WO2015069877A1

WO2015069877A1 - Method for treating osteogenesis imperfecta type v

Info

Publication number: WO2015069877A1
Application number: PCT/US2014/064323
Authority: WO
Inventors: Pierre Moffatt
Original assignee: Shriners Hospitals for Children
Current assignee: Shriners Hospitals for Children
Priority date: 2013-11-06
Filing date: 2014-11-06
Publication date: 2015-05-14
Anticipated expiration: 2016-05-06

Abstract

The present application relates to methods of inhibiting the activity or expression of BRIL, and to methods of treating diseases related to expression of a mutant BRIL. In certain embodiments, the disease is osteogenesis imperfecta (OI) type V. The present application is also directed to methods of identifying compounds that inhibit the activity or expression of BRIL.

Description

METHOD FOR TREATING OSTEOGENESIS IMPERFECTA TYPE V

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional Application Serial No. 61/900,872, filed November 6, 2013, which is incorporated by reference in its entirety.

1. INTRODUCTION

The present application relates to methods of inhibiting the activity and/or expression of bone-restricted IFITM-like protein (BRIL), and to methods of treating diseases related to mutant BRIL, including, but not limited to, osteogenesis imperfecta type V. The application also relates to in vitro and in vivo methods of identifying compounds that inhibit the activity and/or expression of BRIL.

2. BACKGROUND The gene encoding for BRIL (bone-restricted IFIT -like) is expressed in bone forming cells in the skeleton (osteoblasts), and in cells that form the dentin of teeth (odontoblasts). While the absence of BRIL in the skeleton has no impact on bone quality and function, a mutation in the human BRIL gene has been identified as the cause of osteogenesis imperfecta (OI) type V. There is no current cure for OI, although there are partially effective treatments such as the administration of bisphosphonates.

Most cases of autosomal dominant OI are caused by mutations in the genes encoding al or a2 chains of type I collagen (COL1A1 and COLl A2).

However, one class of OI patients without any pathogenic mutation in either COLl A 1 or COLl A2 exhibited characteristics such as radioulnar interosseous membrane ossification (RUIMO), radial head dislocation (RHD), hyperplastic callus formation (HC), metaphyseal radiodense band (MRB), autosomal dominant inheritance, and mesh-like lamellar pattern on histopathologic examination. (Glorieux et al, J Bone Miner Res. Sep 2000; 15(9): 1650-1658). Using whole exome sequencing and/or linkage analysis of these patients, a recurrent heterozygous point mutation of - 14C>T at the 5' untranslated region (UTR) of the gene encoding IFITM5 (interferon-induced transmembrane protein 5), also named as BRIL, was found to be a common mutation in 19 Korean patients, 2 German patients, and in 42 patients of diverse geographic origins including European, Mediterranean, Arabic, Asian, and North, Central and South Americans. These patients are classified as having Ol type V. It is unique that all the 01 type V patients share the same mutation in IF1TM5 without any exception so far. This mutation introduces a new start codon upstream of the coding region in frame with it, and the mutant protein is predicted to have five additional amino acids (Met-Ala-Leu-Glu-Pro) at the N-terminus. An additional mutation in the coding region of BRIL has also been identified in which a serine at amino acid position 40 is converted to a leucine {see Farber et al., "A Dominant Mutation of IFITM5 in Severe Osteogenesis imperfecta Implicates an Interaction between Bril and PEDF in Bone." Abstracts of the 2012 meeting of the American Society of Bone and Mineral Research, J Bone Miner Res 27 (Suppl 1 )). 3. SUMMARY

The present application relates to compositions for the treatment of osteogenesis imperfecta (Ol) type V comprising a compound that can inhibit or reduce the activity of a BRIL (bone-restricted IFITM-like) protein, inhibit or reduce membrane localization of a BRIL protein, and/or inhibit or reduce transcription, translation or expression of a nucleic acid encoding a BRIL protein.

The present application also relates to methods for the treatment of Ol type V, by administering to a subject in need of such treatment a composition that can inhibit or reduce the activity or expression of a BRIL protein. In certain

embodiments, the composition is administered to a subject in an amount effective to inhibit or reduce membrane localization of a BRIL protein. In certain embodiments, the composition is administered to a subject in an amount effective to inhibit or reduce the transcription and/or translation of a nucleic acid encoding a BRIL protein.

The present application also provides a method of inhibiting the activity of BRIL, by contacting the BRIL with a compound of the present application in an amount effective to inhibit the activity of BRIL. In certain non-limiting embodiments, the BRIL protein is a mutant BRIL protein. In certain non-limiting embodiments, the mutant BRIL protein comprises a -14C>T mutation at the 5' untranslated region (UTR) of the BRIL gene. In certain non-limiting embodiments, the mutant BRIL protein comprises five additional amino acids at the N-terminus compared to a wild-type BRIL protein. In certain non-limiting embodiments, the five additional amino acids are Met-Ala-Leu- Glu-Pro.

In certain embodiments, the compositions of the present application comprise an antibody that specifically binds to the N-terminus of BRIL. In certain embodiments, the antibody specifically binds to an amino acid sequence comprising the amino acids DTSYPREDPRAPSS (SEQ ID NO:5). In certain embodiments, the antibody is an antibody described by Moffatt et ah, J Bone Miner Res. Sep

2008;23(9): 1497-1508.

In certain embodiments, the compositions of the present application comprise an antibody that specifically binds to the C-terminus of BRIL. In certain embodiments, the antibody specifically binds to an amino acid sequence comprising the amino acids SKLAKDSAAFFSTKFD (SEQ ID NO:6).

In certain embodiments, the compositions of the present application comprise an antibody that specifically binds to a mutant BRIL protein. In certain embodiments, the antibody specifically binds to an amino acid sequence comprising the amino acid sequence MALEP (SEQ ID NO:7). In certain embodiments, the antibody specifically binds to an amino acid sequence comprising the amino acid sequence MALEPMDT (SEQ ID NO:8). In certain embodiments, the antibody is raised against an amino acid sequence comprising a wild-type BRIL portion and a non-wild type BRIL portion, such as a mutant BRIL portion. In certain embodiments, the antibody is raised against the amino acid sequence MALEPMDT (SEQ ID NO: 8).

In certain embodiments, the compositions of the present application comprise a compound that inhibits palmitoylation of a cysteine amino acid of a BRIL protein. In certain embodiments, the cysteine amino acid is located at position 52, position 53 and/or position 86 relative to a wild-type BRIL amino acid sequence. In certain embodiments, the compound is a palmitoyl transferase enzyme inhibitor. In certain embodiments, the compound is an acyltransferase inhibitor. In certain embodiments, the acyltransferase is a DHHC acyltransferase. In certain

embodiments, the compound is 2-bromopalmitate.

In certain embodiments, the compositions of the present application comprise a compound that can inhibit or reduce BRIL transcription and/or translation. In certain embodiments, the compound comprises an antisense molecule, RNAi molecule, shRNA molecule or siRNA molecule, in certain embodiments, the antisense, RNAi, shRNA molecule or siRNA molecule is complementary to a segment or region of a BRIL mRNA transcript. In certain embodiments, the antisense, RNAi, shRNA molecule or siRNA molecule hybridizes to and inhibits or reduces translation of BRIL mRNA. In certain embodiments, the composition comprises an shRNA molecule described by Moffatt et ah, J Bone Miner Res. Sep 2008;23(9): 1497- 1508.

In certain embodiments, the compound that inhibits or reduces BRIL transcription or translation comprises an inhibitor or antagonist of the Hedgehog signaling pathway. In certain embodiments, the agent inhibits or reduces the activity of Spl , Sp3 and/or GLI2 transcription factors.

The present application further provides a recombinant host cell that expresses a BRIL protein, for example, a mutant BRIL protein, expressed from an expression vector introduced into the host cell. Also provided is a method of screening for compounds that inhibit or reduce the activity of a BRIL protein, membrane localization of a BRIL protein, transcription of a BRIL nucleic acid, and/or translation of BRIL nucleic acid. Such methods comprise, for example, contacting a candidate compound to a host cell expressing the BRIL protein, and determining a reduction in BRIL protein activity, membrane localization, transcription and/or translation. In certain embodiments, a candidate compound that reduces BRIL protein activity, membrane localization of a BRIL protein, transcription of a BRIL nucleic acid and/or translation of BRIL nucleic acid can be used in the methods and compositions of the present application. The present application further provides a transgenic mouse that expresses a BRIL protein, for example, a mutant BRIL protein, expressed from an expression vector introduced into the mouse. Also provided is a method of screening for compounds that inhibit or reduce the activity of a BRIL protein, membrane localization of a BRIL protein, transcription of a BRIL nucleic acid, and/or translation of BRIL nucleic acid in cells of the transgenic mouse. Such methods comprise, for example, contacting a candidate compound to a cell expressing the BRIL protein, and determining a reduction in BRIL protein activity, membrane localization of a BRIL protein, transcription of a BRIL nucleic acid, and/or translation of BRIL nucleic acid. In certain embodiments, the methods are performed in vivo. In certain embodiments, the methods are performed in vitro. In certain embodiments, a candidate compound that reduces BRIL protein activity, membrane localization of a BRIL protein, transcription of a BRIL nucleic acid and/or translation of BRIL nucleic acid can be used in the methods and compositions of the present application.

