as set forth in Table 12, with the proviso that the antibody does not comprise the signal peptide. In some embodiments, the MMP9 antibody comprises only a light chain. [000153] In some embodiments, the MMP9 antibody comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3 comprising about 80% or more identity, about 85% or more identity, about 90% or more identity, about 92% or more identity, about 95% or more identity, about 98% or more identity, about 99% or more identity, or 100% identity to the light chain CDR1, the light chain CDR2, and the light chain CDR3 set forth in Table 13, respectively: Table 13 Light Chain CDR1[000154] In some embodiments, the MMP9 antibody comprises a light chain variable region comprising about 80% or more identity, about 85% or more identity, about 90% or more identity, about 92% or more identity, about 95% or more identity, about 98% or more identity, about 99% or more identity, or 100% identity to light chain variable region set forth in Table 14: Table 14 Light chain variable region L31 53121668.1Attorney Docket No.046483-7440WO1(03745) IYSASYRFSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSYPYT FGGGTKLEIK (SEQ ID NO:48) [000sing about 80% or more identity, about 85% or more identity, about 90% or more identity, about 92% or more identity, about 95% or more identity, about 98% or more identity, about 99% or more identity, or 100% identity to the light chain set forth in Table 15: Table 15 Light Chain QVFVYMLLWLSGVDGDIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQK F V[000156] In other embodiments, the MMP 9 antibody comprises an amino acid sequence as set forth in Table 15, with the proviso that the antibody does not comprise the signal peptide. [000157] In another embodiment, the MMP9 antibody comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, set forth in Table 13, wherein the antibody comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences as set forth in Tables 14 and 15. [000158] In another embodiment, the MMP9 antibody comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, set forth in Table 13, wherein the antibody comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences as set forth in Table 15, with the proviso that the antibody does not comprise the signal peptide. [000159] In some embodiments, the CDR sequences, the heavy / light chain variable region sequences, and / or the heavy / light chain sequences are similar to those described above but 32 53121668.1Attorney Docket No.046483-7440WO1(03745) not identical (i.e., do not share 100% sequence identity). In some embodiments, the sequence identity of the CDR sequences, the heavy / light chain variable region sequences, and / or the heavy / light chain sequences of the MMP9 antibody herein is about 80% or higher, such as about 85% or higher, about 90% or higher, about 95% or higher, about 98% or higher, or about 99% or higher to those described above. [000160] In other embodiments, the MMP9 antibody comprises a CDR comprising a CDR sequence as set forth in any one of Tables 1, 5, and 10, or a CDR sequence as set forth in any one of Tables 1, 5, and 10 with one or more (e.g., one, two, three, four, five or more) substitutions, insertions, deletions, and / or additions (and combinations thereof) as compared to the CDR sequence as set forth in any one of Tables 1, 5, and 10, wherein the MMP9 antibody is capable of binding to MMP9. [000161] In some embodiments, the MMP9 antibody comprises variants of the antibodies described above, such as recombinantly produced antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, an antibody heavy chain monomer, an antibody light chain dimer, an antibody heavy chain dimer, an antibody light chain- antibody heavy chain pair, intrabodies, hetero-conjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fv (scFv), nanobodies, intracellular antibodies, intrabodies, camelized antibodies, camelid antibodies, IgNAR antibodies, affybodies, Fab fragments, F(ab') fragments, F(ab)2, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies (including, e.g., anti-anti-Id antibodies), and antigen-binding fragments of any of the above. Furthermore, the sequences of the MMP9 antibodies herein can be engineered into any type (e.g., IgG, IgE, IgM, IgD, IgA or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2), or any subclass (e.g., IgG2a or IgG2b). [000162] In some embodiments, the protein inhibitor of MMP-9 includes non-antibody protein or peptide inhibitor of MMP-9. Non-limiting examples of non-antibody protein or peptide inhibitors of MMP-9 include proteins from the tissue inhibitors of metalloproteinase (TIMP) family such as TIMP-1, TIMP-3 and the like, FFAGLDD peptide, FFAGLDD TFA, cyclic CTTHWGFTLC, cyclic CTTHWGFTLC TFA, and the like. RNA Interference Nucleic Acids for Downregulate MMP-9 and Vectors Expressing the Same [000163] In some embodiments, the compound that down-regulates MMP-9 level and / or 33 53121668.1Attorney Docket No.046483-7440WO1(03745) activity includes a nucleic acid that downregulates MMP-9 level by RNA interference, and / or an expression vector expressing the nucleic acid. [000164] In some embodiments, the nucleic acid that downregulates the level of MMP-9 by the means of 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 miRNAs) or antisense molecule, which inhibits MMP-9 expression and / or activity. In yet other embodiments, the nucleic acid comprises a promoter / regulatory sequence, such that the nucleic acid is preferably capable of directing expression of the nucleic acid. Thus, the instant specification provides expression vectors and methods for the introduction of exogenous DNA into cells with concomitant expression of the exogenous DNA in the cells such as those described, for example, in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York) and as described elsewhere herein. [000165] In certain embodiments, siRNA is used to decrease the level of MMP-9. RNA interference (RNAi) is a phenomenon in which the introduction of double-stranded RNA (dsRNA) into a diverse range of organisms and cell types causes degradation of the complementary mRNA. In the cell, long dsRNAs are cleaved into short 21-25 nucleotide small interfering RNAs, or siRNAs, by a ribonuclease known as Dicer. The siRNAs subsequently assemble with protein components into an RNA-induced silencing complex (RISC), unwinding in the process. Activated RISC then binds to complementary transcript by base pairing interactions between the siRNA antisense strand and the mRNA. The bound mRNA is cleaved and sequence specific degradation of mRNA results in gene silencing. See, for example, U.S. Patent No.6,506,559; Fire et al., 1998, Nature 391(19):306-311; Timmons et al., 1998, Nature 395:854; Montgomery et al., 1998, TIG 14 (7):255-258; Engelke, Ed., RNA Interference (RNAi) Nuts & Bolts of RNAi Technology, DNA Press, Eagleville, PA (2003); and Hannon, Ed., RNAi A Guide to Gene Silencing, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2003). Soutschek et al. (2004, Nature 432:173-178) describes a chemical modification to siRNAs that aids in intravenous systemic delivery. Optimizing siRNAs involves consideration of overall G / C content, C / T content at the termini, Tm and the nucleotide content of the 3' overhang. See, for instance, Schwartz et al., 2003, Cell, 115:199-208 and Khvorova et al., 2003, Cell 115:209-216. Therefore, the instant specification also includes methods of decreasing levels of MMP-9 using RNAi technology. [000166] In certain embodiments, the instant specification provides a vector comprising an siRNA or antisense polynucleotide. In other embodiments, the siRNA or antisense 34 53121668.1Attorney Docket No.046483-7440WO1(03745) polynucleotide inhibits the expression of MMP-9. The incorporation of a desired polynucleotide into a vector and the choice of vectors is well-known in the art. [000167] In certain embodiments, the expression vectors described herein encode a short hairpin RNA (shRNA) inhibitor. shRNA inhibitors are well known in the art and are directed against the mRNA of a target, thereby decreasing the expression of the target. In certain embodiments, the encoded shRNA is expressed by a cell, and is then processed into siRNA. For example, in certain instances, the cell possesses native enzymes (e.g., dicer) that cleaves the shRNA to form siRNA. [000168] The siRNA, shRNA, or antisense polynucleotide can be cloned into a number of types of vectors as described elsewhere herein. For expression of the siRNA or antisense polynucleotide, at least one module in each promoter functions to position the start site for RNA synthesis. [000169] To assess the expression of the siRNA, shRNA, or antisense polynucleotide, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected using a viral vector. In certain embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers are known in the art and include, for example, antibiotic-resistance genes, such as neomycin resistance and the like. [000170] Following the generation of the siRNA polynucleotide, a skilled artisan will understand that the siRNA polynucleotide has certain characteristics that can be modified to improve the siRNA as a therapeutic compound. Therefore, in some embodiments, the siRNA polynucleotide is further designed to resist degradation by modifying it to include phosphorothioate, or other linkages, methylphosphonate, sulfone, sulfate, ketyl, phosphorodithioate, phosphoramidate, phosphate esters, and the like (see, e.g., Agrwal et al., 1987, Tetrahedron Lett.28:3539-3542; Stec et al., 1985 Tetrahedron Lett.26:2191-2194; Moody et al., 1989 Nucleic Acids Res.12:4769-4782; Eckstein, 1989 Trends Biol. Sci. 14:97-100; Stein, In: Oligodeoxynucleotides. Antisense Inhibitors of Gene Expression, Cohen, ed., Macmillan Press, London, pp.97-117 (1989)). [000171] Any polynucleotide may be further modified to increase its stability in vivo. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and / or 3' ends; the use of phosphorothioate or 2' O-methyl rather than phosphodiester 35 53121668.1Attorney Docket No.046483-7440WO1(03745) linkages in the backbone; and / or the inclusion of nontraditional bases such as inosine, queosine, and wybutosine and the like, as well as acetyl- methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine, and uridine. [000172] In certain embodiments, an antisense nucleic acid sequence expressed by a plasmid vector is used to inhibit MMP-9 protein expression. The antisense expressing vector is used to transfect a mammalian cell or the mammal itself, thereby causing reduced endogenous expression of MMP-9. [000173] Antisense molecules and their use for 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, as that term is defined elsewhere herein, to at least a portion of a specific mRNA molecule (Weintraub, 1990, Scientific American 262:40). In the cell, antisense nucleic acids hybridize to the corresponding mRNA, forming a double-stranded molecule thereby inhibiting the translation of genes. [000174] The use of antisense methods to inhibit the translation of genes is known in the art, and is described, for example, in Marcus-Sakura (1988, Anal. Biochem.172:289). Such antisense molecules may be provided to the cell via genetic expression using DNA encoding the antisense molecule as taught by Inoue, 1993, U.S. Patent No.5,190,931. [000175] Alternatively, antisense molecules of the instant specification may be made synthetically and then provided to the cell. Antisense oligomers of between about 10 to about 30, and more preferably about 15 nucleotides, can be used, since they are easily synthesized and introduced into a target cell. Synthetic antisense molecules contemplated by the instant specification include oligonucleotide derivatives known in the art which have improved biological activity compared to unmodified oligonucleotides (see U.S. Patent No.5,023,243). Ribozyme that Downregulates MMP-9 and Vectors Expressing the Same [000176] In some embodiments the compound that down-regulates MMP-9 level and / or activity includes a ribozyme that downregulates MMP-9, and / or a vector expressing the ribozyme. [000177] A ribozyme is used to inhibit MMP-9 protein expression. Ribozymes useful for inhibiting the expression of a target molecule may be designed by incorporating target sequences into the basic ribozyme structure which are complementary, for example, to the mRNA sequence encoding MMP-9. Ribozymes are antisense RNAs which have a catalytic site capable of specifically cleaving complementary RNAs. Therefore, ribozymes having 36 53121668.1Attorney Docket No.046483-7440WO1(03745) sequence complementary to MMP-9mRNA sequences can downregulate the expression of MMP-9by reduces the level of MMP-9 mRNA. Ribozymes targeting MMP-9, may be synthesized using commercially available reagents (Applied Biosystems, Inc., Foster City, CA) or they may be genetically expressed from DNA encoding them. In some embodiments, the DNA encoding the ribozymes are incorporated in a vector described elsewhere herein. Compounds that Downregulate MMP-9 by CRISPR Knockout / Knockdown and Other Knockout / Knockdown Techniques [000178] In some embodiments the compound that down-regulates MMP-9 level and / or activity includes a compound that downregulates MMP-9 by CRISPR knockout / knockdown and other knockout / knockdown techniques. In some embodiments, the compound includes an expression vector including an expression cassette, wherein the expression cassette expresses CRISPR components that downregulate MMP-9 by CRISPR knockout or CRISPR knockdown. [000179] In some embodiments, the compound that down regulates the activity or level of MMP-9 comprises a CRISPR / Cas9 system for knocking out MMP-9. [000180] The CRISPR / Cas9 system is a facile and efficient system for inducing targeted genetic alterations. Target recognition by the Cas9 protein requires a “seed” sequence within the guide RNA (gRNA) and a conserved di-nucleotide containing protospacer adjacent motif (PAM) sequence upstream of the gRNA-binding region. The CRISPR / Cas9 system can thereby be engineered to cleave virtually any DNA sequence by redesigning the gRNA in cell lines (such as 293T cells), primary cells, and CAR T cells. The CRISPR / Cas9 system can simultaneously target multiple genomic loci by co-expressing a single Cas9 protein with two or more gRNAs, making this system uniquely suited for multiple gene editing or synergistic activation of target genes. [000181] The Cas9 protein and guide RNA form a complex that identifies and cleaves target sequences. Cas9 is comprised of six domains: REC I, REC II, Bridge Helix, PAM interacting, HNH, and RuvC. The RecI domain binds the guide RNA, while the Bridge helix binds to target DNA. The HNH and RuvC domains are nuclease domains. Guide RNA is engineered to have a 5' end that is complementary to the target DNA sequence. Upon binding of the guide RNA to the Cas9 protein, a conformational change occurs activating the protein. Once activated, Cas9 searches for target DNA by binding to sequences that match its protospacer adjacent