Use of miRNA inhibitors to treat osteogenesis imperfecta

Abstract

As epigenetic regulators of gene expression, circulating microRNAs (miRNAs) have been reported as potential prognostic markers in several bone diseases. The inventors' objective was to identify circulating miRNAs that may be associated with the severity of osteogenesis imperfecta (OI) in three steps. The inventors screened for microRNAs (miRNAs) with significantly different expression levels in the serum of a small group of OI patients and a control group using RNA sequencing, followed by a validation step of RT-qPCR analysis of serum from a larger patient population. In the first step of miROI, the inventors identified 79 miRNAs with significantly different expression levels. Therefore, the inventors selected 19 of these as the most relevant. In the second stage, the inventors were able to verify the significant overexpression of eight miRNAs (miR-106b-3p, miR-191-5p, miR-363-3p, miR-451a, miR-93-3p, miR-93-5p, let-7g-5p, miR-15b-5p) in a larger OI group. Finally, the inventors investigated the relationship between the variability levels of the verified miRNAs and the clinical characteristics of OI. The inventors found significant differences in the expression of two microRNAs in patients with dentinogenesis imperfecta. After reviewing the literature, the inventors found that six of the eight miRNAs are already known to have a direct effect on bone homeostasis. Furthermore, using a miRNA-gene interaction prediction model, it was revealed that there is a 100% probability of interaction between two of the eight identified miRNAs (miR93 and miR-106b) and COL1A1 and / or COL1A2. This is the first study to establish miRNA signatures in OI and shows significant changes in the expression of miRNAs that may be involved in the regulation of genes involved in the pathophysiology of OI.

Description

[Technical Field]

[0001] Field of Invention This invention relates to medicine, particularly to the field of bone diseases.

[0002] Background of the Invention Osteogenesis imperfecta (OI) is a rare, heterogeneous genetic connective tissue disorder that causes damage to the bone matrix, leading to fragility and deformity. (1、2) The prevalence of this disease is estimated to be 1 in 10,000 people. This disease is the most common hereditary connective tissue disorder. (3、4) In approximately 85% of affected patients, OI is caused by one or more pathogenic mutations in either COL1A1 or COL1A2, the two genes that encode the α1 and α2 chains of type I procollagen, respectively. The mode of inheritance is autosomal dominant. This leads to a decrease in type I collagen synthesis or structural degradation, resulting in quantitative or qualitative deficiencies of type I collagen. Quantitative damage is thought to be associated with the mildest forms of OI, while qualitative damage is preferentially associated with the most severe forms of OI. The location of the mutation on the nucleotide chain is also an important factor; mutations located at the N-terminus tend to cause relatively mild damage, while mutations located at the C-terminus often cause more lethal damage. (2) Finally, the functional use of collagen in the matrix involves a specific and crucial region called the ligand-binding region (MLBR). (5~7) Over the past 15 years, at least 20 novel mutant genes have been discovered that account for 15% of remaining OI cases. These affect bone mineralization, post-translational modification of type I collagen, collagen processing and cross-linking, and osteoblast differentiation and function. These discoveries have identified different transmission modes for these forms of OI, primarily autosomal recessive or, rarely, X-linked. (2、8、9)Although the identification of various mutations involved in diseases has progressed, the association between the type of mutation and the severity of clinical symptoms remains unclear. There is a lack of correlation between genetics and the diversity of extra-skeletal symptoms of OI that affect severity and prognosis, such as hearing loss, cardiovascular disease, hyperlaxity, scoliosis, dentinogenesis imperfecta, and ophthalmological disorders (10) Furthermore, gene expression is also strongly modulated by epigenetic regulation, which results in characteristic gene expression profiles associated with clinical outcomes. Alterations in the expression of microRNAs (miRNAs), which are endogenous regulators of transcription, may at least partially explain phenotypic variability (11) miRNAs are short (18 to 25 nucleotides) non-coding single-stranded RNAs, which are important epigenetic regulators of many physiological and pathophysiological processes by regulating gene expression (11) It is estimated that more than 60% of human protein-coding genes have binding affinity for miRNAs. This is generally conserved in most mammalian species (12) In bone, miRNAs have been shown to play an essential role in regulating osteoblastogenesis, osteoclastogenesis, mesenchymal stem cell differentiation, and more generally, bone formation and bone homeostasis (13、14) Studying the regulation of miRNA expression and its activity in diseases paves the way for potential therapies by specifically targeting tissue sites using synthetic small RNAs that mimic or antagonize endogenous miRNAs to restore or inhibit their functions, respectively (15、16) Abnormal levels and activities of miRNAs have been consistently reported in bone marrow and bone matrix, mesenchymal stem cells, osteoblasts, osteoclasts, as well as in blood-release in multiple bone-related diseases, such as osteoporosis, ectopic calcification, fibrous dysplasia of bone, and bone metastasis (17~21) For example, miR-100 is upregulated in bone tissue in osteoporosis, as well as in osteoporotic osteoblasts and osteoclasts (22)Such correlations between miRNA levels and bone-related gene expression and the clinical manifestations of OI may help elucidate the pathophysiological processes of the disease.