In certain embodiments, the present disclosure provides for a kit for use in the treatment of 01 type V, or for use in the inhibition or reduction of BRIL activity, membrane localization of BRIL, transcription of BRIL nucleic acid and/or translation of BRIL nucleic acid, wherein the kit comprises at least one compound according to the present application that inhibits or reduces the activity of BRIL protein, membrane localization of a BRIL protein, transcription of a BRIL nucleic acid and/or translation of BRIL nucleic acid. 4. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the genomic organization of the Ifitm gene cluster on mouse chromosome 7, and a comparison of the amino acid sequences of the corresponding proteins.

Figure 2 shows that knockdown of BRIL in C3T3 cells inhibits mineralization in vitro as described by Moffatt et al., J Bone Miner Res. Sep

2008;23(9): 1497- 1508.

Figure 3 shows that BRIL-knockout mice do not display gross abnormalities in long bones at 9-weeks of age.

Figure 4 shows the 5' -14C>T mutation in human BRIL that causes osteogenesis imperfecta type V. Figure 5 shows characteristic of osteogenesis imperfecta type V including hyperplasic callus, calcification of interosseous forearm, and mesh-type lamellation pattern.

Figure 6A-C shows fluorography analysis (A), western blot analysis (B) and coomassie blue analysis (C) of MC3T3 cells expressing a wild type mouse BRIL protein that was incubated with (³H)-palmitic acid.

Figure 7 shows antibody staining of WT mouse BRIL expressed in MC3T3 cells that were or were not permeabilized with triton X-100.

Figure 8 shows antibody staining of OI type V BRIL mutant (MALEP-mBRIL) expressed in MC3T3 cells that were or were not permeabilized with triton X-100.

Figure 9 shows antibody staining of wild type (WT) mouse BRIL (mBRIL) expressed in C3T3 cells that were permeabilized with triton X-100 or digitonin. Figure 10 shows antibody staining of wild type (WT) mouse BRIL

(mBRIL), or mBRIL fused to osteocrin signal peptide (OSTN) expressed in MC3T3 cells.

Figure 11 shows an in silico prediction of palmitoylation sites on BRIL polypeptide at cysteines 52, 53, and 86. Figure 12 shows amino acid sequence of a wild type (WT) mouse

BRIL (mBRIL) that have amino acid changes at cysteine 52, 53, 52+53, or 86, for a glycine residue.

Figure 13 shows antibody staining of wild type (WT) mouse BRIL (mBRIL), or mBRIL with changes at cysteine 52, 53, 52+53, or 86, for a glycine residue in MC3T3 cells.

Figure 14 shows antibody staining of wild type (WT) mouse BRIL (mBRIL) in MC3T3 cells after treatment with 2-bromopalmitate and permeabilized with digitonin. Figure 15 shows antibody staining of mutant (-14C>T) human BRIL protein (MALEP-BRIL) in HEK293 cells after treatment with 2-bromopalmitate and permeabilized with digitonin.

Figure 16 shows the Hedgehog signaling pathway in osteogenesis. Figure 17 shows that mutation of 2 nucleotides within the core GLI binding element of BRIL abolishes activation by GLI2.

Figure 18 shows that an agonist of Smoothened (purmorphamine) and recombinant Indian Hedgehog stimulate BRIL expression in MC3T3 osteoblasts.

Figure 19 shows a schematic representation of mouse wild type BRIL and regions recognized by the antibodies described by Example 3.

Figure 20A-B shows anti-N-terminus and anti-C-terminus antibody staining of wild type human BRIL expressed in MC3T3 cells that were or were not permeabilized with digitonin.

Figure 21 shows anti-N-terminus and anti-C-terminus antibody staining of mutant human MALEP-BRIL expressed in MC3T3 cells that were or were not permeabilized with digitonin.

Figure 22 shows anti-C-terminus antibody staining of wild type mouse BRIL or L101>R-mouse BRIL expressed in MC3T3 cells that were or were not permeabilized with digitonin. Figure 23 shows anti-MALEP antibody staining of mutant human

MALEP-BRIL expressed in MC3T3 cells that were or were not permeabilized with digitonin.

Figure 24 shows Western blot analysis of protein from MC3T3 cells that were transiently transfected with the plasmid encoding either the human wild type (WT) or the mutant MALEP-BRIL using the anti-MALEP antibody.

Figure 25 shows anti-N-terminus and anti-C-terminus antibody staining of wild type and mutant S40L-BR1L expressed in MC3T3 cells that were or were not permeabilized with digitonin. Figure 26 shows the PCR product results of the PCR-based strategy used to identify mice carrying the transgenic constructs described by Example 6. Positive founders are indicated by arrows, and exhibit a 415 bp PCR product.

Figure 27 shows wild type and MALEP-BR1L expressed by MC3T3 cells that were metabolically labeled with ³H-palmitate.

Figure 28 shows transgene BRIL mRNA expression in calvaria of transgenic and wild type mice by real-time qPCR.

Figure 29 shows BRIL production in transgenic calvaria by SDS- PAGE and western blotting. Figure 30A-C shows (A) the trypsin cleavage sites on the Wt BRIL protein, (B) detection of WT BRIL protein expressed by MC3T3 and UMR106 cells using an anti-N terminus BRIL antibody after treatment of the cells with and without trypsin, and (C) detection of WT protein expressed by MC3T3 and UMR106 cells using an anti-C terminus BRIL antibody after treatment of the cells with and without trypsin.

Figure 31A-B shows (A) detection of WT BRIL and mutant MALEP- BRIL protein expressed by MC3T3 cells using an anti-N terminus BRIL antibody after treatment of the cells with and without trypsin, and (B) the topology of MALEP- BRIL at the cell surface. Figure 32A-C shows (A) the sequence of the WT BRIL protein and the KDEL-mutant BRIL, wherein the last four amino acids of the WT protein were mutated from EDYN to KDEL. (B) shows detection of WT BRIL and KDEL-mutant BRIL protein expressed by MC3T3 cells using an anti-C terminus BRIL antibody and an anti-N terminus BRIL antibody after the cells were or were not permeabilized with digitonin. (C) shows Western blot of WT BRIL and KDEL-mutant BRIL expressed by MC3T3 cells that were or were no treated with trypsin.

Figure 33A-E shows (A) a schematic representation of the mouse wild type and mutant BRIL constructs used to generate the transgenic mice described by Example 6. (B) a quantitative PCR-based copy-number-assay to estimate the numbers of integrated transgene copies in the transgenic founder mice. (C) Northern blot analysis of Bril expression in 6-week-old WT, Tg Wt-Bril and Tg MALEP-BRIL mice. (D) Western blot analysis of BRIL protein expressed by 6-week old mice from mouse line 335 (Tg WT-BriT) and mouse line 694 (Tg MALEP-Bril). Anti-N terminus BRIL antibody was used for the blots. (E) Femurs of 6-week old transgenic (Tg) mice expressing MALEP-BRIL, and wild type littermates. Graphs display bone volume, tissue volume, and bone volume/tissue volume (%) comparison of the Tg MALEP-BRIL and wild type mice.

Figure 34A-C shows (A) the strategy to create the MALEP-Bril KI (knockin) mouse. (B) Southern blot analysis of Rl ES cells expressing the knockin I allele. (C) PCR amplification and sequencing of ES clone I-C3 confirming that the KI substitution was correctly introduced in one allele.

Figure 35 shows detection of BRIL in MC3T3 cells expressing wild type mouse BRIL that were labeled with anti-N terminus BRIL antibody and anti- COLl A1 antibody (an endoplasmic reticulum marker). Cells were treated with brefeldin A (BFA) or DMSO for 6h prior to immunostaining.

5- DETAILED DESCRIPTION

The present application is based, at least in part, on the discovery that both wild type BRIL and mutant MALEP-BRIL mostly localize inside a ceil, and further, comprise a single transmembrane domain, wherein the N-terminus of the proteins are intracellular, and the C-terminus of the proteins are extracellular

(luminal). Furthermore, the application is based, at least in part, on studies that utilize antibodies that specifically bind to the N-terminus or C-terminus of wild type BRIL, as well as the identification of an antibody that specifically binds to the mutant MALEP-BRIL protein. The present application is also based, at least in part, on the identification that palmitoylation of cysteine residues at positions 52 and 53, relative to a wild-type BRIL amino acid sequence, are necessary for plasma membrane localization of the BRIL protein. In light of the role mutant MALEP-BRIL plays in 01 type V, the compounds of the instant application that inhibit or reduce BRIL protein activity, translation and/or transcription can be used to inhibit or reduce BRIL activity levels and thereby ameliorate pathologies associated with diseases relating to mutant MALEP-BRIL. For clarity and not by way of limitation, this detailed description is divided into the following sub-portions:

(i) BRIL;

(ii) BRIL inhibitors;

(iii) methods of treatment; and

(iv) pharmaceutical compositions.