motif (PAM) sequence. A PAM is a two or three nucleotide base sequence within one nucleotide downstream of the region complementary to the guide RNA. In one non- 37 53121668.1Attorney Docket No.046483-7440WO1(03745) limiting example, the PAM sequence is 5'-NGG-3'. When the Cas9 protein finds its target sequence with the appropriate PAM, it melts the bases upstream of the PAM and pairs them with the complementary region on the guide RNA. Then the RuvC and HNH nuclease domains cut the target DNA after the third nucleotide base upstream of the PAM. [000182] One non-limiting example of a CRISPR / Cas system used to inhibit gene expression, CRISPRi, is described in U.S. Patent Appl. Publ. No. US2014 / 0068797. CRISPRi induces permanent gene disruption that utilizes the RNA-guided Cas9 endonuclease to introduce DNA double stranded breaks which trigger error-prone repair pathways to result in frame shift mutations. A catalytically dead Cas9 lacks endonuclease activity. When coexpressed with a guide RNA, a DNA recognition complex is generated that specifically interferes with transcriptional elongation, RNA polymerase binding, or transcription factor binding. This CRISPRi system efficiently represses expression of targeted genes. [000183] CRISPR / Cas gene disruption occurs when a guide nucleic acid sequence specific for a target gene and a Cas endonuclease are introduced into a cell and form a complex that enables the Cas endonuclease to introduce a double strand break at the target gene. In certain embodiments, the CRISPR / Cas system comprises an expression vector, such as, but not limited to, an pAd5F35-CRISPR vector. In other embodiments, the Cas expression vector induces expression of Cas9 endonuclease. Other endonucleases may also be used, including but not limited to, T7, Cas3, Cas8a, Cas8b, Cas10d, Cse1, Csy1, Csn2, Cas4, Cas10, Csm2, Cmr5, Fok1, other nucleases known in the art, and any combinations thereof. [000184] In certain embodiments, inducing the Cas expression vector comprises exposing the cell to an agent that activates an inducible promoter in the Cas expression vector. In such embodiments, the Cas expression vector includes an inducible promoter, such as one that is inducible by exposure to an antibiotic (e.g., by tetracycline or a derivative of tetracycline, for example doxycycline). However, it should be appreciated that other inducible promoters can be used. The inducing agent can be a selective condition (e.g., exposure to an agent, for example an antibiotic) that results in induction of the inducible promoter. This results in expression of the Cas expression vector. [000185] In certain embodiments, guide RNA(s) and Cas9 can be delivered to a cell as a ribonucleoprotein (RNP) complex. RNPs are comprised of purified Cas9 protein complexed with gRNA and are well known in the art to be efficiently delivered to multiple types of cells, including but not limited to neurons, stem cells and immune cells (Addgene, Cambridge, MA, Mirus Bio LLC, Madison, WI). [000186] The guide RNA is specific for a genomic region of interest and targets that region 38 53121668.1Attorney Docket No.046483-7440WO1(03745) for Cas endonuclease-induced double strand breaks. The target sequence of the guide RNA sequence may be within a loci of a gene 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. [000187] Guide RNA (gRNA), also referred to as "short guide RNA" or "sgRNA", provides both targeting specificity and scaffolding / binding ability for the Cas9 nuclease. The gRNA can be a synthetic RNA composed of a targeting sequence and scaffold sequence derived from endogenous bacterial crRNA and tracrRNA. gRNA is used to target Cas9 to a specific genomic locus in genome engineering experiments. Guide RNAs can be designed using standard tools well known in the art. [000188] In the context of formation of a CRISPR complex, "target sequence" refers to a sequence to which a guide sequence is designed to have some complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. A target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides. In certain embodiments, a target sequence is located in the nucleus or cytoplasm of a cell. In other embodiments, the target sequence may be within an organelle of a eukaryotic cell, for example, mitochondrion or nucleus. Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near (e.g., within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs) the target sequence. As with the target sequence, it is believed that complete complementarity is not needed, provided this is sufficient to be functional. [000189] In certain embodiments, one or more vectors driving expression of one or more elements of a CRISPR system are introduced into a host cell, such that expression of the elements of the CRISPR system direct formation of a CRISPR complex at one or more target sites. For example, a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector. CRISPR system elements that are combined in a single vector may be arranged in any suitable 39 53121668.1Attorney Docket No.046483-7440WO1(03745) orientation, such as one element located 5' with respect to ("upstream" of) or 3' with respect to ("downstream" of) a second element. The coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element, and oriented 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 of the guide sequence, tracr mate sequence (optionally operably linked to the guide sequence), and a tracr sequence embedded within one or more intron sequences (e.g., each in a different intron, two or more in at least one intron, or all in a single intron). [000190] In certain embodiments, the CRISPR enzyme is part of a fusion protein comprising one or more heterologous protein domains (e.g. about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition to the CRISPR enzyme). A CRISPR enzyme fusion protein may comprise any additional protein sequence, and optionally a linker sequence between any two domains. Examples of protein domains that may be fused to a CRISPR enzyme include, without limitation, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity. Additional domains that may form part of a fusion protein comprising a CRISPR enzyme are described in U.S. Patent Appl. Publ. No. US20110059502, incorporated herein by reference. In certain embodiments, a tagged CRISPR enzyme is used to identify the location of a target sequence. [000191] Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids in mammalian and non-mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding components of a CRISPR system to cells in culture, or in a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (e.g., a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell (Anderson, 1992, Science 256:808-813; and Yu, et al., 1994, Gene Therapy 1:13-26). [000192] In certain embodiments, the CRISPR / Cas is derived from a type II CRISPR / Cas system. In other embodiments, the CRISPR / Cas system is derived from a Cas9 protein. The Cas9 protein can be from Streptococcus pyogenes, Streptococcus thermophilus, or other species. 40 53121668.1Attorney Docket No.046483-7440WO1(03745) [000193] In general, Cas proteins comprise at least one RNA recognition and / or RNA binding domain. RNA recognition and / or RNA binding domains interact with the guiding RNA. Cas proteins can also comprise nuclease domains (i.e., DNase or RNase domains), DNA binding domains, helicase domains, RNAse domains, protein-protein interaction domains, dimerization domains, as well as other domains. The Cas proteins can be modified to increase nucleic acid binding affinity and / or specificity, alter an enzymatic activity, and / or change another property of the protein. In certain embodiments, the Cas-like protein of the fusion protein can be derived from a wild type Cas9 protein or fragment thereof. In other embodiments, the Cas can be derived from modified Cas9 protein. For example, the amino acid sequence of the Cas9 protein can be modified to alter one or more properties (e.g., nuclease activity, affinity, stability, and so forth) of the protein. Alternatively, domains of the Cas9 protein not involved in RNA-guided cleavage can be eliminated from the protein such that the modified Cas9 protein is smaller than the wild type Cas9 protein. In general, a Cas9 protein comprises at least two nuclease (i.e., DNase) domains. For example, a Cas9 protein can comprise a RuvC-like nuclease domain and a HNH-like nuclease domain. The RuvC and HNH domains work together to cut single strands to make a double-stranded break in DNA. (Jinek, et al., 2012, Science, 337:816-821). In certain embodiments, the Cas9-derived protein can be modified to contain only one functional nuclease domain (either a RuvC-like or a HNH-like nuclease domain). For example, the Cas9-derived protein can be modified such that one of the nuclease domains is deleted or mutated such that it is no longer functional (i.e., the nuclease activity is absent). In some embodiments in which one of the nuclease domains is inactive, the Cas9-derived protein is able to introduce a nick into a double-stranded nucleic acid (such protein is termed a "nickase"), but not cleave the double-stranded DNA. In any of the above-described embodiments, any or all of the nuclease domains can be inactivated by one or more deletion mutations, insertion mutations, and / or substitution mutations using well- known methods, such as site-directed mutagenesis, PCR-mediated mutagenesis, and total gene synthesis, as well as other methods known in the art. [000194] In one non-limiting embodiment, a vector drives the expression of the CRISPR system. The art is replete with suitable vectors that are useful in the instant specification. The vectors to be used are suitable for replication and, optionally, integration in eukaryotic cells. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence. The vectors of the instant specification may also be used for nucleic acid standard gene delivery protocols. Methods for gene delivery are known in the art (U.S. Patent Nos.5,399,346, 41 53121668.1Attorney Docket No.046483-7440WO1(03745) 5,580,859 & 5,589,466, incorporated by reference herein in their entireties). [000195] Further, the vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (4thEdition, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 2012), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, Sindbis virus, gammaretrovirus and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No.6,326,193). [000196] In some embodiments, the compound that down regulates the activity or expression level of MMP-9 comprises a nucleic acid that down regulates the expression level of MMP-9 by the means of CRISPR knockdown. CRISPR knockdown includes, but not limited to, CRISPRCas13 knockdown. (See e.g., 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 entireties of which are incorporated herein by reference). [000197] In some embodiments, the present invention includes any other methods for effecting gene knockdown and / editing, which allow for deletion and / or inactivation of MMP-9, such as but not limited to those described in WO 2018 / 236840 (which is incorporated herein in its entirety by reference). Compounds that Downregulate MMP-9 by Inactivating and / or Sequestering [000198] 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 sequestering MMP-9. In some embodiment, the compound includes a nucleic acid that express the protein that downregulates the activity of MMP-9 by inactivating and / or sequestering MMP-9. In some embodiments, the compound includes an expression vector that express the protein that downregulates the activity of MMP-9 by inactivating and / or sequestering MMP-9 (see “Vector” section for non-limiting descriptions on vectors). [000199] 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 a vector expressing the trans-dominant negative mutant of MMP-9. [000200] In some embodiments, the term "treatment" or "treating" includes the application or administration of a therapeutic agent, e.g., a compound useful within the disclosure (alone or 42 53121668.1Attorney Docket No.046483-7440WO1(03745) in combination with another pharmaceutical agent), to a subject, or application or administration of the therapeutic agent to an isolated tissue or cell line from a subject (e.g., for diagnosis or ex vivo applications), who has a disease or disorder and / or a symptom of a disease or disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder and / or the symptoms of the disease or disorder. Such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics. [000201] The terms “fibrodysplasia ossificans progressiva” or “FOP,” as used herein, refer to a genetic disorder of congenital skeletal malformations and progressive heterotopic ossification (HO). [000202] In some embodiments, “heterotopic ossification” or “HO,” as used herein, refer to the formation of extraskeletal bone in muscle and soft tissues. HO is one of the post-natal features of FOP, and is a common occurrence after trauma in non-FOP patients. Details of heterotopic ossification is described in, for example, Meyers et al. (JBMR Plus 2019, 3: e10172) and Dey et al., (Transl Res.2017 Aug; 186: 95–111). [000203] In some embodiments, the term “flare-up,” when used in connection to FOP herein, refers to inflammatory soft tissue swellings experienced by most FOP patients, which may begin during early childhood and progress throughout life. Flare-ups include unpredictable episodes of soft tissue swelling, pain, reduced movement, and / or stiffness. These flare-ups usually result in extra bone formation, but not always. Details of flare-ups in FOP is described in, for example, Pignolo et al. (J Bone Miner Res.2016 Mar;31(3):650-6). Method for Treating, Ameliorating and / or Preventing Non-FOP Heterotopic Ossification [000204] The present study discovered that down-regulating MMP9 level and / or activity can reduce heterotopic bone formation in transgenic mice harboring R206H or Q207D mutation in the Acvr1 gene. These transgenic mice are widely accepted in the art as animal models for the study of heterotopic ossification (HO) in general. This is especially true for the Q207D transgenic mice, as the mutation does not cause spontaneous heterotopic ossification but rather results in HO in response to injury only and produces very robust post-traumatic HO. As such, the method herein is also applicable to non-FOP HO. [000205] Accordingly, in some aspects, the present invention is directed to a method of treating, ameliorating and / or preventing non-FOP heterotopic ossification (HO) in a subject in need thereof. [000206] In some embodiments, the method includes administering to the subject a 43 53121668.1Attorney Docket No.046483-7440WO1(03745) compound that down-regulates MMP-9 level and / or activity in the subject. [000207] In some embodiments, the compound that down-regulates MMP-9 level and / or activity is the same as or similar to those as described elsewhere herein, such as in the “Method of Treating, Ameliorating and / or Preventing Fibrodysplasia Ossificans Progressiva” section. [000208] In