5.1 BRIL

The terms used in this specification generally have their ordinary meanings in the art, within the context of this application and in the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the compositions and methods of the application and how to make and use them.

As used herein the term "wild-type gene" refers to a nucleic acid sequences which encodes a protein capable of having normal biological functional activity in vivo. The wild-type nucleic acid sequence may contain nucleotide changes that differ from the known, published sequence, as long as the changes result in amino acid substitutions having little or no effect on the biological activity.

As used herein, the term "wild-type protein" refers to any protein encoded by a wild-type gene that is capable of having functional biological activity when expressed or introduced in vivo. The term "normal wild-type activity" refers to the normal physiological function of a protein in a cell. Such functionality can be tested by any means known to establish functionality of a protein.

The terms "BRIL," "bone-restricted IFITM-like protein," "IFITM5" and "interferon-induced transmembrane protein 5" refer to a polypeptide that is a positive modulator of mineralization. In certain embodiments, BRIL is a human BRIL encoded by a human BRIL gene (OMIM accession number MIM:614757; and/or GenBank accession number NM_001025295). In certain embodiments, BRIL is a human BRIL encoded by a human BRIL gene as described by SEQ ID NO: ] . In certain embodiments, such a human BRIL is a non-mutant wild type human BRIL. Alternatively, BRIL can be encoded by any nucleic acid molecule exhibiting at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or up to 100% homology to the BRIL gene (as determined by standard software, e.g. BLAST or FASTA), and any sequences which hybridize under standard conditions to these sequences. The BRIL may be a recombinant BRIL polypeptide encoded by a recombinant nucleic acid, for example, a recombinant DNA molecule.

In certain embodiments, BRIL refers to a human BRIL polypeptide (GenBank accession number NP 001020466). In certain embodiments, BRIL refers to a polypeptide comprising an amino acid sequence described by SEQ ID NO:2. Alternatively, BRIL can be a polypeptide exhibiting at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or up to 100% homology to the BRIL polypeptide (as determined by standard software, e.g. BLAST or FASTA).

As used herein, the terms "mutant" and "mutation" mean any detectable change in genetic material, e.g., DNA, or any process, mechanism or result of such a change. This includes gene mutations, in which the structure (e.g., DNA sequence) of a gene is altered, any gene or DNA arising from any mutation process, and any expression product (e.g., RNA, protein or enzyme) expressed by a modified gene or DNA sequence.

The terms "BRIL mutation" or "mutant BRIL" refers to a mutation in the gene encoding BRIL. In one non-limiting embodiment, the expression of a BRIL mutation may result in OI type V in a subject when compared to BRIL expression from a wild type BRIL gene that does not have the mutation. In certain non-limiting embodiments, the mutant BRIL is encoded by a nucleic acid sequence comprising a cytidine to thymidine point mutation fourteen nucleotides prior to the ATG start codon of a wild type BRIL (i.e., -14C>T). In certain embodiments, the mutant BRIL is encoded by a nucleic acid sequence described by SEQ ID NO:3.

In certain embodiments, a mutant BRIL refers to a polypeptide comprising an amino acid sequence comprising MALEP (SEQ ID NO:7).

In certain embodiments, a mutant BRIL refers to a polypeptide comprising an amino acid sequence described by SEQ ID NO:4, In certain embodiments, expression vectors or constructs may be utilized to produce BRIL protein products, including fragments or isoforms of BRIL, which can then be purified and, for example, be used to generate antisera or a monoclonal antibody which may be used according to the methods of the present application. Expression requires that appropriate signals be provided in the vectors, and which include various regulatory elements, such as enhancers/promoters from viral and mammalian sources, as appropriate that drive expression of the genes of interest in host cells.

As used herein, the term "expression construct" or "expression vector" is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed. The transcript may be translated into a protein, or protein fragment, but it need not be. In certain embodiments, expression includes both transcription of a gene and translation of mRNA into a gene product. In other embodiments, expression only includes transcription of the nucleic acid encoding a gene of interest.

In certain embodiments, the nucleic acid encoding a gene product is under transcriptional control of a promoter. A "promoter" refers to a DNA sequence recognized by the transcriptional machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrase "under transcriptional control" means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene. Typically, the promoter is selected for high level or controlled expression, such as lac inducible promoter for use in E. coli, alcohol oxidase for yeast, CMV IE for various mammalian systems, or the polyhedron promoter for

Baculovirus. Other elements include polyadenylation signals, origins of replication, internal ribosome entry sites (IRES) and selectable markers (e.g., neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol).

In certain embodiments, the present application provides for a host cell expressing a BRIL protein, for example, a recombinant BRIL protein. The term "host cell" means any cell of any organism that is selected, modified, transformed, grown, or used or manipulated in any way, for the production of a substance by the cell, for example, the expression by the cell of a gene, a DNA or RNA sequence, a protein or an enzyme. In one embodiment, the present application provides for a host cell that is transfected with an expression vector encoding a BRIL protein that can be used in a method of identifying a compound of the present application. In one non-limiting embodiment, the recombinant protein is expressed by a cell line in vitro. In another non-limiting embodiment, the host cell is a mammalian cell. In another non-limiting embodiment, the host cell may be a CHO cell, HeLa cell, HE -293 cell, 293T cell, COS cell, COS-7 cell, mouse primary myoblast, MC3T3 cell, NIH 3T3 cell, human SaOS-2 osteosarcoma cell, or rat UMR! 06 osteosarcoma cell. In certain

embodiments, the host cell constitutively expresses BRIL. In certain embodiments, the present application provides for transgenic animals expressing a BRIL protein, for example, a recombinant BRIL protein. In certain embodiments, the transgenic animal is a transgenic mouse.

In certain embodiments, the transgenic mouse is a transgenic knockout mouse which comprises a disruption in the endogenous BRIL gene, in one non- limiting embodiment, the disruption can be introduced by homologous recombination of the endogenous BRIL gene. In another non-limiting embodiment, the disruption can be made by introducing a gene encoding antisense, RNAi, shRNA molecule or siRNA targeted to a BRIL gene. The disruption of the BRIL gene results in decreased expression of endogenous BRIL protein. In certain embodiments, the transgenic mouse is a transgenic knock-in mouse which comprises a disruption in the endogenous BRIL gene. In one non- limiting embodiment, the disruption can be introduced by homologous recombination of the endogenous BRIL gene. In further non-limiting embodiments, a BRIL gene comprising a mutation in the BRIL gene nucleic acid sequence is homologously recombined with an endogenous BRIL gene. In certain embodiments, one or more nucleotide bases in the BRIL gene is substituted with a nucleotide that is different than the endogenous BRIL gene nucleic acid sequence. In certain embodiments, one nucleotide base in the BRIL gene is substituted at -14C>T. The disruption of the BRIL gene results in decreased expression of endogenous BRIL protein, and/or expression of a BRIL protein with reduced BRIL activity as compared to a wild type BRIL protein. 5.2 BRIL Inhibitors

The present application provides for methods for inhibiting or reducing BRIL activity, membrane localization, transcription and/or translation, for example, but not limited to, in the treatment of 01 type V, According to one embodiment of the present application, inhibiting BRIL activity, membrane localization, transcription and/or translation comprises administering a compound to a subject or patient that inhibits BRIL activity, membrane localization, transcription and/or translation.

In one non-limiting embodiment of the application, the compound is an antibody that specifically binds to and inhibits, blocks or reduces the activity or membrane localization of a BRIL protein. The antibody may be a monoclonal or a polyclonal antibody. Such antibodies may also include but are not limited to chimeric, human, humanized, single chain, Fab fragments, and a Fab expression library. Means for preparing and characterizing antibodies are well known in the art and can be readily prepared through use of well-known techniques, such as those exemplified in U.S. Pat. No. 4,1 6,265, incorporated herein by reference (see also, e.g., Kohler and Milstein, Nature, 1975; 256:495-497; and Kohler and Milstein, Eur. J. Immunol., 1976; 6:51 1-519; Goding, Monoclonal Antibodies: Principles and Practice, 2d ed., Academic Press, Orlando, Fla., pp 60-61, 71-74, 1986; and Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory, pp 139- 281 , 1988). Specifically, techniques developed for the production of chimeric and humanized antibodies have been described by Neuberger, et al., Nature, 1984;

312:604-608; Bruggemann et ah, Proc Natl Acad Sci U S A, 1989; 86(17): 6709- 6713; and Takeda et al., Nature, 1985; 314:452-454. Various techniques have been described for the production of single chain antibodies including those in U.S. Pat, No. 5,476,786; 5,1 2,405; 4,946,778 each of which is incorporated herein by reference, and these techniques can be adapted to produce for example, BRIL protein- specific single chain antibodies. An additional embodiment of the disclosure may utilize the techniques described for the construction of Fab expression libraries of Huse et al., Science, 1989; 246: 1275-1281 , to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for a BRIL protein.

In certain embodiments, the compound of the present application comprises an antibody that specifically binds to the N-terminus of BRIL. In certain embodiments, the antibody specifically binds to an amino acid sequence comprising the amino acids DTS YPREDPRAPSS .

In certain embodiments, the compound of the present application comprises an antibody that specifically binds to the C-terminus of BRJL. In certain embodiments, the antibody specifically binds to an amino acid sequence comprising the amino acids SKLAKDSAAFFSTKFD.

In certain embodiments, the compound of the present application comprises an antibody that specifically binds to a mutant BRIL protein. In certain embodiments, the antibody specifically binds to an amino acid sequence comprising the amino acid sequence MALEP.

In certain embodiments, the compound is an antisense molecule, RNAi molecule, shRNA molecule or siRNA molecule. In certain embodiments, the antisense, RNAi, shRNA molecule or siRNA molecule is complementary to a segment or region of a BRIL mRNA transcript. In certain embodiments, the antisense, RNAi, shRNA molecule or siRNA molecule hybridizes to and inhibits or reduces translation of BRJL mRNA. In certain embodiments, the antisense, RNAi, shRNA molecule or siRNA molecule hybridizes to BRIL mRNA and increases degradation of the BRIL mRNA.

In certain embodiments, the compound that inhibits or reduces BRIL transcription or translation comprises an inhibitor or antagonist of the Hedgehog signaling pathway. In certain embodiments, the agent inhibits or reduces the activity of Spl , Sp3 and/or GLI2 transcription factors. In certain embodiments, such compounds include, for example, cyclopamine, saridegib (IPI-926), itraconazole, LDE-225 (Novartis), TAK-441 (Millennium Pharmaceuticals), BMS-833923

(XL 139) (Exelixis/Bristol-Myers Squibb), PF-04449913 (Pfizer), and/or Vismodegib (IPI-926; Erivedge: Genentech).

In certain embodiments, the composition of the present application comprises a compound that inhibits palmitoylation of a cysteine amino acid of a BRIL protein. In certain embodiments, the cysteine amino acid is located at position 52, position 53 and/or position 86 relative to a wild-type BRIL amino acid sequence. In certain embodiments, the compound is a palmitoyl transferase enzyme inhibitor. In certain embodiments, the compound is an acyltransferase inhibitor. In certain embodiments, the acyltransferase is a DHHC acyltransferase. In certain

embodiments, the compound is 2-bromopalmitate. In certain embodiments, the compound is one or more of the DHHC inhibitors described in Jennings et al., J Lipid Res. 2009 Feb;50(2);233-42; Resh, Methods, 2006 Oct;40(2): 191 -7; Ducker et ah, Mol Cancer Ther. 2006 Jul;5(7): 1647-59; Korycka et al., Eur J cell Biol. 2012 Feb;91(2): 107-17; and Leong et al, PloS One. 2009;4(l):e4135.

In certain embodiments, the composition of the present application comprises a compound that inhibits BRIL from interacting with, for example, binding to, one or more other proteins. In certain embodiments, the one or more other proteins is FK506 binding protein 1 1 (F BP19) which is expressed by the gene FKBP11, tetraspanin protein CD9, and/or tetraspanin protein CD81 .

In other non-limiting embodiments, the composition of the present application is a molecule, compound or drug that, for example, inhibits, blocks or reduces the functionality of a BRIL protein, BRIL membrane localization, BRIL transcription and/or BRIL translation. In certain embodiments, the molecule, compound or drug comprises an inhibitor of a peptidyl-prolyl cis-trans isomerase. In certain embodiments, the molecule, compound or drug comprises an inhibitor of an F BP protein. In certain embodiments, the molecule, compound or drug comprises an inhibitor of FKBP 19 protein. In certain embodiments, the molecule, compound or drug comprises FK506 (i.e., Tacrolimus).

In further embodiments, the application provides kits comprising the compositions of the present application for use in treating a subject diagnosed with, or at risk of developing, OI type V. In certain embodiments, such kits will generally comprise one or more antisense, RNAi, shRNA molecule, siRNA and/or antibody that have specificity for BRIL nucleic acids or BRIL proteins. In certain embodiments, such kits will generally comprise one or more compositions that inhibits or reduces BRIL protein activity, membrane localization, transcription and/or translation.

In certain embodiments, the present application provides for a method of identifying a compound that inhibits BRIL protein activity, membrane localization of BRIL protein, transcription of a BRIL nucleic acid and/or translation of a BRIL nucleic acid. Such methods comprise (i) providing a host cell expressing a BRIL protein; (ii) administering a candidate compound to the host cell; and (iii) measuring BRIL protein activity, membrane localization of BRIL protein, transcription of a BRIL nucleic acid and/or translation of a BRIL nucleic acid, where a decrease in the level of BRIL protein activity, membrane localization of BRIL protein, transcription of a BRIL nucleic acid and/or translation of a BRIL nucleic acid compared to such levels in a host cell expressing a BRIL protein not contacted with the candidate compound indicates that the candidate compound is suitable for use according to the methods of the present application. In other non-limiting embodiments, the method of identifying a compound that inhibits BRIL protein activity, membrane localization of BRIL protein, transcription of a BRIL nucleic acid and/or translation of a BRIL nucleic acid comprises administering a candidate compound to a transgenic anima, for example, but not limited to, a transgenic mouse, expressing a recombinant BRIL protein, and measuring BRIL protein activity, membrane localization of BRIL protein,

transcription of a BRIL nucleic acid and/or translation of a BRIL nucleic acid in a cell of the transgenic animal, where a decrease in the level of BRIL protein activity, membrane localization of BRIL protein, transcription of a BRIL nucleic acid and/or translation of a BRIL nucleic acid compared to such levels in a cell expressing a BRIL protein not contacted with the candidate compound indicates that the candidate compound is suitable for use according to the methods of the present application. In certain embodiments, such methods are performed in vitro. In other embodiments, the methods are conducted in vivo.

5.3 Methods of Treatment In certain non-limiting embodiments, the present application provides for methods for inhibiting or reducing the activity of a BRIL protein, inhibiting or reducing membrane localization of a BRIL protein, inhibiting or reducing

transcription of a nucleic aid encoding a BRIL protein, and/or inhibiting or reducing the translation of a BRIL nucleic acid, wherein the method comprises administering one or more compounds of the present application to a subject in need thereof in an amount effective to inhibit or reduce BRIL protein activity, membrane localization of BRIL protein, transcription of a nucleic aid encoding a BRIL protein, and/or translation of a BRIL nucleic acid. In certain embodiments, the BRIL is a mutant BRIL.

In one non-limiting embodiment, the subject or patient has been diagnosed with, or has been identified as having an increased risk of developing, 01 type V.

According to the application, a "subject" or "patient" is a human or non-human animal. Although the animal subject is preferably a human, the compounds and compositions of the application have application in veterinary medicine as well, e.g. , for the treatment of domesticated species such as canine, feline, and various other pets; farm animal species such as bovine, equine, ovine, caprine, porcine, etc.; wild animals, e.g., in the wild or in a zoological garden; and avian species, such as chickens, turkeys, quail, songbirds, etc.

in other non-limiting embodiments, the present application provides for methods of reducing the risk of damage resulting from diseases related to expression of a mutant BRIL protein to a tissue of a subject comprising administering to the subject, an effective amount of a composition according to the present application.

The present application provides for methods of treating diseases related to expression of a mutant BRIL in a subject in need of such treatment by administration of a therapeutic formulation which comprises a compound of the present application. In particular embodiments, the formulation may be administered to a subject in need of such treatment in an amount effective to inhibit or reduce BRIL protein activity, membrane localization of a BRIL protein, transcription of a BRIL nucleic acid and/or translation of a BRIL nucleic acid. Where the formulation is to be administered to a subject in vivo, the formulation may be administered systemically (e.g. by intravenous injection, oral administration, inhalation, etc.), or may be administered by any other means known in the art. The amount of the formulation to be administered may be determined using methods known in the art, for example, by performing dose response studies in one or more model system, followed by approved clinical testing in humans.

According to the present application, the compositions of the present application can be introduced by intravenous, intra-arteriole, intramuscular, intradermal, transdermal, subcutaneous, oral, intraperitoneal, intraventricular, and intrathecal administration. In yet another embodiment, the compositions can be delivered in a controlled or sustained release system. For example, a composition may be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump may be used (see, e.g., Sefton, 1987, C C Crit. Ref. Biomed. Eng. 14:201 ; Buchwald et al„ 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med.

321 :574). in another embodiment, polymeric materials can be used (see, e.g., Langer and Wise eds., 1974, Medical Applications of Controlled Release, CRC Press: Boca Raton, Fla; Smolen and Ball eds., 1984, Controlled Drug Bioavailability, Drug Product Design and Performance, Wiley, N.Y.; Ranger and Peppas, 1983, J.