some embodiments, the non-FOP HO is caused by trauma or injury in the bone, severe burns, stroke, head trauma, paralysis, orthopaedic procedures, hip replacements, severe crush injury, explosive wounds (such as those from improvised explosive), war wounds, severe civilian trauma, polio, tetanus, syringomyelia, myelodysplastic syndromes, multiple sclerosis (MS), carbon monoxide poisoning, or tumors in the spine. Kit or Composition for Treating, Ameliorating and / or Preventing Fibrodysplasia Ossificans Progressiva and / or Non-FOP Heterotopic Ossification [000209] In some aspects, the present invention is directed to a kit for treating, ameliorating and / or preventing fibrodysplasia ossificans progressiva (FOP), or for treating, ameliorating and / or preventing a non-FOP HO in a subject in need thereof. [000210] In some aspects, the present invention is directed to a kit for treating, ameliorating and / or preventing heterotopic ossification in a subject in need thereof. [000211] In some embodiments, the kit includes a compound that down-regulates MMP-9 level and / or activity in the subject; and a manual instructing that an effective amount of the compound be administered to the subject. In some embodiments, the compound that down- regulates MMP-9 level and / or activity in the subject is the same as or similar to those as described elsewhere herein, such as in the “Method of Treating, Ameliorating and / or Preventing Fibrodysplasia Ossificans Progressiva” section. [000212] In some aspects, the present invention is directed to a composition, such as a pharmaceutical composition for treating, ameliorating and / or preventing fibrodysplasia ossificans progressiva (FOP), or for treating, ameliorating and / or preventing a non-FOP HO in a subject in need thereof. [000213] In some embodiments, the composition includes a compound that down-regulates MMP-9 level and / or activity in the subject, and a pharmaceutically acceptable carrier. In some embodiments, the compound that down-regulates MMP-9 level and / or activity in the subject is the same as or similar to those as described elsewhere herein, such as in the “Method of Treating, Ameliorating and / or Preventing Fibrodysplasia Ossificans Progressiva” section. The pharmaceutically acceptable carrier is described elsewhere herein. 44 53121668.1Attorney Docket No.046483-7440WO1(03745) Vectors [000214] Vectors can increase the stability of the nucleic acids, make the delivery easier, or allow the expression of the nucleic acids or protein products thereof in the cells. [000215] Therefore, in some embodiments, the protein inhibitors or the nucleic acids that that down regulates the activity or expression level of MMP-9 is incorporated into a vector. [000216] In some embodiments, the instant specification relates to a vector, including the nucleic acid sequence of the instant specification or the construct of the instant specification. The choice of the vector will depend on the host cell in which it is to be subsequently introduced. In certain embodiments, the vector of the instant specification is an expression vector. Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. In certain embodiments, the expression vector is selected from the group consisting of a viral vector, a bacterial vector and a mammalian cell vector. Prokaryote- and / or eukaryote-vector based systems can be employed for use with the instant specification to produce polynucleotide, or their cognate polypeptides. Many such systems are commercially and widely available. [000217] In some embodiments, the vector is a viral vector. Viral vector technology is well known in the art and is described, for example, in virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. (See, e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No.6,326,193. [000218] In some embodiments, the viral vector is a suitable adeno-associated virus (AAV), such as the AAV1-AAV8 family of adeno-associated viruses. In some embodiments, the viral vector is a viral vector that can infect a human. The desired nucleic acid sequence, such as the nucleic acids that downregulates MMP-9 described above, can be inserted between the inverted terminal repeats (ITRs) in the AAV. In various embodiments, the viral vector is an AAV2 or an 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 expression in the bone marrow, the lymphoid tissues, the connective tissues, the kidney, the urinary bladder and other tissues where MMP-9 is expressed. In some embodiments, the promoter is a neuron-selective promoter or a neuron-specific promoter. The AAV can be a recombinant AAV, in which the capsid comes from one AAV serotype 45 53121668.1Attorney Docket No.046483-7440WO1(03745) and the ITRs come from another AAV serotype. In various embodiments, the AAV capsid is selected from the group consisting of a AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and a AAV8 capsid. In various embodiments, the ITR in the AAV is at least one ITR selected from the group consisting of a AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and an AAV8 ITR. In various embodiments, the instant specification contemplates an AAV8 viral vector (recombinant or non-recombinant) containing a desired nucleic acid expression sequence and at least one promoter sequence that, when administered to a subject, causes elevated systemic expression of the desired nucleic acid. In some embodiments, the viral vector is a recombinant or non-recombinant AAV2 or AAV5 containing any of the desired nucleic acid expression sequences described herein. [000219] In some embodiments, the vector in which the nucleic acid sequence is introduced is a plasmid that is or is not integrated in the genome of a host cell when it is introduced in the cell. Illustrative, non-limiting examples of vectors in which the nucleotide sequence of the instant specification or the gene construct of the instant specification can be inserted include a tet-on inducible vector for expression in eukaryote cells. [000220] The vector may be obtained by conventional methods known by persons skilled in the art (Sambrook et al., 2012). In certain embodiments, the vector is a vector useful for transforming animal cells. [000221] In certain embodiments, the recombinant expression vectors may also contain nucleic acid molecules which encode a peptide or peptidomimetic inhibitor of the instant specification, described elsewhere herein. [000222] A promoter may be one naturally associated with a gene or polynucleotide sequence, as may be obtained by isolating the 5c non-coding sequences located upstream of the coding segment and / or exon. Such a promoter can be referred to as "endogenous." Similarly, an enhancer may be one naturally associated with a polynucleotide sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding polynucleotide segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a polynucleotide sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a polynucleotide sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not "naturally occurring," i.e., containing 46 53121668.1Attorney Docket No.046483-7440WO1(03745) different elements of different transcriptional regulatory regions, and / or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and / or nucleic acid amplification technology, including PCR™, in connection with the compositions disclosed herein (U.S. Patent 4,683,202, U.S. Patent 5,928,906). Furthermore, it is contemplated the control sequences that direct transcription and / or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well. [000223] It will be important to employ a promoter and / or enhancer that effectively directs the expression of the DNA segment in the cell type, organelle, and organism chosen for expression. Those of skill in the art of molecular biology generally know how to use promoters, enhancers, and cell type combinations for protein expression. The promoters employed may be constitutive, tissue-specific, inducible, and / or useful under the appropriate conditions to direct high-level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous. [000224] The recombinant expression vectors may also contain a selectable marker gene which facilitates the selection of transformed or transfected host cells. Suitable selectable marker genes are genes encoding proteins such as G418 and hygromycin which confer UHVLVWDQFH^WR^FHUWDLQ^GUXJV^^ȕ-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or an immunoglobulin or portion thereof such as the Fc portion of an immunoglobulin preferably IgG. The selectable markers may be introduced on a separate vector from the nucleic acid of interest. Combination Therapies [000225] In some embodiments, the method of treating, ameliorating, and / or preventing the disease and / or disorder contemplated herein includes administering to the subject the effective amount of at least one compound and / or composition contemplated within the disclosure. [000226] In some embodiments, the subject is further administered at least one additional agent that treats, ameliorates, and / or prevents the disease and / or disorder contemplated herein. In other embodiments, the compound and the at least one additional agent are co- administered to the subject. In yet other embodiments, the compound and the at least one additional agent are co-formulated. [000227] The compounds contemplated within the disclosure are intended to be useful in 47 53121668.1Attorney Docket No.046483-7440WO1(03745) combination with one or more additional compounds. These additional compounds may comprise compounds of the present disclosure and / or at least one additional agent for treating neurodegenerative conditions, and / or at least one additional agent that treats one or more diseases or disorders contemplated herein. [000228] A synergistic effect may be calculated, for example, using suitable methods such as, for example, the Sigmoid-Emaxequation (Holford & Scheiner, 1981, Clin. Pharmacokinet. 6:429-453), the equation of Loewe additivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol.114:313-326) and the median-effect equation (Chou & Talalay, 1984, Adv. Enzyme Regul.22:27-55). Each equation referred to above may be applied to experimental data to generate a corresponding graph to aid in assessing the effects of the drug combination. The corresponding graphs associated with the equations referred to above are the concentration-effect curve, isobologram curve and combination index curve, respectively. Administration / Dosage / Formulations [000229] The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations contemplated within the disclosure may be administered to the subject either prior to or after the onset of a disease and / or disorder contemplated herein. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations contemplated within the disclosure may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation. [000230] Administration of the compositions contemplated within the disclosure to a patient, preferably a mammal, more preferably a human, may be carried out using known procedures, at dosages and for periods of time effective to treat a disease and / or disorder contemplated herein in the patient. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the state of the disease or disorder in the patient; the age, sex, and weight of the patient; and the ability of the therapeutic compound contemplated within the disclosure to treat a disease and / or disorder contemplated herein in the patient. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non- limiting example of an effective dose range for a therapeutic compound contemplated within the disclosure is from about 1 and 5,000 mg / kg of body weight / per day. One of ordinary skill 48 53121668.1Attorney Docket No.046483-7440WO1(03745) in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation. [000231] Actual dosage levels of the active ingredients in the pharmaceutical compositions contemplated within the disclosure may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. [000232] In particular, the selected dosage level depends upon a variety of factors including the activity of the particular compound employed, the time of administration, the rate of excretion 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 like factors well, known in the medical arts. [000233] A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds contemplated within the disclosure employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. [000234] In particular embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms contemplated within the disclosure are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding / formulating such a therapeutic compound for the treatment of a disease and / or disorder contemplated herein. [000235] In certain embodiments, the compositions of the disclosure are formulated using one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the pharmaceutical compositions of the disclosure comprise a therapeutically effective amount of a compound of the disclosure and a pharmaceutically acceptable carrier. [000236] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and 49 53121668.1Attorney Docket No.046483-7440WO1(03745) the like), suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin. [000237] In certain embodiments, the compositions of the disclosure are administered to the patient in dosages that range from one to five times per day or more. In another embodiment, the compositions of the disclosure are administered to the patient in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks. It is readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the disclosure varies from individual to individual depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the disclosure should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient is determined by the attending physical taking all other factors about the patient into account. [000238] Compounds of the disclosure for administration may be in the range of from about 1 ^J^WR^DERXW^^^^^^^^PJ^^DERXW^^^^^J^WR^DERXW^^^^^^^PJ^^DERXW^^^^^J^WR^DERXW^^^^^^^PJ^^DERXW^ ^^^^J^WR^DERXW^^^^^^^PJ^^DERXW^^^^^^J^WR^DERXW^^^^^^^PJ^^DERXW^^^^^^J^WR^DERXW^^^^^^ mg, DERXW^^^^^^^J^WR^DERXW^^^^^^^PJ^^DERXW^^^^^^J^WR^DERXW^^^^^^^PJ^^DERXW^^^^^^J^WR^DERXW^ 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. [000239] In some embodiments, the dose of a compound of the disclosure is from about 1 mg and about 2,500 mg. In some embodiments, a dose of a compound of the disclosure used in 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 50 53121668.1Attorney Docket No.046483-7440WO1(03745) about 200 mg, or less than about 50 mg. Similarly, in some embodiments, a dose of a second compound as 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. [000240] In certain embodiments, the present 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 pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of neurodegenerative conditions in a patient. [000241] Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for intracranially, intrathecal , oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and / or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic agents. [000242] Routes of administration of any of the compositions of the disclosure include oral, nasal, rectal, intravaginal, parenteral, buccal, sublingual or topical. The compounds for use in the disclosure may be formulated for administration by any suitable route, such as for oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration. [000243] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical 51 53121668.1Attorney Docket No.046483-7440WO1(03745) administration and the like. It should be understood that the formulations and compositions that would be useful in the present disclosure are not limited to the particular formulations and compositions that are described herein. Oral Administration [000244] For oral application, particularly suitable are tablets, dragees, liquids, drops, suppositories, or capsules, caplets and gelcaps. The compositions intended for oral use may be prepared according to any method known in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutically excipients that are suitable for the manufacture of tablets. Such excipients include, for example an inert diluent such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate. The tablets may be uncoated or they may be coated by known techniques for elegance or to delay the release of the active ingredients. Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert diluent. [000245] For oral administration, the compounds of the disclosure may be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., polyvinylpyrrolidone, hydroxypropylcellulose or hydroxypropylmethylcellulose); fillers (e.g., cornstarch, lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrates (e.g., sodium starch glycollate); or wetting agents (e.g., sodium lauryl sulphate). If desired, the tablets may be coated using suitable methods and coating materials such as OPADRY™ film coating systems available from Colorcon, West Point, Pa. (e.g., OPADRY™ OY Type, OYC Type, Organic Enteric OY-P Type, Aqueous Enteric OY-A Type, OY-PM Type and OPADRY™ White, 32K18400). Liquid preparation for oral administration may be in the form of solutions, syrups or suspensions. The liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agent (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxy benzoates or sorbic acid). [000246] The present disclosure also includes a multi-layer tablet comprising a layer providing for the delayed release of one or more compounds of the disclosure, and a further layer providing for the immediate release of another medication. Using a wax / pH-sensitive polymer mix, a gastric insoluble composition may be obtained in which the active ingredient 52 53121668.1Attorney Docket No.046483-7440WO1(03745) is entrapped, ensuring its delayed release. Parenteral Administration [000247] For parenteral administration, the compounds of the disclosure may be formulated for injection or infusion, for example, intravenous, intramuscular or subcutaneous injection or infusion, or for administration in a bolus dose and / or continuous infusion. Suspensions, solutions or emulsions in an oily or aqueous vehicle, optionally containing other formulatory agents such as suspending, stabilizing and / or dispersing agents may be used. Additional Administration Forms [000248] Additional dosage forms of this disclosure include dosage forms as described in U.S. Patents Nos.6,340,475; 6,488,962; 6,451,808; 5,972,389; 5,582,837; and 5,007,790. Additional dosage forms of this disclosure also include dosage forms as described in U.S. Patent Applications Nos.20030147952; 20030104062; 20030104053; 20030044466; 20030039688; and 20020051820. Additional dosage forms of this disclosure also include dosage forms as described in PCT Applications 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. Controlled Release Formulations and Drug Delivery Systems [000249] In certain embodiments, the formulations of the present disclosure may be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations. [000250] The term sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period. The period of time may be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form. [000251] For sustained release, the compounds may be formulated with a suitable polymer or hydrophobic material which provides sustained release properties to the compounds. As such, the compounds for use the method of the disclosure may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation. [000252] In certain embodiments of the disclosure, the compounds of the disclosure are 53 53121668.1Attorney Docket No.046483-7440WO1(03745) administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation. [000253] The term delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that mat, although not necessarily, includes a delay of from about 10 minutes up to about 12 hours. [000254] The term pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration. [000255] The term immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration. [000256] As used herein, short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration. [000257] As used herein, rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration. Dosing [000258] The therapeutically effective amount or dose of a compound of the present disclosure depends on the age, sex and weight of the patient, the current medical condition of the patient and the progression of the neurodegenerative condition in the patient being treated. The skilled artisan is able to determine appropriate dosages depending on these and other factors. [000259] A suitable dose of a compound of the present disclosure may be in the range of from about 0.01 mg to about 5,000 mg per day, such as from about 0.1 mg to about 1,000 mg, for example, from about 1 mg to about 500 mg, such as about 5 mg to about 250 mg per day. The dose may be administered in a single dosage or in multiple dosages, for example from 1 to 4 or more times per day. When multiple dosages are used, the amount of each dosage may be the same or different. For example, a dose of 1 mg per day may be administered as two 0.5 mg doses, with about a 12-hour interval between doses. [000260] It is understood that the amount of compound dosed per day may be administered, 54 53121668.1Attorney Docket No.046483-7440WO1(03745) in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on. [000261] In the case wherein the patient's status does improve, upon the doctor's discretion the administration of the modulator of the disclosure is optionally given continuously; alternatively, the dose of drug being administered is temporarily reduced or temporarily suspended for a certain length of time (i.e., a "drug holiday"). The length of the drug holiday optionally varies 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. The dose reduction during a drug holiday includes from 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%. [000262] Once improvement of the patient's conditions has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, is reduced, as a function of the patient's condition, to a level at which the improved disease is retained. In certain embodiments, patients require intermittent treatment on a long- term basis upon any recurrence of symptoms and / or infection. [000263] The compounds for use in the method of the disclosure may be formulated in unit dosage form. The term "unit dosage form" refers to physically discrete units suitable as unitary dosage for patients undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier. The unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose. [000264] Toxicity and therapeutic efficacy of such therapeutic regimens are optionally determined in cell cultures or experimental animals, including, but not limited to, the determination of the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD50 and ED50. Capsid assembly modulators exhibiting high therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are optionally used in formulating a 55 53121668.1Attorney Docket No.046483-7440WO1(03745) range of dosage for use in human. The dosage of such capsid assembly modulators lies preferably within a range of circulating concentrations that include the ED50with minimal toxicity. The dosage optionally varies within this range depending upon the dosage form employed and the route of administration utilized. [000265] Those skilled in the art recognizes, or is able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents were considered to be within the scope of this disclosure and covered by the claims appended hereto. For example, it should be understood, that modifications in assay and / or reaction conditions, with art-recognized alternatives and using no more than routine experimentation, are within the scope of the present application. [000266] It is to be understood that wherever values and ranges are provided herein, all values and ranges encompassed by these values and ranges, are meant to be encompassed within the scope of the present disclosure. Moreover, all values that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present application. Examples [000267] The instant specification further describes in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless so specified. Thus, the instant specification should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein. Example 1: MMP-9 Deficiency Confers Resilience in Fibrodysplasia Ossificans Progressiva [000268] Single case studies of extraordinary disease resilience may provide therapeutic insight into conditions where no definitive treatments exist. A healthy 35-year-old man (Patient-R) with the canonical pathogenic ACVR1R206Hvariant and the classic congenital great toe malformation of fibrodysplasia ossificans progressiva (FOP) had extreme paucity of post-natal heterotopic ossification (HO) and nearly normal mobility. It was hypothesized that patient-R had a deficient post-natal inflammatory trigger for HO. A plasma biomarker survey revealed a reduction in total matrix metaloproteinase-9 (MMP-9) compared to healthy controls or individuals with quiescent FOP. Exome sequencing of genomic DNA and family 56 53121668.1Attorney Docket No.046483-7440WO1(03745) studies revealed that patient-R was a compound heterozygote for MMP-9 (A20V; D165N). Structural analysis of the MMP-9D165Nvariant predicted both decreased MMP-9 secretion and activity, confirmed by ELISA and gelatin zymography. Further, proinflammatory macrophages from both MMP-9 variants of patient-R produced significantly less Activin A, an obligate ligand for HO in FOP, compared to MMP-9 wildtype controls. Importantly, MMP-9 inhibition by genetic, biologic, or pharmacologic means in multiple FOP mouse models abrogated trauma-induced HO and led to regeneration of injured skeletal muscle. The data herein suggest that MMP-9 provides a druggable node between inflammation and HO, plays an existential role in the pathogenesis of FOP and illustrates that a single resilient individual can reveal molecular mechanisms of disease that unveil novel treatment strategies. [000269] The progressive transformation of one tissue into another is a fundamental signature of fibrodysplasia ossificans progressiva (FOP; MIM#135100), an ultra-rare condition and the most catastrophic form of extraskeletal bone formation in humans. Individuals with FOP appear normal at birth except for characteristic malformations of the great toes that are present in all classically affected individuals. During the first decade of life, episodic soft tissue swellings (or flare-ups) arise in the neck and back and undergo pathological metamorphosis into mature heterotopic bone through an endochondral pathway. Minor trauma such as intramuscular immunizations, mandibular blocks for dental work, muscle over-exertion, blunt muscle trauma, bumps, bruises, falls, or influenza-like viral illnesses can trigger flare-ups of FOP that lead to progressive heterotopic ossification (HO). [000270] Most patients are immobilized by age 30 and require lifelong assistance with activities of daily living. The median estimated lifespan is 56 years; death often results from complications of thoracic insufficiency syndrome. Definitive treatments are not yet available for FOP, and there is a critical unmet need for effective therapies. Standard-of-care medical management is currently supportive. [000271] Heterozygous missense germline pathogenic variants in activin receptor A type I / Activin-like kinase 2 (ACVR1 / ALK2), a bone morphogenetic protein (BMP) type I receptor, have been identified in all individuals with sporadic or familial. Pathogenic variants render ACVR1 susceptible to dysregulated BMP pathway signaling. Importantly, activin A, a member of the transforming growth factor-ȕ^^7*F-ȕ^^IDPLO\^RI^PROHFXOHV^HQKDQFHV^%03^ pathway signaling in fibroadipogenic progenitor cells (FAPs) harboring the ACVR1R206Hmutation and drives heterotopic bone formation in FOP. [000272] The pathogenic ACVR1R206Hvariant is necessary for the myriad developmental features of classic FOP but may not be sufficient to induce the bulk of inflammatory flare-ups 57 53121668.1Attorney Docket No.046483-7440WO1(03745) that lead to disabling post-natal HO. FOP flare-ups strongly implicate an underlying inflammatory trigger, and global immune suppression leads to decreased heterotopic ossification in FOP but has severe attendant risks. [000273] Here, the present study reports a 35-year-old man (patient-R) who has classic developmental features of FOP and the canonical pathogenic ACVR1R206Hvariant but has extreme resilience to the post-natal features of FOP including disabling flare-ups and severe progressive HO. The presence of classic developmental anomalies of FOP and the paradoxical paucity of post-natal HO led to the hypothesis that patient-R had an insufficient inflammatory trigger for flare-up initiation and subsequent HO. Biomarker analysis and genetic studies in patient-R and family members respectively revealed decreased basal secretion and activity of matrix metalloproteinase-9 (MMP-9) as well as compound heterozygosity for MMP-9. Those observations prompted further studies in FOP mouse models that revealed that even partial inhibition of MMP-9 activity by genetic, pharmacologic or biologic means potently inhibited HO and unveiled an unexpected molecular target in FOP and in possibly more common forms of HO. Example 2: Case Report [000274] An asymptomatic 21-year-old man (patient-R) was seen in the FOP clinic. He was the product of a full-term pregnancy and a caesarian section delivery due to a nuchal cord. Short malformed great toes and thumbs with absence of interphalangeal joints were noted early in life. He had routine childhood intramuscular immunizations without flare-ups. At four years of age, he suffered a venomous Loxosceles spider bite on his left thigh. There was no dermonecrosis, but he developed a five mm. hard nodule in his left quadriceps which did not affect movement. He enjoyed an active