Macromol. Sci. Rev, Macromol. Chem., 23:61 ; Levy et al., 1985, Science 228: 190; During et al., 1989, Ann. Neurol., 25:351 ; Howard et al., 9189, J.Neurosurg. 71 : 105). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, e.g., a long bone, thus requiring only a fraction of the systemic dose (see, e.g. , Goodson, 1984, in Medical Applications of Controlled Release, supra, Vol. 2, pp. 1 15-138). Other controlled release systems known in the art may also be used.

In certain non-limiting embodiments, the application provides a method for inhibiting or reducing the activity of a BRIL protein which comprises contacting the BRIL protein with a compound of the present application in an amount effective to inhibit or reduce BRIL activity.

According to the application, an effective amount is an amount of a compound of the present application which inhibits or reduces BRIL protein activity, membrane localization of BRIL protein, transcription of a BRIL nucleic acid and/or translation of a BRIL nucleic acid, or which reduces the clinical symptoms of diseases related to expression of a mutant BRIL, lor example, OI type V. For example, an effective amount is an amount of a compound of the present application that reduces radioulnar interosseous membrane ossification (RUIMO), radial head dislocation (RHD), hyperplastic callus formation (HC), metaphyseal radiodense band (MRB), and/or mesh-like lamellar pattern on histopathologic examination.

In a further non-limiting embodiment, the effective amount of a compound of the present application may be determined via an in vitro assay. By way of example, and not of limitation, such an assay may utilize a host cell expressing a BRIL protein, for example, a mutant BRIL protein, wherein the compound is contacted to the cell, and the amount of compound required to inhibit or reduce BRIL protein activity, inhibit or reduce membrane localization of BRIL protein, inhibit or reduce translation of a BRIL nucleic acid, and/or inhibit or reduce transcription of a BRIL nucleic acid is determined. In certain embodiments, the amount of compound required to achieve such an effect is a therapeutically effective amount.

In certain non-limiting embodiments, an effective amount of a compound of the present application can be correlated with the compound's ability to inhibit or reduce BRIL activity, membrane localization, transcription and/or translation by at least about 5-10%, or from at least about 10-20%, or from at least about 20-30%, or from at least about 30-40%, or from at least about 40-50%, or from at least about 50-60%, or from at least about 60-70%, or from at least about 70-80%, or from at !east about 80-90%, or from at least about 90-100%, when the compound is administered in an in vitro assay, wherein a greater level of BRIL inhibition at a lower concentration in the in vitro assay is correlative with the compound's therapeutic efficacy,

5.4 Pharmaceutical Compositions

The compounds and compositions of the application may be formulated as pharmaceutical compositions by admixture with a pharmaceutically acceptable carrier or excipient.

In one non-limiting embodiment, the pharmaceutical composition may comprise an effective amount of a compound of the application and a physiologically acceptable diluent or carrier. The pharmaceutical composition may further comprise a second drug, for example, but not by way of limitation, a bisphosphonate.

The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable when administered to a subject. Preferably, but not by way of limitation, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, or, for solid dosage forms, may be standard tabletting excipients. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable phannaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin, 18th Edition, or other editions.

In certain embodiments, the present application provides for a kit for use in the treatment of 01 type V, or for use in the inhibition or reduction of BRIL activity, membrane localization of BRIL, transcription of BRIL nucleic acid and/or translation of BRIL nucleic acid, wherein the kit comprises at least one compound according to the present application that inhibits or reduces the activity of BRIL protein, membrane localization of a BRIL protein, transcription of a BRIL nucleic acid and/or translation of BRIL nucleic acid.

6. Examples

EXAMPLE 1 : Blocking BRIL palmytoilation inhibits membrane localization

Experiment 1 : Metabolic labeling of MC3T3 osteoblastic cells with 3H-palmitate shows that BRIL is palmytoilated

• MC3T3 osteoblastic cells were transfected with a control plasmid or a plasmid encoding wild type mouse BRIL

• 24h later, cells were incubated for 3h with charcoal-stripped fetal bovine serum containing 0.25mCi/ml of tritiated (³H)-palmitic acid

· Cell extract were separated on 15% SDS-PAGE for 3 different analyses: o fluorography (to detect radiolabed proteins (BRIL))

o western blotting to detect total BRIL

o coomassie blue (to visualize all proteins)

As shown in Figure 6, cells expressing BRIL that were incubated with (³H)-palmitic acid exhibited an increase in radiolabeled BRIL protein.

Experiment 2: Inhibiting BRIL palmytoilation at cysteine 52 or 53 inhibits BRIL membrane localization

• MC3T3 osteoblast were transfected with plasmids encoding wild type (WT) mouse BRIL (mBRIL), or with p!asmids encoding mBRIL mutants that have amino acid change at cysteine 52, 53, 52+53, or 86, for a glycine residue (see Figure 12). • 24h later cells were washed with PBS, fixed with 3% paraformaldehyde, permeabilized with digitonin, and processed for immunofluorescence detection of BRIL with the anti-N terminus BRIL antibody (RED) described by Example 3. Nuclei were stained with DAPI (BLUE). Other wells were processed in parallel for western blotting.

As shown in Figure 13, mutation of cysteines C52 or C53, abrogates membrane localization of mBRIL in MC3T3 cells. Cellular levels of mutant mBRIL with C52, C53, and C52+C53 are reduced. Mutation of C86 does not significantly reduce the plasma membrane localization of mBRIL but slightly reduces its production. Thus, C52 or C53 are required for addition of a palmitoyl moiety to BRIL and for the membrane localization and production of BRIL.

Experiment : Treatment of MC3T3 osteoblast with 2-bromopalmitate (2-BP) a palmitate transferase inhibitor, inhibits BRIL production

• MC3T3 osteoblast were transfected with plasmids encoding wild type (WT) mouse BRIL (mBRIL)

• 24h later cells were treated or not with 1 ΟΟμΜ 2-bromopalmitate (2-BP)

• 24h later cells were washed with PBS, fixed with 3% paraformaldehyde, permeabilized with digitonin, and processed for immunofluorescence detection of BRIL with the anti-N terminus BRIL antibody (RED) described by Example 3. Nuclei were stained with DAPI (BLUE). Other wells were processed in parallel for western blotting

As shown in Figure 14, treatment of MC3T3 osteoblast with 2-BP dramatically decreased the membrane localization of BRIL, and the cellular levels of BRIL. A new form of BRIL appeared at around 25kDa in cells treated with 2-BP. 2- BP treatment can prevent production and membrane localization of BRIL in osteoblasts. The size of BRIL after 2-BP treatment fits with the addition of a ubiquitin molecule (around 7kDa). This ubiquitination of BRIL would likely send it to degradation through the proteasome machinery.

Experiment 4: 2-BP treatment inhibits plasma membrane localization and production of mutant MALEP-human BRIL in stably transfected HE 193 cells • HEK293 stably expressing the mutant (-14C>T) human BRIL protein

(MALEP-BRIL)

• Cells were seeded, grown for 24h, and treated for an additional 24h with

ΙΟΟμΜ 2-BP

· Cells were washed with PBS, fixed with 3% paraformaldehyde, permeabilized with digitonin, and processed for immunofluorescence staining with the anti-N terminus BRIL antibody (red) described by Example 3. Nuclei were stained with DAPI (BLUE). Another set of wells were processed for Western blotting.

As shown by Figure 15, membrane localization of MALEP-hBRIL is abrogated by 2-BP in stably expressing HEK293 cells. Production of cellular

MALEP-hBRIL is also reduced. Thus, 2-BP treatment reduces cellular content and changes localization of MALEP-hBRIL.

Experiment 5 : Cells expressing the mutant (-14C>T) human BRIL protein (MALEP-BRIL) will be metabolically labeled with an analog of palmitate (Alkl 6) to show that BRIL is palmitoylated at cysteine residue 52 and 53

• HEK293 cells stably expressing the wild type human BRIL (WT-hBRIL) or the mutant (-14C>T) MALEP-hBRIL will be prepared, seeded and grown for 24h

• MC3T3 osteoblasts will be transfected with wild type (WT) mouse BRIL, or with constructs having cysteine to glycine mutation

• Cells will be metabolically labeled with an analog of 2-BP, Alkl 6 (Sigma 08382) (50μΜ) ίθΓ 41ι

• BRIL will be immunoprecipitated and labeled with rhodamine using the

'click-chemistry' as described (Yount JS, Zhang MM, Hang HC (201 1 ) Curr Protoc Chem Biol 3:65-79)

• Labeled BRIL will be image using a Typhoon Phosphorimager

Experiment 6: Cells expressing wild type or the mutant (-14C>T) human BRIL protein (MALEP-BRIL) were metabolically labeled with 3H-palmitic acid to show that BRIL is palmitoylated • MC3T3 osteoblasts were transfected with wild type (WT) BRIL, or with MALEP-BRIL

• Cells were metabolically labeled with H-palmitate

• BRIL was immunoprecipitated and, and labeled BRIL was detected (Western blot and fluorography)

As shown in Figure 27, both wild type and MALEP-BRIL were metabolically labeled with ³H-palmitate.