childhood playing soccer and baseball but experienced no flare-ups or loss of movement. He had two fractured teeth following a fall followed by root canal surgery under local anesthesia without flare-ups. He and his parents denied any history of short stature during childhood or lower extremity bowing. He had no hearing problems. He had always done well in school. He took multivitamins daily but no medications or nutritional supplements. His diet was unremarkable. There was no family history of skeletal malformations or HO. [000275] At 20 years of age, he spontaneously developed a two-centimeter nodule on the right side of his neck. Excisional biopsy revealed a fibroproliferative lesion and he was diagnosed with aggressive fibromatosis. A non-restricting ribbon of HO developed at the operative site. Clinical evaluation and radiographs of the feet revealed malformed great toes 58 53121668.1Attorney Docket No.046483-7440WO1(03745) with absence of the interphalangeal joints (Figs.1A-1B). [000276] Physical examination revealed a healthy 21-year-old male, 183 centimeters in height and 78 kilograms in weight with no abnormalities other than the absence of interphalangeal joints of the thumbs and great toes (Figs.1A-1B) and bilateral proximal medial tibial osteochondromas. He had normal range of motion of all other joints of the axial and appendicular skeleton, with the exception of slight decreased range of motion of the cervical spine and chest wall. He had a normal gait, no scoliosis and full muscle strength. There were no dental abnormalities, alopecia, or cardiac findings. His Cumulative Analogue Joint Involvement Scale (CAJIS) score, a validated tool for assessing FOP disease burden was 2 / 30 (age related range: 12-22; median: 18). Patient-R participated in a sponsored natural history study of classically-affected FOP patients in which low dose whole body computed tomography (WBCT) was performed to evaluate the total body burden of HO. A WBCT at 28 years of age revealed a small amount of asymptomatic HO in the lower lumbar spine (Fig. 1C, arrow). The WBCT volume of HO for patient-R was 47,000 mm3and remained constant over the three years of the natural history study (mean for 13 subjects between 25-35 years of age: 434,000 mm3). Over the next seven years, the FOP did not progress although there was some evanescent soft tissue swelling at the site of the previous biopsy without subsequent bone formation or loss of mobility. At the latest examination, the patient was 35 years old and the CAJIS remained at 2 / 30. Genetic testing confirmed the classic heterozygous pathogenic FOP variant (ACVR1 c.617G>A; R206H). There was no evidence of mosaicism (Fig.1D). The patient’s asymptomatic parents had normal ACVR1 alleles. [000277] Patient-R has the mildest form of classic FOP reported to date for a patient of his age. He had one minor post-traumatic flare-up of FOP and approximately 90 percent less heterotopic bone compared to age-matched controls. The presence of classic developmental features of FOP (Figs.1A-1C) suggested that FOP was active during embryogenesis. However, the paucity of postnatal inflammatory flare-ups of FOP and subsequent HO as well as the extraordinary preservation of mobility in adulthood suggested to us that he might have an insufficient inflammatory trigger for post-natal flare-ups and HO. [000278] One objective of the present study was to identify factors that contributed to the resilience to postnatal flare-ups and HO in this unique individual with FOP and to determine whether those factors were causally related to HO in genetically accurate mouse models of FOP. The identification of such resiliency factors could unveil previously unknown protective mechanisms in FOP and herald new therapeutic strategies for limiting and treating the inexorable progression of this disabling condition. 59 53121668.1Attorney Docket No.046483-7440WO1(03745) Example 3: Methods Institutional Review Board [000279] All clinical evaluations and imaging studies were obtained as part of routine clinical care. The proposal to publish these observations was reviewed by the Institutional Review Board of the University of Pennsylvania and was exempted from human subject research. Genotyping and Exome sequencing [000280] Genomic DNA from peripheral blood of the patient and family members was examined by DNA sequence analysis of the ACVR1 gene by PCR amplification using exon- flanking primers. [000281] Exome sequencing of genomic DNA was performed commercially and detected the MMP-9 variants of unknown significance (VUS) reported here. The presence of MMP-9 variants was confirmed by DNA sequence analysis of the MMP-9 gene by using PCR amplicons with primers for 59C>T 5’-GAGTCAGCACTTGCCTGTCA-3’ (forward, SEQ ID NO:50) and 5’-GATACGCCCATCACCACCAA-3’ (reverse, SEQ ID NO:51), and 493G>A 5’-CCTTCCTCTGGCTCTTACGC-3’ (forward, SEQ ID NO:52), 5’- GGCAGGAGGGGCCGTATAA-3’ (reverse, SEQ ID NO:53). DNA sequence analysis of ACVR1 and MMP-9 was conducted through the University of Pennsylvania Perelman School of Medicine DNA Sequencing Facility. Blood Samples [000282] Blood samples were collected from FOP patients, their family members (unaffected controls), and patient-R with informed consent as part of routine clinical care visits in the Department of Orthopaedic Surgery at the Perelman School of Medicine of the University of Pennsylvania. Blood was collected in 10 ml K2 EDTA tubes (BD, #366643) for adults and children more than two years old or 4 ml K2 EDTA tubes (BD, #367861) for children under two years of age. Samples were maintained at room temperature until processing 2-24 hours later. Human Plasma and Peripheral Blood Mononuclear Cells (PBMC) Preparation [000283] Undiluted blood was layered over Ficoll-Paque (GE Healthcare, #17-1440-02) in 15 ml tubes and centrifuged at room temperature for 30 min at 400 x g with no brake. Plasma was collected from the top layer (leaving behind PBMCs, red blood cells, and Ficoll) and 60 53121668.1Attorney Docket No.046483-7440WO1(03745) then transferred to a new 15 ml tube. The plasma was centrifuged for 10 min at 1400 x g with brake to remove residual cells, and the supernatant was aliquoted into 1.5 ml cryovials. Samples were stored at -80oC. [000284] The mononuclear cell layer with PBMCs was carefully collected after Ficoll-Paque separation as above and washed two times by centrifugation with PBS (5 min at 300 x g). PBMC were aliquoted, frozen, and stored in liquid nitrogen. Plasma Biomarker Analysis Multiplex Luminex analysis was performed by Myriad RBM as described in Pignolo (J Bone Miner Res.2022 Mar;37(3):475-483). Briefly, plasma analytes of inflammation were chosen from among 113 biomarkers and independently of the original analysis compared patient-R to a group of 11 other FOP patients who had quiescent flare-up status at the time of blood collection (quiescent status was defined when the last flare-up occurred more than 2 years before sample collection). The present study compared 38 FOP patients and 39 controls with patient-R (obtained at two different visits, one year apart). Mice [000285] Acvr1Q207D / +mice were a gift from Dr. Yuji Mishina (University of Michigan). To induce expression of ACVR1Q207Dfollowed by HO, AV-Cre (University of Pennsylvania Vector Core; 1 × 1011particles per mouse) was injected together with 50 μL of 10 μM cardiotoxin into the popliteal fossa of Acvr1Q207D / +mice at 3-4 weeks of age. All animal procedures were reviewed and approvedAnimal Care and Use Committee at the University of Pennsylvania. [000286] Acvr1ARC-R206H / +mice were used for breeding with Gt(ROSA26)SorCreERT2 / CreERT2mice (The Jackson Laboratory, #008463) to generate tamoxifen-inducible global Acvr1ARC-R206Hallele expression after recombination by Cre recombinase in produced Acvr1ARC-R206H / +;Gt(ROSA26)SorCreERT2 / +(Acvr1ARC-R206H / +;CreERT2- / +) mice.5 times over 2 weeks (starting at 4 weeks of age) with tamoxifen in corn oil (100 mg / kg body weight, 10 mg / ml stock solution, Sigma, St. Louis, MO, USA; #T5648) to induce expression of Acvr1ARC-R206Hfollowed by injecting 50 μL of 10 μM cardiotoxin from Naja mossambica (Sigma-Aldrich, St. Louis, MO, USA; #C9759) into muscles for inducing HO. CRISPR / Cas-9 MMP-9 Gene Editing 61 53121668.1Attorney Docket No.046483-7440WO1(03745) [000288] Gene editing was used to insert separately MMP-959C>Tand MMP-9493G>Ainto human THP-1 cells. gRNA and dDNA were designed with TrueDesign Genome Editor (https: / / www.thermofisher.com / us / en / home / life-science / genome-editing / invitrogen- truedesign-genome-editor.html). [000289] Briefly, THP-1 cells were seeded at 50 x 103cells / well one day before transfection in 24-well plates. Transfection was performed using 2,000 ng of TrueCut HiFi Cas9 Protein (Invitrogen, #A50574), 12.0 pM of guide RNA, 18.0 pM donor DNA per 50 x 103cells with Neon Transfection System (Invitrogen, #MPK5000) according to protocol from ThermoFisher Scientific. THP-1 cells after transfection were expanded for seven days and single cell clones were obtained after limiting dilution cloning in 96-well plates and screened for MMP-959C>Tand MMP-9493G>Areplacements using PCR primers 5’- GAGTCAGCACTTGCCTGTCA-3’ (forward, SEQ ID NO:50), 5’- GATACGCCCATCACCACCAA-3’ (reverse, SEQ ID NO:51) and 5’- CCTTCCTCTGGCTCTTACGC-3’ (forward, SEQ ID NO:52), 5’- GGCAGGAGGGGCCGTATAA-3’ (reverse, SEQ ID NO:53) respectively, and the PCR products were Sanger-sequenced to confirm the replacement. Cell Culture [000290] PBMCs were defrosted, suspended in AIM-V medium (Gibco, #12055091) and plated at a density of 2.5×106cells / ml in 24-well plates with 20 ng / ml of TNF-Į^DQG^ incubated at 370C in a 5% CO2atmosphere. Cell culture supernatants were collected at 72 hours, centrifuged, aliquoted and stored at -80oC. Human monocytic cells deriving from an acute monocytic leukemia patient THP-1 were obtained from ATCC (#TIB-202) and cultured in RPMI-1640 (Gibco, #11875085) with 10% of FBS, including 50 μM of 2-mercaptoetanol (Gibco, #21985023), 10mM HEPES (Gibco, #15630080), 1mM sodium pyruvate (Gibco, #11360070), and antibiotic-antimycotic (Gibco, #15240096). THP-1 cells were centrifuged and transferred at 100 x 103cells per well of 24-well plate in AIM-V medium (Giboco, #31035025), to differentiate into M0 macrophages by 72 h incubation with 10 nM of phorbol 12-myristate 13-acetate (PMA, Sigma, #P8139) followed by 24 h incubation in fresh AIM-V medium. M0 macrophages were polarized into M1-like macrophages with 20 ng / ml of IFN-g (PeproTech, #300-02) and 10 pg / ml of LPS (Sigma, #8630). M2a-like macrophages were obtained by incubation with 20 ng / ml of IL-4 (PeproTech, #200-04) and 20 ng / ml of IL-13 (PeproTech, #200-13) (Genin, 2015). Cell culture supernatants were collected at indicated in figures time-points, centrifuged, aliquoted and stored at -80oC. Cells after lysis with RIPA 62 53121668.1Attorney Docket No.046483-7440WO1(03745) Lysis and Extraction buffer (ThermoFisher Scientific, Grand Island, NY, USA; # 89901) with 1x proteinase inhibitor cocktail (ThermoFisher Scientific, Grand Island, NY, USA; #78429) were collected for analysis also as cell culture supernatants. ELISA [000291] Total MMP9 protein (pro-, active, and TIMP-complexed MMP-9) was determined by sandwich ELISA (Mouse Total MMP-9 Quantikine ELISA Kit, MMPT90, R&D Systems, Minneapolis, MN, USA) in heparin treated platelet-poor plasma. Human Activin A was measured by Human Activin A ELISA Kit (ThermoFisher Scientific, #EHACTIVINA) and mouse Activin A was measured by Mouse Activin A ELISA Kit (ThermoFisher Scientific, #EM3RB). MMP-9 and Activin A in cell culture supernatants were normalized to protein in cell lysates. Gelatin Zymography [000292] Gelatin zymography was performed using cell supernatants for measurement of active MMP9 with Novex Zymogram Plus Gels according manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA, ZY00100BOX). The gels were scanned, the images were inverted, and densitometry levels were determined by using the ImageJ software (https: / / imagej.nih.gov / ij / index.html). The generated profile yielded peaks corresponding to each band. The quantity of MMP-9 in cell culture supernatants was normalized to protein in cell lysates. Micro-Computerized Tomography [000293] Micro computerized WRPRJUDSK\^^^&7^^ZDV^SHUIRUPHG^RQ^KLQG^OLPEV^IURP^PLFH^ obtained 14 days after cardiotoxin or adenovirus-Cre / cardiotoxin injection using a Scanco VivaCT 40 device (Bruettisellen, Switzerland) to determine the volume of heterotopic bone and obtain a two-dimensional image of the medial sagittal plane of each limb. Scanning was SHUIRUPHG^XVLQJ^D^VRXUFH^YROWDJH^RI^^^^N9^^D^VRXUFH^FXUUHQW^RI^^^^^^$^^DQG^DQ^LVRWURSLF^ YR[HO^VL]H^RI^^^^^^^P^^%RQH^ZDV^GLIIHUHQWLDWHG^IURP^³QRQ-bone” by an upper threshold of 1000 Hounsfield units and a lower threshold of 150 Hounsfield units. Structural-Functional Analyses of MMP-9 Variants [000294] Bioinformatic analysis of the MMP-9A20Vvariant was conducted with the SignalP server (http: / / www.cbs.dtu.dk / services / SignalP / . High-resolution crystal structures of MMP-9 63 53121668.1Attorney Docket No.046483-7440WO1(03745) deposited in the Protein Data Bank (PDB) were viewed and superposed with the graphics program PyMOL. Statistical Analysis [000295] All experiments were performed with at least three biological replicates, with sample sizes indicated in the figure descriptions. GraphPad Prism 8.0 software (San Diego, CA, USA) was used for statistical analysis. Data were analyzed using an unpaired t-test or t- test with Mann-Whitney test when two groups were being compared. One-way Anova with Tukey’s multiple comparisons test or two-way analysis of variance was used when more than two groups were compared, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 were considered significant. Data are represented as the means ± standard error of the mean (SEM). Example 4 [000296] Based on the index case findings, patient-R has developmental findings of classic FOP but a paucity of post-natal HO. He has the classic FOP pathogenic variant (ACVR1R206H) with no evidence of mosaicism. An early clue to his resiliency was uncovered when the present study compared 113 analytes between patient-R and eleven other adults with classic FOP who were also quiescent (no flare-ups for at least two years) at the time of plasma sample collection. Example 5: Patient-R has Suppressed Plasma MMP-9 Levels and Decreased Enzymatic Activity of MMP-9 Compared to Other FOP Patients and Controls [000297] There were no statistically significant differences in MMP-9 levels between unaffected individuals (no FOP) and those with genetically confirmed classic FOP (Fig.2A). However, compared to other patients with classic FOP, patient-R had dramatically lower levels of MMP-9 (mean 186 ± 15), at least four-fold less than the concentration found in other FOP patients (mean 807 ± 103) (Fig.2B). Importantly, gelatin zymography revealed statistically significant lower MMP-9 activity in PBMC culture supernatants from Patient-R compared to unaffected