EXAMPLE 2: BRIL localization in MC3T3 osteoblast cells is mostly located to the plasma membrane (and Golgi membrane), wherein the N-terminus is intracellular to the plasma membrane

Experiment 1

• MC3T3 were transfected with plasmids encoding wild type (WT) mouse BRIL (mBRIL)

• 24h later cells were washed with PBS, fixed with 3% paraformaldehyde, permeabilized or not with triton X-l 00, and processed for

immunofluorescence detection of BRIL with the anti-N terminus BRIL antibody (RED) described by Example 3. Nuclei are stained with DAPI (BLUE)

As shown in Figure 7, WT mBRIL is detected only in a punctate and patchy manner in non permeabilized cells, but is detected throughout the plasma membrane in Triton XI 00 permeabilized cells (also visible in the permeabilized cells is intracellular organelles staining, likely Golgi apparatus). Thus, permeabilization is required to detect the majority of BRIL suggesting the N-terminus is facing the cytoplasmic side.

Experiment 2

• MC3T3 were transfected with plasmids encoding OI type V BRIL mutant (MALEP-mBRIL)

• 24h later cells were washed with PBS, fixed with 3% paraformaldehyde, permeabilized or not with triton X-l 00, and processed for 24h later cells were washed with PBS, fixed with 3% paraformaldehyde, permeabilized or not with triton X-l 00, and processed for

• immunofluorescence detection of BRIL with an anti-N terminus BRIL

antibody (RED) described by Example 3. Nuclei are stained with DAPI (BLUE)

As shown in Figure 8, MALEP-mBRIL is detected oniy in a punctate and patchy manner in non permeabilized cells, as the N-terminus is not readily accessible to the externally applied antibody. However, MALEP-mBRIL is detected throughout the plasma membrane in Triton X100 permeabilized cells (also visible is intracellular organelles staining, likely Golgi apparatus). Permeabilization is required to detect the majority of BRIL suggesting the N-terminus is facing the cytoplasmic side. Additionally, as shown by Figures 7 and 8, the staining of MALEP-mBRIL is indistinguishable from the WT-mBRIL, suggesting that the mutation does not affect its cellular localization. Experiment 3

• 24h later cells were washed with PBS, fixed with 3% paraformaldehyde, permeabilized either with triton X100 or digitonin, and processed for immunofluorescence detection of BRIL with the anti-N terminus BRIL antibody (GREEN) described by Example 3. Nuclei are stained with DAPI (BLUE)

As shown in Figure 9, the same signal for BRIL was detected whether cells were permeabilized with triton X-l 00 or digitonin. Because digitonin only creates small pores in the plasma membrane and does not permeabilize intracellular organelles (Golgi, endoplasmic reticulum), the signal observed for BRIL detected in cells permeabilized with digitonin comes only from the cytoplasm.

BRIL N-terminus is not present in the lumen of ER-Golgi, and does not have its N-terminus facing the extracellular milieu.

Experiment 4 • MC3T3 osteoblast were transfected with plasmids encoding wild type (WT) mouse BRIL (mBRIL), or with a fusion of the osteocrin signal peptide (OSTN) to mBRIL

• 24h later cells were washed with PBS, fixed with 3% paraformaldehyde, and processed for immunofluorescence detection of BRIL with the anti-BRIL antibody (RED). Nuclei are stained with DAPI (BLUE). Other wells were processed for western blotting

As shown in Figure 10, cellular localization of mBRIL in intact cells (non permeabilized) is changed from punctate/patchy signal to the entire plasma membrane by the addition of a cieavable OSTN signal peptide. The molecular weight increases due to the additional OSTN peptide fused to BRIL.

Fusion of an heterologous signal sequence at the N-terminus of BRIL changes its topology from a Type II transmembrane protein with a single

transmembrane domain and an intracellular N-terminus, to a transmembrane protein with two transmembrane domains and an N-terminus and C-terminus that are both extracellular.

Experiment 5

• MC3T3 cells were transfected with plasmids encoding wild type (WT) BRIL, or the mutant (-140T) MALEP-hBRIL. UMR106 cells, that endogenously express BRIL, were also used in the study

• Cells were collected with NP40 lysis buffer (-) or with trypsin (+). Cell

lysates were analyzed by Western blotting with anti-N terminus and anti-C terminus BRIL antibodies.

Figure 3 OA shows the trypsin cleavage sites on the Wt BRIL protein. Treating the cells with trypsin results in cleavage of BRIL at the extracellular cleavage sites. As shown in Figure 30B, trypsin digestion resulted in a shift in size of the digested BRIL protein from the MC3T3 cells expressing WT protein, and the UMR106 cells endogenously expressing WT BRIL, to a lower molecular weight when probed with the N-terminus antibody. The anti-N terminus antibody detected the cleaved BRIL without the C-terminus. The majority of BRIL is located at he plasma membrane, as there is little residual signal of uncleaved protein after treatment with trypsin. As shown in Figure 30C, when probed with the C-terminus antibody, an almost total loss of signal occurred in trypsin treated cells, as the anti-C terminus antibody only detects the non-cleaved BRIL with an intact C-terminus.

As shown in Figure 31 A, trypsin digestion of MC3T3 cells expressing WT BRIL or MALEP-BRIL resulted in a shift in size (but not intensity) of the protein to a lower molecular weight when probed with the N-terminus antibody.

Both WT and MALEP-BRIL exhibit the same topology, wherein the protein is mostly located at the plasma membrane, with the N-terminus located on the intracellular side of the plasma membrane. Additionally, both the WT and MALEP- BRIL proteins were expressed at similar levels in the MC3T3 cells.

Experiment 6

BRIL is targeted to the rER (rough endoplasmic reticulum) en route to the cell membrane

• The last 4 amino acids of mouse BRIL (EDYN) were mutated to create an rER-retention motif, DEL, as shown in Figure 32A

• Plasmids encoding Wt-BRIL and KDEL-mutant BRIL were transfected into MC3T3 cells.

• 24 hours after transfection, cells were fixed with 3% PFA, and processed for immunofluorescence staining. Cells used for immunofluorescence staining were either permeabilized with digitonin, or non-permeabilized. Parallel cultures were processed for collection with or without trypsin digestions. Cellular extracts were analyzed with Western blotting.

Figure 32B shows positive staining of the extracellular BRIL C- terminus of wild-type BRIL in non-permeabilized cells with the anti-C terminus BRIL antibody. However, this staining is abolished in the KDEL mutant. In cells permeabilized with digitonin, probing cells expressing wild-type BRIL with the anti- N terminus resulted in positive staining of the cell membrane and intracellular N- terminus. However, digitonin permeabilized cells expressing the KDEL mutant exhibited staining localized to rER-like intracellular staining. These results demonstrate that the KDEL-mutant BRIL cannot exit the rER and does not reach the plasma membrane, and that the initial passage of BRIL is through the rER

As shown by Figure 32C, digestion of wild-type BRIL with trypsin produced a BRIL with a lower molecular weight due to the cleavage of the

extracellular C-terminus. However, the molecular weight of the KDEL-mutant BRIL expressed from cells that were treated with trypsin was the same as KDEL-mutant BRIL form cells not treated with trypsin. These results demonstrate that the C- terminus of the KDEL-mutant BRIL was not extracellular, and not available for trypsin digestion.

Experiment 7

BRIL is targeted to the endoplasmic reticulum en route to the cell membrane

• MC3T3 cells were transfected with plasmid expressing wild type mouse BRIL

• 18h later, cells were treated for 6h in absence (DMSO used as a control) or presence of brefeldin A (BFA) (18uM) for 6h. (BFA disrupts Golgi structures, resulting in retention of secretory products in the endoplasmic reticulum).

• Cells were fixed, permeabilized with triton XI 00, and immunolabeled with the anti-N anti-BRIL and anti-COLlAl (which labels endoplasmic reticulum) antibodies

Figure 35 shows that BRIL and COL1A1 co-localizes perfectly in BFA treated cells only. Thus, BFA causes a shift in the cellular localization of BRIL from plasma membrane/Golgi to the endoplasmic reticulum.

Experiment 8

The S40L BRIL mutation (wherein a serine at amino acid position 40 is converted to a leucine in the coding region of BRIL, (see Farber et al., Abstracts of the 2012 meeting of the American Society of Bone and Mineral Research, J Bone Miner Res 27 (Suppl 1)) is trapped in the Golgi apparatus, and very little can reach the plasma membrane.

• MC3T3 osteoblast were transfected (1 ug/well) with plasmids encoding wild type ( WT) or S40L-BRIL • 24h later cells were fixed with 3% paraformaldehyde and processed for immunolabeling with the anti-N terminus BRIL antibody or anti-C terminus BRIL antibody (Green) described by Example 3. Nuclei are stained with DAPI (BLUE)

• Cells were either left intact (non permeabilized) or permeabilized with

digitonin

As shown in Figure 25, WT-BRIL is only detected at the cell surface with the anti-C terminus BRIL antibody in non-permeabilized cells. WT-BRIL is also detected at the cell surface and Golgi in digitonin permeabililed cells with the anti-N terminus BRIL antibody.

In contrast S40L-BRIL is not detected at the cell surface with the anti- C terminus BRIL antibody in non-permeabilized cells (an no signal is detectable). S40L-BRIL is mostly detected in the Golgi (arrows) in digitonin permeabilized cells using the anti-N terminus BRIL antibody.

EXAMPLE 3: Immunofluorescence labeling of BRIL with antibodies to BRIL N-terminus and C-terminus shows that the C-terminus of wild type human BRIL and mutant human MALEP-BRIL is extracellular

Antibodies to BRIL

• Current polyclonal anti-N and anti-C antibodies have been raised in rabbits against peptides corresponding to region [2-15] and [1 14-129], respectively. (See Figure 19).

• Peptides used to immunize rabbits had an exogenous cysteine (C) for

coupling, and in some exogenous glycine (G) to act as a spacer:

o Anti-N: D²TSYPREDPRAPSS¹⁵C

o Anti-C: CGS ^{1 i4}KLAKDS AAFFSTKFD ¹²⁹

• The third antibody was also generated in rabbits against the mutant form of BRIL and the peptidic sequence used to immunize the rabbits was:

o Anti-MALEP: MALEPMDTGGC

• All antibodies recognize equally well the mouse and human BRIL. • They were all purified on their respective peptide-affinity chromatography from the crude antiserum.

Immunofluorescence labeling of BRIL with anti-N and anti-C specific antibodies shows that the C-terminus of wild type human BRIL is extracellular

• MC3T3 cells were transiently transfected with plasmids encoding wild type human BRIL

• Immunofiuoresence staining was performed 24h later on non-permeabilized cells, or after permeabihzation with digitonin (digitonin only creates small wholes in the plasma membrane, but not in intracellular organelles)

• Pictures taken at 40X magnification

• BLUE is DAPI staining of the nuclei

As shown in Figure 20A-B the anti-N-terminus antibody did not result in any significant staining in non-permeabilized cells, but did exhibit staining at the membrane and on Golgi upon permeabilization with digitonin. In contrast, the anti-C- terminus antibody exhibited detectable signal when used to stain non-permeabilized cells, and also on cellular plasma membrane in permeabilized cells. Thus, as shown in Figure 20A-B, the C-terminus of BRIL is extracellular, while the N-terminus is cytoplasmic.

Immunofluorescence labeling of BRIL with anti-N and anti-C specific antibodies shows that the C-terminus of mutant human MALEP-BRIL is extracellular

• MC3T3 cells were transiently transfected with plasmids encoding the mutant human MALEP-BRIL

• Pictures taken at 40X magnification

• BLUE is DAPI staining of the nuclei

As shown in Figure 21 , mutant MALEP-BRIL does not shown any appreciable difference in localization compared to wild type BRIL (Figure 20).

Additionally, both wild type BRIL and mutant MALEP-BRIL are recognized by the anti-N-terminus and anti-C-terminus antibodies. EXAMPLE 4: Mutation of a single leucine (L) at position 101 into an arginine (R) (within the putative transmembrane segment) abolishes transmembrane localization of BRIL

• MC3T3 cells were transiently transfected with plasmids encoding either wild type mouse BRIL or L101 >R-mouse BRIL

• Immunofluoresence staining was performed 24h later on non-permeabilized cells, or after permeabilization with digitonin

• Antibody used is the anti-C-terminus antibody, as described in Example 3

• Pictures taken at 40X magnification

• BLUE is DAPI staining of the nuclei

As shown in Figure 22, labeling non-permeabilized cells showed that the C-terminus of wild type mouse BRIL was extracellular, while the C-terminus of the L101>R-mouse BRIL was not detected in the non-permeabilized cells. However, the L101>R-mouse BRIL did localize to the internal surface of plasma membrane and Golgi surface in digitonin permeabilized cells.

EXAMPLE 5: Validation of the antibody raised against the mutant MALEP

• C3T3 cells were transiently transfected with the plasmid encoding the

human mutant MALEP-BRIL

• Antibody used is the anti-MALEP, as described in Example 3

• Pictures taken at 40X magnification

• BLUE is DAPI staining of the nuclei

As shown in Figure 23, the anti-MALEP antibody detects the mutant human BRIL only when cells are permeabilized (as shown in Figure 19, the MALEP amino acid sequence is located at the N-terminus of the protein). These results are consistent with the topological demonstration that the N-terminus is intracellular (even for the mutant protein). This also shows that the mutant protein localizes identically to the wild type.

The antibody raised against the mutant MALEP BRIL is selective for the mutant protein • MC3T3 cells were transiently transfected with the plasmid encoding either the human wild type (WT) or the mutant MALEP-BRIL

• Cellular extract were prepared and analyzed by SDS-PAGE (1 %) and

western blotting with the 3 different antibodies

As shown in Figure 24, the anti-N and anti-C antibodies detect equally well the wild type and mutant BRIL (The slight shift in size for the mutant protein compared to wild type is consistent with the additional 5 residues at it's N-terminus (0.5kDa more)). However, the anti-MALEP antibody detects only the mutant- MALEP BRIL at the correct molecular weight. Additionally, as discussed above in Experiment 5 of Example 2, both WT BRIL and mutant MALEP-BRIL were equally sensitive to trypsin digestion of live cells expressing either of the proteins, demonstrating that both proteins have an extracellular C-terminus, and an intracellular N-terminus that is not accessible to trypsin digestions. (See Figures 30 and 31 ).

EXAMPLE 6: Generation of transgenic mouse models expressing either the mouse wild type BRIL (WT-BRIL) or the mutant MALEP-BRIL

Expression vectors used to create transgenic mice

• In each case, the transgene BRIL cDNA (WT or Mutant) is driven by the

mouse collagen type 1 alpha 1 promoter regulatory sequence that will direct expression only in osteoblast cells (See Figure 33A)

• Other elements in the transgene constructs include the upstream rabbit beta globin intron (to increase mRNA translation), and the downstream simian virus 40 poly adenylation sequence

• The underlined sequence in the Bril cDNA represents the 5' untranslated

region, with the wild type (WT) sequence, or with the -14C>T mutation (same as that reported in patients with osteogenesis imperfecta type V)

• Only part of the coding sequence is shown for simplicity

• The introduction of the -14C>T mutation was accomplished by standard

molecular biology techniques

• The construct DNA was injected into fertilized B6C3F1 eggs and implanted into pseudopregnant female mice Identification of founder mice carrying the transgene DNA by polymerase chain reaction (PCR) on isolated genomic DNA (gDNA)

• At weaning (3-weeks of age), tails were clipped and gDNA was extracted

• A PCR-based strategy was used to identify mice carrying the transgenic

construct using primers internal for the BRIL cDNA

• PCR products were visualized after agarose gel electrophoresis and ethidium bromide staining (See Figure 26)

• Positive PCR product= 415 base pairs (bp) (positive founders are indicated by arrows in Figure 26)

• Endogenous BRIL gene also gives rise to a 1.1 kilobase product with the primers used

Confirmation of transgene integration by Southern blotting and by PCR copy number assay

• gDNA was digested with Mfel and electrophoresed on agarose gel

• DNA was transferred to a nylon membrane and hybridized with a Bril probe (469bp) radiolabed with a³²P-dCTP

• As shown in Figure 33, autoradiography revealed the correct size fragment of variable intensity in the transgenic founder mice

• As shown in Figure 33, a quantitative PCR-based copy-number-assay was performed to estimate the numbers of integrated transgene copies in the transgenic founder mice (wild type used as controls).

Assessment of transgene BRIL mRNA expression in calavaria of transgenic and wild type mice by real-time qPCR

• At 5-weeks of age, total RNA was extracted from calvaria of mice

• RNA was converted to cDNAs by reverse transcription and used for real-time quantitative PCR to estimate expression levels

• All values are normalized to the level of endogenous beta-actin • As shown in Figure 28, transgenic mice wtBRIL#62 and mutBR!L#559 showed a significant increase over the endogenous levels of BRIL (also detected by this method)

• Expression of Bril was also determined by Northern blot analysis. Total RNA extracted from calvaria of 6-week-old WT, Tg Wt-Bril and Tg MALEP-Bril mice was analyzed by Northern blot, as shown in Figure 33C. Expression was very high in mouse 335 (Tg Wt-Bril) and high in mouse 394 (Tg MALEP- Bril).