controls or other individuals with classic FOP (Figs.2C-2D). Example 6: Structural Analysis Predicts Decreased Enzymatic Activity of MMP- 9D165Nin Patient-R [000298] Bioinformatic analysis with the SignalP server indicates that residue 20 of MMP-9 64 53121668.1Attorney Docket No.046483-7440WO1(03745) is predicted to be contained within the signal peptide cleavage motif, comprising the N- terminus of the secreted polypeptide. However, the efficiency of secretion of the MMP-9A20Vvariant is unlikely to be disrupted since signal peptide cleavage is calculated to be comparable to that of wildtype. [000299] In contrast, the MMP-9D165Nvariant was predicted by a suite of computational approaches to be highly deleterious to MMP-9 structure and function and to result in a non- functional variant. The MMP-9D165Nvariant corresponds to the zinc binding region of the catalytic domain of MMP-9, thus is likely defective in its enzymatic activity. [000300] In addition, inspection of the three-dimensional structure of the MMP-9 proprotein revealed that Asp165 forms a robust ion pair with His118 (Figs.4A-4C). An asparagine sidechain at residue 165 would disrupt the linkage, a key structural element that might serve manifold functional roles. Along with deficient enzymatic activity per above, the MMP- 9D165Nvariant is predicted to be highly unstable, which would diminish folding efficiency in the endoplasmic reticulum, a site of quality control regulating the trafficking of properly folded secretory proteins. Furthermore, since the ion pair stabilizes a loop just several residues C-terminal of the site of MMP-9 cleavage and activation (Figs.4B-4C), the loop of the MMP-9D165Nprotein might not adopt a conformation optimal for proteolysis by the activating enzyme plasminogen. Example 10: MMP-9 Blockade with Minocycline Leads to Decreased Injury- induced HO in FOP Mouse Models [000301] Acvr1Q207D / +mice were treated twice daily with a pharmacologic inhibitor of MMP- 9 (minocycline) and two weeks post-injury HO was assessed and quantified. Minocycline treatment resulted in statistically significant decreases in HO in the mice (Fig.5). Further testing in the Acvr1Q207D / +mice indicated that minocycline could be administered at a dose as low as 5mg / kg and still abrogate formation of HO (Fig.6). Example 11: MMP-9 Blockade with a Monoclonal Antibody to MMP-9 Leads to Decreased Injury-induced HO in FOP Mouse Models [000302] Minocycline may have off-target effects, and so the present study used a specific monoclonal antibody against MMP-9 and demonstrated similar efficacy in reducing HO in the FOP Acvr1ARC-R206H / +;CreERT2- / +and Acvr1Q207D / +mouse models (Figs.7A-7B). Example 12: MMP-9 Variants in Patient-R Impairs Activin A Release from 65 53121668.1Attorney Docket No.046483-7440WO1(03745) Polarized M1-like Macrophages [000303] Activin A is the primary driver of BMP pathway signaling leading to HO in FOP. Macrophages are a key inflammatory component and is a major source for both MMP-9 and Activin A, where M1-like macrophages are known to produce relatively greater amounts of Activin A compared to M2-like macrophages. CRISPR Cas9 MMP-9 gene editing was used to insert MMP-959C>Tor MMP-9493G>A, the patient-R variants corresponding to MMP-9 A20V and MMP-9 D165N, respectively, into human THP-1 cells. Analysis of MMP-9 enzymatic activity by gelatin zymography revealed statistically significant lower activity in cell culture supernatants from D165N macrophages after polarization into M1-like and M2- like cells (Figs.8A-8B). Compared to M1-like and M2-like wild-type macrophages, the levels of Activin A from cell culture supernatants of A20V and D165N macrophages were significantly reduced (Fig.8C). These results suggest that MMP-9 variants in patient-R have an inhibitory effect on Activin A secretion from M1-like macrophages. This inhibitory effect of MMP-9 down-regulation on Activin A secretion was further supported by MMP-9 inactivation by minocycline in THP-1 derived M1-like macrophages (Fig.10). Example 13 [000304] The present study shows that MMP-9 plays a critical role in the pathogenesis of FOP and illustrates that the study of a single resilient individual can reveal new mechanisms of disease that unveil novel treatment strategies. The presence of the classic great toe malformation of FOP along with an extreme paucity of post-natal HO in patient-R suggested to us that FOP was active during embryogenesis but relatively inactive post-natally, possibly due to the paucity of a proinflammatory trigger for HO. The findings of significantly decreased levels and activity of MMP-9 in patient-R along with the noted genetic variants in MMP-9 drew the attention to MMP-9 as a putative trigger of HO in FOP which was validated by in vivo functional studies in FOP mouse models and with detailed functional studies in human cells. Taken together, these results support that MMP-9 mediates HO in FOP by regulating an inflammatory response to tissue injury. [000305] MMP-9 (or gelatinase B) belongs to a multi-gene family of more than 20 matrix metalloproteinases that are highly conserved throughout the animal kingdom. MMP-9 is expressed and secreted by neutrophils, mast cells, and macrophages, regulates and is regulated by inflammatory cytokines including IL-^ȕ^^7*)-ȕ^DQG^9(*)^DQG^PRGXODWHV multiple immunoregulatory pathways, impairs neutrophil chemoattraction, lymphocyte infiltration, and monocyte differentiation, degrades and remodels the extracellular matrix and 66 53121668.1Attorney Docket No.046483-7440WO1(03745) orchestrates the migration of stem cells and tissue progenitor cells in a large spectrum of physiologic and pathological processes. [000306] MMP-9 induces endochondral ossification both developmentally and in fracture healing, and is expressed at elevated levels in ischemic and hypoxic skeletal muscle which exists in early FOP flare-ups. Although MMP-2 and MMP-9 share the same substrates in vitro, they are not redundant in vivo, and their individual roles are uniquely regulated in osteogenesis. MMP-2 is exclusively involved in intramembranous osteogenesis while MMP- 9 is uniquely involved in endochondral osteogenesis – the latter exclusively involved in the HO of FOP. Additionally, MMP-9 regulates chondrogenic and osteogenic cell differentiation during early stages of fracture repair, a process nearly identical histologically to HO in FOP. [000307] Studies in mice and humans show that MMP-9 is a predictive diagnostic biomarker for early HO, but its role, if any, in the pathogenesis of FOP has not been investigated. The clinical, biochemical and genetic findings in patient-R and the supporting data in FOP mice suggest that even a partial reduction in MMP-9 levels protects against HO in the presence of the classic ACVR1R206Hmutation. What is particularly intriguing is that haploinsufficiency of MMP-9 also seems to be protective of HO in FOP mice. This unexpected finding is consistent with the upregulation of MMP-9 expression during the early stages of traumatic HO and suggests that basal levels of MMP-9 may be sufficient for normal skeletal development but insufficient to drive the pathologic process of HO in FOP. The observations in patient-R and the in vivo studies in multiple FOP mouse models confirm this hypothesis and demonstrate that MMP-9 plays a pivotal role in the pathogenesis of HO in FOP. Collectively, these data support a hypothesis that pharmacologic reduction in tissue levels of MMP-9 in patients with FOP may protect against injury-induced HO. [000308] While the MMP-9D165Nvariant discovered in patient-R is rare and non-pathogenic in the general population, this variant has been studied extensively. Importantly, the specific enzymatic activity (total activity / secreted protein amount) for the MMP-9D165Nvariant is significantly lower than that for wild-type MMP-9 suggesting a substantial effect of the amino acid substitution on the enzymatic activity of MMP-9 in addition to a decrease in secreted protein. Consistent with this finding, the MMP-9D165Nvariant is predicted to be protective in patients with aortic aneurisms and possibly other disorders of dysregulated inflammation. The MMP-9 variant (D165N) detected in patient-R presents a structural basis for loss-of-function of secreted MMP-9 and has important implications for suppression of HO in FOP (Figs.4A-4C). [000309] Bolstered by the protection from HO afforded by even heterozygous deficiency of 67 53121668.1Attorney Docket No.046483-7440WO1(03745) MMP-9 in the genetic knockout model, the present study inquired about the availability of approved medications to therapeutically lower MMP-9 levels that could potentially be used in those with FOP who do not harbor rare, hypomorphic variants in MMP-9 as in patient-R. The attention was directed to the tetracycline class of pharmaceuticals, which independently of their antibiotic properties, are inhibitors of MMP-9 transcription, translation, secretion and activation at sub-antimicrobial doses. Interestingly, tetracyclines confer resilience to diverse conditions in which MMP-9 has a pathogenic role including dermonecrosis from Loxosceles spider bites, multiple sclerosis, atherosclerosis, myocardial infarction, thoracic and abdominal aortic aneurism, glomerulonephritis, Duchenne muscular dystrophy and ischemic skeletal muscle injury. Importantly, inhibition of MMP-9 by doxycycline or monoclonal antibodies against MMP-9 improves muscle regeneration, and that suppression of MMP-9 by minocycline diminishes HO in a BMP-implant model. [000310] As pharmaceutical inhibition of HO by tetracyclines may not work exclusively through MMP-9 blockade, the present study also investigated whether a monoclonal antibody that specifically blocks MMP-9 might be effective in reducing HO. The present study showed proof-of-concept that a blocking monoclonal antibody to MMP-9 reduced HO in FOP mice. [000311] The mechanisms, by which MMP-9 blockade confers resilience against HO in FOP, and possibly in more common forms of HO, are likely multi-factorial and complex. The three cardinal requirements for HO in any context are 1) an inductive signal, 2) a receptive progenitor cell, and 3) a conducive microenvironment. MMP-9 likely functions in all three domains in FOP. [000312] The elimination of macrophages abrogates HO and proinflammatory macrophages are a potent source of both MMP-9 and Activin A. Furthermore, Activin A, an obligate ligand for HO in FOP, which is rapidly upregulated by inflammatory macrophages and other activated immune cells, leads to production of pro-inflammatory cytokines, such as TNFĮ^^ IL-1E, and IL-6, and the recruitment of mast cells, thereby initiating HO pathogenesis. The data in patient-R suggests that MMP-9 stimulates the secretion of Activin A. MMP-9 secreted by pro-inflammatory macrophages degrades the heparan sulfate proteoglycan perlecan to which Activin A is bound, thus releasing it in active form from the matrix and allowing it to bind to the mutant ACVR1signaling complex expressed in FAPs and signal through the SMAD 1, 5 complex. Importantly, TGF-ȕ^SRWHQWO\^LQGXFHV^$FWLYLn A production in fibroblasts from patients with FOP. Taken together, these data provide a plausible mechanism by which MMP-9 and TGF-ȕ^PD\^UHJXODWH^WKH^$FWLYLQ^$^OHYHOV^LQ^D^SDUDFULQH^PDQQHU^LQ^)23^ 68 53121668.1Attorney Docket No.046483-7440WO1(03745) thus contributing to HO. Remarkably, the present study found that both the MMP-9A20Vand the MMP-9D165Nvariants from patient-R produce significantly less activin A in inflammatory macrophages than MMP-9WT. Interestingly, minocycline targets both MMP-9 and activin A in inflammatory macrophages and may potentially afford a beneficial and redundant mode of protection from HO, particularly in FOP. [000313] The fact that MMP-9 blockade in the constitutively active and engineered ACVR1Q207Dsurrogate mouse model of FOP as well as in the BMP implant model protects against the formation of HO suggests that the reduction of Activin A may not be the only mechanism by which HO in FOP is inhibited by MMP-9 deficiency. Recent studies indicate that Activin A may promote but does not drive the development of acquired HO. Previous studies show that Activin A inhibition is incapable of inhibiting HO in ACVR1Q207Dmice, thus suggesting that MMP-9 inhibition of HO acts in part through ligand independent as well as ligand dependent mechanisms, even in FOP and may likely be a therapeutic target in non- genetic forms of HO. [000314] Extracellular matrix (ECM) degradation by inflammatory cells also plays a vital role in osteogenesis. Macrophage-released MMP-9 degrades multiple ECM proteins through proteolytic cleavage and increases the availability of a variety of bioactive molecules that have profound effects on stem cells that regulate critical biological processes, such as angiogenesis, fibrosis, chondrogenesis and osteogenesis, all essential for HO. Additionally, differences in ECM composition and collagen organization affected by MMP-9 following injury of ACVR1R206H / +muscle could enable ACVR1R206H / +cells to misinterpret ECM rigidity and facilitate HO. [000315] Our data suggest a hypothetical model in which MMP-9 plays a central role in the pathogenesis of HO in FOP (Fig.11). This model posits that trauma-induced flare-ups of FOP are triggered by inflammation and stimulate the secretion of MMP-9 from proinflammatory macrophages as well as elevated Activin A production. MMP-9 secreted and activated in the lesional microenvironment would facilitate the egress of inflammatory cells from hypoxic capillaries, which further degrades extracellular matrix, releases activin A and VEGF, recruits inflammation-activated fibroadipogenic progenitor cells (FAPs) from the connective tissue stem cell niche and amplifies the downstream cascade of Smad 1 / 5 signaling leading to HO in FOP. [000316] Our model does not rule-out the possibility that the secretion of MMP-9 could also be amplified by connective tissue progenitor cells such as FAP cells and thus participate directly in remodeling the extracellular matrix during regeneration of skeletal muscle, 69 53121668.1Attorney Docket No.046483-7440WO1(03745) however the observation that bone marrow transplantation corrects the skeletal phenotype in MMP-9 null mice suggests that targeted ex-vivo gene knockdown of MMP-9 in autologous hematopoietic stem cells may be a tantalizing therapeutic possibility to explore in FOP pre- clinical studies. [000317] An important question clinically is: when does MMP-9 act in the formation of HO in FOP mice? The data in genetically correct FOP mouse models suggest that MMP-9 acts very early in the process of HO, likely in the first three days. The data do not exclude the possibility that MMP-9 may also play a late role in the process of HO in FOP, yet the histology clearly indicates that the process of HO and regeneration diverge in the genetically correct mouse model of FOP by day five post injury. [000318] In summary, the present study identified MMP-9 as a critical modulator of HO in FOP and shown how the systematic