BRIL protein production in transgenic calvaria by SDS-PAGE and western blotting

At 5-weeks of age, total protein was extracted from calvarial samples

Proteins were separated on SDS-PAGE (15%) and detected by western blotting using the anti-N antibody (see Figure 29)

Ponceau red staining (lower panel) of the nitrocellulose membrane indicates equal amounts of protein loaded on gel

6 different genotype were analyzed (see legend)

In transgenic mice, endogenous protein production is still present to normal levels

Expression of BRIL in mouse line 335 (Tg WT-Bril) and mouse line 694 (Tg MALEP-Bril) was also determined by Western blot analysis. Calvaria protein extracts from 6-week-old mice were separated on 15% SDS-PAGE and blotted with the anti-N terminus BRIL antibody. As shown in Figure 33D, mouse lines 335 and 394 expressed Tg WT-BRIL and Tg MALEP-BRIL, respectively, at high levels.

The cross of the mutant BRIL transgenic mouse to a BRIL-knockout mouse generated a transgenic mouse with half the amount of endogenous BRIL

Generation of a MALEP-Bril knockin mouse A "knockin" mouse model is being developed to mimic OI-V patients.

In this model, one nucleotide base in the Bril gene is being substituted at -14C>T. As shown in Figure 34A, the targeting vector was engineered with a -14C>T substitution in the Bril gene, and 5' and 3' homology arms of 3.9 and 4.1 kb, respectively. The phosphogly cerate kinase 1 promoter (PGK)-Neo resistance cassette was introduced in the 3' intergenic region, and is flanked by LoxP sites for later excision with Cre recombinase. As shown in Figure 34B, the recombinant knockin allele was successfully integrated into the DNA of Rl ES cells, as measured by Southern blot analysis. Figure 34C confirms through nucleic acid sequencing that that the recombinant allele was successfully integrated into the DNA of one of the ES cells (ES clone 1-C3). "Chimeric" mice will next be checked for germline transmission to the progeny.

Sequences

Wild Type Human BRIL DNA (GenBank accession number

NM _001025295.2) (SEQ ID NO: 1 )

1 accagtctga gtgtggaaga gacggcgctg gaacccatgg acacggcgta tccecgcgag

61 gacacccggg cccccacgcc cagcaaggcc ggtgcccaca cagccctcac actgggggcc

121 ccgcaccccc cgcctcgaga ccacttgatc tggtcggtgt tcagcaccct ctacctgaat

181 ctgtgttgcc tcggcttcct ggcgctggcc tactccatca aggcccgaga tcagaaggtg

241 gttggtgacc tggaagcggc ccggcgtttt ggctccaaag ccaagtgcta caacatcctg

301 gccgcgatgt ggacgctggt gccgccactg ctgctcctgg ggctggtggt gactggtgcc

361 ctgcacctgg cccggctggc caaggactct gccgccttct tcagcaccaa gtttgatgac

421 gcggactatg actgacaggc tgggtcctga tctggggcac tagccccagg acactgaccc

481 caggctgctg cccctggggc ccaatactga ctccccggag cctggccctc cttctgtggg

541 gcctccatcc ctgccccatc ctgatcctgg ggccctccag ccccaacatg ggcacctaag

601 gctgaaccag tcagaccccg gggtcttcac cctaacccga gagttcccgg gccctaactc

661 tgcccccgat cctgcccctc cctcctcaca ctccaggccc ctcggttcca cgattaaaag

721 tgcctggttc cagaaaaaaa aaaaaaaaaa a

Wild Type Human BRIL protein (GenBank accession number NPJ)01020466) (SEQ ID NO:2) 1 mdtaypredt raptpskaga htaltlgaph ppprdhliws vfstlylnlc clgflalays

61 ikardqkvvg dleaarrfgs kakcynilaa mwtlvpplll lglvvtga!h larlakdsaa

121 ffstkfddad yd Mutant Human BRIL DNA (MALEP BRIL) (SEQ ID NO:3)

1 accagtctga gtgtggaaga gatggcgctg gaacccatgg acacggcgta tccccgcgag

61 gacacccggg cccccacgcc cagcaaggcc ggtgcccaca cagccctcac actgggggcc 121 ccgcaccccc cgcctcgaga ccacttgatc tggtcggtgt tcagcaccct ctacctgaat

181 ctgtgttgcc tcggcttcct ggcgctggcc tactccatca aggcccgaga tcagaaggtg

241 gttggtgacc tggaagcggc ccggcgtttt ggctccaaag ccaagtgcta caacatcctg

301 gccgcgatgt ggacgctggt gccgccactg ctgctcctgg ggctggtggt gactggtgcc

361 ctgcacctgg cccggctggc caaggactct gccgccttct tcagcaccaa gtttgatgac

421 gcggactatg actgacaggc tgggtcctga tctggggcac tagccccagg acactgaccc

481 caggctgctg cccctggggc ccaatactga ctccccggag cctggccctc cttctgtggg

541 gcctccatcc ctgccccatc ctgatcctgg ggccctccag ccccaacatg ggcacctaag

601 gctgaaccag tcagaccccg gggtcttcac cctaacccga gagttcccgg gccctaactc

661 tgcccccgat cctgcccctc cctcctcaca ctccaggccc ctcggttcca cgattaaaag

721 tgcctggttc cagaaaaaaa aaaaaaaaaa a

Mutant Human BRIL protein (MALEP BRIL) (SEQ ID NO:4) malepmdtaypredt raptpskaga htaltlgaph ppprdhliws vfstlylnlc clgflalays ikardqkvvg dleaarrfgs kakcynilaa mwtlvppll! Iglvvtgalh !arlakdsaa

ffstkfddad yd

The present application is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the application in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

Patents, patent applications, publications, product descriptions, GenBank Accession Numbers, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purpose.

Claims

WHAT IS CLAIMED IS:

1. A method for reducing the activity of a BRIL protein which comprises contacting the BRIL protein, or a nucleic acid encoding a BRIL protein, with a composition comprising a compound selected from the group consisting of an antisense molecule, RNAi molecule, shRNA molecule, siRNA molecule, 2- bromopalmitate and an antibody, in an amount effective to inhibit BRIL activity.

2. The method of claim 1, wherein the BRIL protein is expressed by a cell.

3. The method of claim 2, wherein the cell is a mammalian cell.

4. The method of claim 2, and wherein the composition reduces localization of BRIL protein to a membrane of the cell.

5. The method of claim 1 , wherein the composition comprises an antibody.

6. The method of claim 5, wherein the antibody selectively binds to the N- terminus of BRIL.

7. The method of claim 6, wherein the antibody selectively binds to an amino acid sequence comprising SEQ ID NO:5.

8. The method of claim 5, wherein the antibody selectively binds to the C- terminus of BRIL.

9. The method of claim 8, wherein the antibody selectively binds to an amino acid sequence comprising SEQ ID NO:6.

10. The method of claim 1, wherein the BRIL is a mutant BRIL comprising an amino acid sequence described by SEQ ID NO:7.

1 1. The method of claim 10, wherein the mutant BRIL comprises an amino acid sequence described by SEQ ID NO:3.

12. The method of claim 10, wherein the composition comprises an antibody that selectively binds to an amino acid sequence comprising SEQ ID NO:7.

13. A method for treating a subject diagnosed with or at risk of developing osteogenesis imperfecta type V, comprising administering to the subject a

composition comprising a compound selected from the group consisting of an antisense molecule, RNAi molecule, shRNA molecule, siR A molecule, 2- bromopalmitate and an antibody, in an amount effective to inhibit BRIL activity or expression.

14. The method of claim 13, wherein the composition comprises an antibody.

15. The method of claim 14, wherein the antibody selectively binds to an amino acid sequence comprising SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7.

16. The method of claim 13, wherein the composition is administered in an amount effective to reduce cell membrane localization of BRIL.

17. A method of identifying a therapeutic compound that reduces BRIL activity or expression comprising

(i) contacting a host cell expressing a recombinant BRIL protein with a candidate compound;

(ii) determining BRIL activity or expression level in the host cell contacted with the candidate compound;

(iii) comparing the activity or expression level with the activity or expression level of BRIL expressed by a second host cell not contacted with the candidate compound; and

(iv) selecting the candidate compound as a therapeutic compound when the BRIL activity or expression level in the host cell contacted with the candidate compound is less than the BRIL activity or expression level in the second host cell not contacted with the candidate compound.

18. A pharmaceutical composition comprising a compound selected from the group consisting of an antisense molecule that selectively binds to a BRIL nucleic acid, RNAi molecule that selectively binds to a BRIL nucleic acid, shRNA molecule that selectively binds to a BRIL nucleic acid, siRNA molecule that selectively binds to a BRIL nucleic acid, 2-bromopalmitate and an antibody that selectively binds to a BRIL protein, and pharmaceutically acceptable salts and prodrugs thereof.

19. The composition of claim 18, wherein the compound is an antibody.

20. The composition of claim 19, wherein the antibody selectively binds acid sequence comprising SEQ ID NO: 5, SEQ ID NO:6 or SEQ ID NO: 7.