study of a single resilient individual can unveil unexpected mechanisms of disease that may lead to novel treatment strategies. These investigations highlight an important finding in the pathogenesis of HO in FOP that is amenable to pharmacologic and / or biologic testing in clinical trials in humans. Because HO is not limited to FOP, there are opportunities to potentially broaden the findings herein to more common forms of HO. Enumerated Embodiments [000319] In some aspects, the present invention is directed to the following non-limiting embodiments: [000320] Embodiment 1: A method of treating, ameliorating, and / or preventing fibrodysplasia ossificans progressiva (FOP) in a subject in need thereof, the method comprising down- regulating matrix metalloproteinase 9 (MMP-9) level and / or activity in the subject. [000321] Embodiment 2: The method of Embodiment 1, wherein down-regulating the MMP- 9 level and / or activity in the subject comprises administering to the subject an effective amount of: a small molecule MMP-9 inhibitor, a protein MMP-9 inhibitor, a nucleic acid (and / or an expression vector expressing the nucleic acid) that downregulates MMP-9 by RNA interference, a ribozyme (and / or a vector expressing the ribozyme) that downregulates MMP- 9, an expression vector comprising an expression cassette, wherein the expression cassette expresses CRISPR components that downregulate MMP-9 by CRISPR knockout and / or CRISPR knockdown, and a trans-dominant negative mutant protein of MMP-9, and / or an expression vector that expresses the trans-dominant negative mutant protein of MMP-9. [000322] Embodiment 3: The method of any one of Embodiments 1-2, wherein the subject 70 53121668.1Attorney Docket No.046483-7440WO1(03745) has a mutant ACVR1 gene. [000323] Embodiment 4: The method of Embodiment 3, wherein the mutant ACVR1 gene encodes a constitutively active ACVR1 polypeptide. [000324] Embodiment 5: The method of Embodiment 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. [000325] Embodiment 6: The method of any one of Embodiments 2-5, wherein the small molecule MMP-9 inhibitor is selected from the group consisting of metformin, doxycycline, incyclinide, and minocycline, or a salt or solvate thereof. [000326] Embodiment 7: The method of any one of Embodiments 2-5, wherein the protein MMP-9 inhibitor is an anti-MMP-9 antibody or an antigen binding fragment thereof. [000327] Embodiment 8: The method of Embodiment 7, wherein the anti-MMP-9 antibody or the antigen binding fragment thereof comprises at least one of the following: (a) a heavy chain complementarity-determining region 1 (CDR1) set forth in SEQ ID NO:3, a heavy chain CDR2 set forth in SEQ ID NO:4, a heavy chain CDR4 set forth in SEQ ID NO:5, a light chain CDR1 set forth in SEQ ID NO:6, a light chain CDR2 set forth in SEQ ID NO:7, and a light chain CDR3 set forth in SEQ ID NO:8; (b) a heavy chain CDR1 set forth in SEQ ID NO:15, a heavy chain CDR2 set forth in SEQ ID NO:16, a heavy chain CDR4 set forth in SEQ ID NO:17, a light chain CDR1 set forth in SEQ ID NO:18, a light chain CDR2 set forth in SEQ ID NO:19, and a light chain CDR3 set forth in SEQ ID NO:20; (c) a heavy chain CDR1 set forth in SEQ ID NO:35, a heavy chain CDR2 set forth in SEQ ID NO:36, a heavy chain CDR4 set forth in SEQ ID NO:37, a light chain CDR1 set forth in SEQ ID NO:38, a light chain CDR2 set forth in SEQ ID NO:39, and a light chain CDR3 set forth in SEQ ID NO:40; (d) a light chain CDR1 set forth in SEQ ID NO:45, a light chain CDR2 set forth in SEQ ID NO:46, and a light chain CDR3 set forth in SEQ ID NO:47. [000328] Embodiment 9: The method of any one of Embodiments 7-8, wherein the anti- MMP-9 antibody or the antigen binding fragment thereof comprises at least one of the following: (a) a heavy chain variable region set forth in SEQ ID NO:9, and a light chain variable region set forth in SEQ ID NO:10; (b) a heavy chain variable region selected from the group consisting of SEQ ID 71 53121668.1Attorney Docket No.046483-7440WO1(03745) NOs:21, 23, 25, 27 and 29, and a light chain variable region selected from the group consisting of SEQ ID NOs:22, 24, 26, 28 and 30; (c) a heavy chain variable region set forth in SEQ ID NO:21 and a light chain variable region set forth in SEQ ID NO:22; (d) a heavy chain variable region set forth in SEQ ID NO:27 and a light chain variable region set forth in SEQ ID NO:30; (e) a heavy chain variable region set forth in SEQ ID NO:41 and a light chain variable region set forth in SEQ ID NO:42; (f) a light chain variable region set forth in SEQ ID NO:48. [000329] Embodiment 10: The method of any one of Embodiments 1-9, further comprising surgically removing an ossified tissue from the subject. [000330] Embodiment 11: The method of Embodiment 10, wherein the surgically removing step is performed after the MMP-9 level or activity in the subject is down-regulated. [000331] Embodiment 12: The method of any one of Embodiments 1-11, wherein the subject is a human. [000332] Embodiment 13: A method of treating, ameliorating and / or preventing a non-FOP heterotopic ossification (HO) in a subject in need thereof, the method comprising down- regulating matrix metalloproteinase 9 (MMP-9) level and / or activity in the subject. [000333] Embodiment 14: The method of Embodiment 13, wherein down-regulating the MMP-9 level and / or activity in the subject comprises administering to the subject an effective amount of: a small molecule MMP-9 inhibitor, a protein MMP-9 inhibitor, a nucleic acid (and / or an expression vector expressing the nucleic acid) that downregulates MMP-9 by RNA interference, a ribozyme (and / or a vector expressing the ribozyme) that downregulates MMP- 9, an expression vector comprising an expression cassette, wherein the expression cassette expresses CRISPR components that downregulate MMP-9 by CRISPR knockout and / or CRISPR knockdown, and a trans-dominant negative mutant protein of MMP-9, and / or an expression vector that expresses the trans-dominant negative mutant protein of MMP-9. [000334] Embodiment 15: The method of Embodiment 14, wherein the small molecule MMP-9 inhibitor is selected from the group consisting of metformin, doxycycline, incyclinide, and minocycline, or a salt or solvate thereof. [000335] Embodiment 16: The method of Embodiment 14, wherein the protein MMP-9 inhibitor is an anti-MMP-9 antibody or an antigen binding fragment thereof. [000336] Embodiment 17: The method of Embodiment 16, wherein the anti-MMP-9 antibody or the antigen binding fragment thereof comprises at least one of the following: 72 53121668.1Attorney Docket No.046483-7440WO1(03745) (a) a heavy chain complementarity-determining region 1 (CDR1) set forth in SEQ ID NO:3, a heavy chain CDR2 set forth in SEQ ID NO:4, a heavy chain CDR4 set forth in SEQ ID NO:5, a light chain CDR1 set forth in SEQ ID NO:6, a light chain CDR2 set forth in SEQ ID NO:7, and a light chain CDR3 set forth in SEQ ID NO:8; (b) a heavy chain CDR1 set forth in SEQ ID NO:15, a heavy chain CDR2 set forth in SEQ ID NO:16, a heavy chain CDR4 set forth in SEQ ID NO:17, a light chain CDR1 set forth in SEQ ID NO:18, a light chain CDR2 set forth in SEQ ID NO:19, and a light chain CDR3 set forth in SEQ ID NO:20; (c) a heavy chain CDR1 set forth in SEQ ID NO:35, a heavy chain CDR2 set forth in SEQ ID NO:36, a heavy chain CDR4 set forth in SEQ ID NO:37, a light chain CDR1 set forth in SEQ ID NO:38, a light chain CDR2 set forth in SEQ ID NO:39, and a light chain CDR3 set forth in SEQ ID NO:40; (d) a light chain CDR1 set forth in SEQ ID NO:45, a light chain CDR2 set forth in SEQ ID NO:46, and a light chain CDR3 set forth in SEQ ID NO:47. [000337] Embodiment 18: The method of any one of Embodiments 16-17, wherein the anti- MMP-9 antibody or the antigen binding fragment thereof comprises at least one of the following: (a) a heavy chain variable region set forth in SEQ ID NO:9, and a light chain variable region set forth in SEQ ID NO:10; (b) a heavy chain variable region selected from the group consisting of SEQ ID NOs:21, 23, 25, 27 and 29, and a light chain variable region selected from the group consisting of SEQ ID NOs:22, 24, 26, 28 and 30; (c) a heavy chain variable region set forth in SEQ ID NO:21 and a light chain variable region set forth in SEQ ID NO:22; (d) a heavy chain variable region set forth in SEQ ID NO:27 and a light chain variable region set forth in SEQ ID NO:30; (e) a heavy chain variable region set forth in SEQ ID NO:41 and a light chain variable region set forth in SEQ ID NO:42; (f) a light chain variable region set forth in SEQ ID NO:48. [000338] Embodiment 19: The method of any one of Embodiments 13-18, wherein the non- FOP HO is caused by a trauma or injury in the bone, a severe burn, a stroke, a head trauma, paralysis, an orthopaedic procedure, a hip replacement, a severe crush injury, an explosive wound, a war wound, a severe civilian trauma, polio, tetanus, a syringomyelia, a myelodysplastic syndrome, multiple sclerosis (MS), carbon monoxide poisoning, or a tumor 73 53121668.1Attorney Docket No.046483-7440WO1(03745) in the spine. [000339] Embodiment 20: The method of any one of Embodiments 13-20, wherein the subject is a human. [000340] Embodiment 21: A kit for treating, ameliorating and / or preventing fibrodysplasia ossificans progressiva (FOP) or a non-FOP heterotopic ossification (HO) in a subject in need thereof, comprising: a compound for down-regulating matrix metalloproteinase 9 (MMP-9) level and / or activity in the subject; and an instruction for administering an effective amount of the compound to the subject. [000341] Embodiment 22: The kit of Embodiment 21, wherein the compound comprises at least one selected from the group consisting of: a small molecule MMP-9 inhibitor, a protein MMP-9 inhibitor, a nucleic acid (and / or an expression vector expressing the nucleic acid) that downregulates MMP-9 by RNA interference, a ribozyme (and / or a vector expressing the ribozyme) that downregulates MMP-9, an expression vector comprising an expression cassette, wherein the expression cassette expresses CRISPR components that downregulate MMP-9 by CRISPR knockout and / or CRISPR knockdown, and a trans-dominant negative mutant protein of MMP-9, and / or an expression vector that expresses the trans-dominant negative mutant protein of MMP-9. [000342] Embodiment 23: The kit of any one of Embodiments 21-22, wherein the kit is for treating, ameliorating and / or preventing FOP, and wherein the subject has a mutant ACVR1 gene. [000343] Embodiment 24: The kit of Embodiment 23, wherein the mutant ACVR1 gene encodes a constitutively active ACVR1 polypeptide. [000344] Embodiment 25: The kit of Embodiment 24, 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. [000345] Embodiment 26: The kit of any one of Embodiments 21-22, wherein the kit is for treating, ameliorating and / or preventing non-FOP HO, and wherein the HO is caused by a trauma or injury in the bone, a severe burn, a stroke, a head trauma, paralysis, an orthopaedic procedure, a hip replacement, a severe crush injury, an explosive wound, a war wound, a severe civilian trauma, polio, tetanus, a syringomyelia, a myelodysplastic syndrome, multiple sclerosis (MS), carbon monoxide poisoning, or a tumor in the spine. [000346] Embodiment 27: The kit of any one of Embodiments 22-26, wherein the small molecule MMP-9 inhibitor is selected from the group consisting of metformin, doxycycline, 74 53121668.1Attorney Docket No.046483-7440WO1(03745) incyclinide, and minocycline. [000347] Embodiment 28: The kit of any one of Embodiments 22-26, wherein the protein MMP-9 inhibitor is an antibody against MMP-9 or an antigen binding fragment thereof. [000348] Embodiment 29: The kit of Embodiment 28, wherein the anti-MMP-9 antibody or the antigen binding fragment thereof comprises at least one of the following: (a) a heavy chain complementarity-determining region 1 (CDR1) set forth in SEQ ID NO:3, a heavy chain CDR2 set forth in SEQ ID NO:4, a heavy chain CDR4 set forth in SEQ ID NO:5, a light chain CDR1 set forth in SEQ ID NO:6, a light chain CDR2 set forth in SEQ ID NO:7, and a light chain CDR3 set forth in SEQ ID NO:8; (b) a heavy chain CDR1 set forth in SEQ ID NO:15, a heavy chain CDR2 set forth in SEQ ID NO:16, a heavy chain CDR4 set forth in SEQ ID NO:17, a light chain CDR1 set forth in SEQ ID NO:18, a light chain CDR2 set forth in SEQ ID NO:19, and a light chain CDR3 set forth in SEQ ID NO:20; (c) a heavy chain CDR1 set forth in SEQ ID NO:35, a heavy chain CDR2 set forth in SEQ ID NO:36, a heavy chain CDR4 set forth in SEQ ID NO:37, a light chain CDR1 set forth in SEQ ID NO:38, a light chain CDR2 set forth in SEQ ID NO:39, and a light chain CDR3 set forth in SEQ ID NO:40; (d) a light chain CDR1 set forth in SEQ ID NO:45, a light chain CDR2 set forth in SEQ ID NO:46, and a light chain CDR3 set forth in SEQ ID NO:47. [000349] Embodiment 30: The kit of any one of Embodiments 28-29, wherein the anti-MMP- 9 antibody or the antigen binding fragment thereof comprises at least one of the following: (a) a heavy chain variable region set forth in SEQ ID NO:9, and a light chain variable region set forth in SEQ ID NO:10; (b) a heavy chain variable region selected from the group consisting of SEQ ID NOs:21, 23, 25, 27 and 29, and a light chain variable region selected from the group consisting of SEQ ID NOs:22, 24, 26, 28 and 30; (c) a heavy chain variable region set forth in SEQ ID NO:21 and a light chain variable region set forth in SEQ ID NO:22; (d) a heavy chain variable region set forth in SEQ ID NO:27 and a light chain variable region set forth in SEQ ID NO:30; (e) a heavy chain variable region set forth in SEQ ID NO:41 and a light chain variable region set forth in SEQ ID NO:42; (f) a light chain variable region set forth in SEQ ID NO:48. [000350] Embodiment 31: The kit of any one of Embodiments 22-30, wherein the instruction 75 53121668.1Attorney Docket No.046483-7440WO1(03745) further instructions for surgically removing an ossified tissue from the subject. [000351] Embodiment 32: The kit of Embodiment 31, wherein the instruction further comprises instructions to perform the surgically removing after the level or the activity of MMP-9 in the subject is down-regulated. [000352] Embodiment 33: The kit of any one of Embodiments 22-32, wherein the subject is a human. [000353] The foregoing outlines features of several embodiments so that those skilled in the art may better understand the aspects of the present disclosure. Those skilled in the art should appreciate that they may readily use the present disclosure as a basis for designing or modifying other processes and structures for carrying out the same purposes and / or achieving the same advantages of the embodiments introduced herein. Those skilled in the art should also realize that such equivalent constructions do not depart from the spirit and scope of the present disclosure, and that they may make various changes, substitutions, and alterations herein without departing from the spirit and scope of the present disclosure. 76 53121668.1

Claims

Attorney Docket No.046483-7440WO1(03745) CLAIMS WHAT IS CLAIMED IS:

1. A method of treating, ameliorating, and / or preventing fibrodysplasia ossificans progressiva (FOP) in a subject in need thereof, the method comprising administering an effective amount of an anti-MMP-9 antibody or an antigen binding fragment thereof to the subject, thereby down-regulating matrix metalloproteinase 9 (MMP-9) level and / or activity in the subject.

2. The method of claim 1, wherein the subject has a mutant ACVR1 gene. The method of claim 2, wherein the mutant ACVR1 gene encodes a constitutively active ACVR1 polypeptide.

4. The method of claim 3, 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.

5. The method of any one of claims 1-4, wherein the anti-MMP-9 antibody or the antigen binding fragment thereof comprises at least one of the following: (a) a heavy chain complementarity-determining region 1 (CDR1) set forth in SEQ ID NO:3, a heavy chain CDR2 set forth in SEQ ID NO:4, a heavy chain CDR4 set forth in SEQ ID NO:5, a light chain CDR1 set forth in SEQ ID NO:6, a light chain CDR2 set forth in SEQ ID NO:7, and a light chain CDR3 set forth in SEQ ID NO:8; (b) a heavy chain CDR1 set forth in SEQ ID NO:15, a heavy chain CDR2 set forth in SEQ ID NO:16, a heavy chain CDR4 set forth in SEQ ID NO:17, a light chain CDR1 set forth in SEQ ID NO:18, a light chain CDR2 set forth in SEQ ID NO:19, and a light chain CDR3 set forth in SEQ ID NO:20; (c) a heavy chain CDR1 set forth in SEQ ID NO:35, a heavy chain CDR2 set forth in SEQ ID NO:36, a heavy chain CDR4 set forth in SEQ ID NO:37, a light chain CDR1 set forth in SEQ ID NO:38, a light chain CDR2 set forth in SEQ ID NO:39, and a light chain CDR3 set forth in SEQ ID NO:40; (d) a light chain CDR1 set forth in SEQ ID NO:45, a light chain CDR2 set forth in 77 53121668.1Attorney Docket No.046483-7440WO1(03745) SEQ ID NO:46, and a light chain CDR3 set forth in SEQ ID NO:

47.

6. The method of any one of claims 1-4, wherein the anti-MMP-9 antibody or the antigen binding fragment thereof comprises at least one of the following: (a) a heavy chain variable region set forth in SEQ ID NO:9, and a light chain variable region set forth in SEQ ID NO:10; (b) a heavy chain variable region selected from the group consisting of SEQ ID NOs:21, 23, 25, 27 and 29, and a light chain variable region selected from the group consisting of SEQ ID NOs:22, 24, 26, 28 and 30; (c) a heavy chain variable region set forth in SEQ ID NO:21 and a light chain variable region set forth in SEQ ID NO:22; (d) a heavy chain variable region set forth in SEQ ID NO:27 and a light chain variable region set forth in SEQ ID NO:30; (e) a heavy chain variable region set forth in SEQ ID NO:41 and a light chain variable region set forth in SEQ ID NO:42; (f) a light chain variable region set forth in SEQ ID NO:

48.

7. The method of any one of claims 1-6, further comprising surgically removing an ossified tissue from the subject.

8. The method of claim 7, wherein the surgically removing step is performed after the MMP-9 level or activity in the subject is down-regulated.

9. The method of any one of claims 1-8, wherein the subject is a human.

10. A method of treating, ameliorating and / or preventing a non-FOP heterotopic ossification (HO) in a subject in need thereof, the method comprising administering an effective amount of an anti-MMP-9 antibody or an antigen binding fragment thereof to the subject, thereby down-regulating matrix metalloproteinase 9 (MMP-9) level and / or activity in the subject.

11. The method of claim 10, wherein the anti-MMP-9 antibody or the antigen binding fragment thereof comprises at least one of the following: (a) a heavy chain complementarity-determining region 1 (CDR1) set forth in SEQ ID 78 53121668.1Attorney Docket No.046483-7440WO1(03745) NO:3, a heavy chain CDR2 set forth in SEQ ID NO:4, a heavy chain CDR4 set forth in SEQ ID NO:5, a light chain CDR1 set forth in SEQ ID NO:6, a light chain CDR2 set forth in SEQ ID NO:7, and a light chain CDR3 set forth in SEQ ID NO:8; (b) a heavy chain CDR1 set forth in SEQ ID NO:15, a heavy chain CDR2 set forth in SEQ ID NO:16, a heavy chain CDR4 set forth in SEQ ID NO:17, a light chain CDR1 set forth in SEQ ID NO:18, a light chain CDR2 set forth in SEQ ID NO:19, and a light chain CDR3 set forth in SEQ ID NO:20; (c) a heavy chain CDR1 set forth in SEQ ID NO:35, a heavy chain CDR2 set forth in SEQ ID NO:36, a heavy chain CDR4 set forth in SEQ ID NO:37, a light chain CDR1 set forth in SEQ ID NO:38, a light chain CDR2 set forth in SEQ ID NO:39, and a light chain CDR3 set forth in SEQ ID NO:40; (d) a light chain CDR1 set forth in SEQ ID NO:45, a light chain CDR2 set forth in SEQ ID NO:46, and a light chain CDR3 set forth in SEQ ID NO:

47.

12. The method of claim 10, wherein the anti-MMP-9 antibody or the antigen binding fragment thereof comprises at least one of the following: (a) a heavy chain variable region set forth in SEQ ID NO:9, and a light chain variable region set forth in SEQ ID NO:10; (b) a heavy chain variable region selected from the group consisting of SEQ ID NOs:21, 23, 25, 27 and 29, and a light chain variable region selected from the group consisting of SEQ ID NOs:22, 24, 26, 28 and 30; (c) a heavy chain variable region set forth in SEQ ID NO:21 and a light chain variable region set forth in SEQ ID NO:22; (d) a heavy chain variable region set forth in SEQ ID NO:27 and a light chain variable region set forth in SEQ ID NO:30; (e) a heavy chain variable region set forth in SEQ ID NO:41 and a light chain variable region set forth in SEQ ID NO:42; (f) a light chain variable region set forth in SEQ ID NO:

48.

13. The method of any one of claims 10-12, wherein the non-FOP HO is caused by a trauma or injury in the bone, a severe burn, a stroke, a head trauma, paralysis, an orthopaedic procedure, a hip replacement, a severe crush injury, an explosive wound, a war wound, a severe civilian trauma, polio, tetanus, a syringomyelia, a myelodysplastic syndrome, multiple sclerosis (MS), carbon monoxide poisoning, or a tumor in the spine. 79 53121668.1Attorney Docket No.046483-7440WO1(03745) 14. The method of any one of claims 10-13, wherein the subject is a human.

15. A kit for treating, ameliorating and / or preventing fibrodysplasia ossificans progressiva (FOP) or a non-FOP heterotopic ossification (HO) in a subject in need thereof, comprising an antibody against MMP-9 or an antigen binding fragment thereof; and an instruction for administering an effective amount of the antibody or antigen binding fragment thereof to the subject.

16. The kit of claim 15, wherein the kit is for treating, ameliorating and / or preventing FOP, and wherein the subject has a mutant ACVR1 gene.

17. The kit of claim 16, wherein the mutant ACVR1 gene encodes a constitutively active ACVR1 polypeptide.

18. The kit of claim 17, 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.

19. The kit of any one of claims 15-18, wherein the kit is for treating, ameliorating and / or preventing non-FOP HO, and wherein the HO is caused by a trauma or injury in the bone, a severe burn, a stroke, a head trauma, paralysis, an orthopaedic procedure, a hip replacement, a severe crush injury, an explosive wound, a war wound, a severe civilian trauma, polio, tetanus, a syringomyelia, a myelodysplastic syndrome, multiple sclerosis (MS), carbon monoxide poisoning, or a tumor in the spine.

20. The kit of any one of claims 15-19, wherein the anti-MMP-9 antibody or the antigen binding fragment thereof comprises at least one of the following: (a) a heavy chain complementarity-determining region 1 (CDR1) set forth in SEQ ID NO:3, a heavy chain CDR2 set forth in SEQ ID NO:4, a heavy chain CDR4 set forth in SEQ ID NO:5, a light chain CDR1 set forth in SEQ ID NO:6, a light chain CDR2 set forth in SEQ ID NO:7, and a light chain CDR3 set forth in SEQ ID NO:8; (b) a heavy chain CDR1 set forth in SEQ ID NO:15, a heavy chain CDR2 set forth in SEQ ID NO:16, a heavy chain CDR4 set forth in SEQ ID NO:17, a light chain CDR1 set 80 53121668.1Attorney Docket No.046483-7440WO1(03745) forth in SEQ ID NO:18, a light chain CDR2 set forth in SEQ ID NO:19, and a light chain CDR3 set forth in SEQ ID NO:20; (c) a heavy chain CDR1 set forth in SEQ ID NO:35, a heavy chain CDR2 set forth in SEQ ID NO:36, a heavy chain CDR4 set forth in SEQ ID NO:37, a light chain CDR1 set forth in SEQ ID NO:38, a light chain CDR2 set forth in SEQ ID NO:39, and a light chain CDR3 set forth in SEQ ID NO:40; (d) a light chain CDR1 set forth in SEQ ID NO:45, a light chain CDR2 set forth in SEQ ID NO:46, and a light chain CDR3 set forth in SEQ ID NO:

47.

21. The kit of any one of claims 15-19, wherein the anti-MMP-9 antibody or the antigen binding fragment thereof comprises at least one of the following: (a) a heavy chain variable region set forth in SEQ ID NO:9, and a light chain variable region set forth in SEQ ID NO:10; (b) a heavy chain variable region selected from the group consisting of SEQ ID NOs:21, 23, 25, 27 and 29, and a light chain variable region selected from the group consisting of SEQ ID NOs:22, 24, 26, 28 and 30; (c) a heavy chain variable region set forth in SEQ ID NO:21 and a light chain variable region set forth in SEQ ID NO:22; (d) a heavy chain variable region set forth in SEQ ID NO:27 and a light chain variable region set forth in SEQ ID NO:30; (e) a heavy chain variable region set forth in SEQ ID NO:41 and a light chain variable region set forth in SEQ ID NO:42; (f) a light chain variable region set forth in SEQ ID NO:

48.

22. The kit of any one of claims 15-21, wherein the instructions are further for surgically removing an ossified tissue from the subject.

23. The kit of claim 22, wherein the instructions further comprise instructions to perform the surgically removing after the level or the activity of MMP-9 in the subject is down- regulated.

24. The kit of any one of claims 15-23, wherein the subject is a human. 81 53121668.1

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