[0003] Summary of the Invention The present invention is defined by the claims. In particular, the present invention relates to the use of a miRNA inhibitor for treating osteogenesis imperfecta, wherein the miRNA inhibitor is selected from the group consisting of miR-93 inhibitors, miR-106b inhibitors, miR-25 inhibitors and miR-182 inhibitors.

[0004] Detailed description of the invention As epigenetic regulators of gene expression, circulating microRNAs (miRNAs) have been reported as potential prognostic markers in several bone diseases. The inventors' objective was to identify circulating miRNAs that may be associated with the severity of osteogenesis imperfecta (OI) in three steps. The inventors screened for microRNAs (miRNAs) with significantly different expression levels in the serum of a small group of OI patients versus a control group using RNA sequencing, and then performed a validation step by analyzing the serum of a larger patient population using RT-qPCR. In the first step of miROI, the inventors found 79 miRNAs with significantly different expression levels. Therefore, the inventors selected 19 of these as the most relevant. In the second stage, the inventors were able to verify significant overexpression of eight miRNAs (miR-106b-3p, miR-191-5p, miR-363-3p, miR-451a, miR-93-3p, miR-93-5p, let-7g-5p, miR-15b-5p) in a larger OI group. Finally, the inventors investigated the relationship between the variability levels of the verified miRNAs and the clinical characteristics of OI. The inventors found significant differences in the expression of two microRNAs in patients with dentinogenesis imperfecta. After reviewing the literature, the inventors found that six of the eight miRNAs are already known to have a direct effect on bone homeostasis. Furthermore, using a miRNA-gene interaction prediction model, it was revealed that there is a 100% probability of interaction between two of the eight identified miRNAs (miR93 and miR-106b) and COL1A1 and / or COL1A2. This is the first study to establish miRNA signatures in OI and demonstrates significant changes in the expression of miRNAs that may be involved in the regulation of genes involved in the pathophysiology of OI.

[0005] The present invention relates to a method for treating osteogenesis imperfecta (OI) in a patient requiring treatment for OI, comprising administering to the patient a therapeutically effective dose of at least one miRNA inhibitor selected from the group consisting of miR-93 inhibitors, miR-106b inhibitors, miR-25 inhibitors, and miR-182 inhibitors.

[0006] As used herein, the terms “osteogenesis imperfecta” or “OI” have their common meaning in the art and refer to a heterogeneous group of rare hereditary connective tissue disorders that cause damage to the bone matrix, resulting in fragility and deformation. 1、2In approximately 90% of cases, OI is caused by monoallelic mutations in the COL1A1 and COL1A2 genes (17q21.33 and 7q21.3), which encode the alpha-1 and alpha-2 chains of type I collagen. These mutations can cause types I through IV of OI. In type V, the mutation is located in the promoter region of the IFITM5 gene (11p15.5). More than 15 causative genes have been identified, and several phenotypes are becoming clear: children with BMP1 mutations may be observed to have high bone mass. Children with homozygous WNT1 mutations have ptosis and neurodevelopmental delay, and craniosynostosis is associated with P4HB or SEC24D mutations. Age at diagnosis depends on the severity of the disease. Five clinically distinct types of osteogenesis imperfecta (OI) have been identified. The most clinically relevant feature of all OI types is bone fragility, which manifests as multiple spontaneous fractures. Type I is mild, without deformities, with normal or short stature, blue sclera, and no dentinogenesis imperfecta (DI). Patients with Type II show multiple rib and long bone fractures, significant deformities, broad long bones, low density on cranial X-rays, and dark sclera at birth. These patients die at birth or shortly after birth. Type III is severe, with the main signs including severe short stature, triangular face, severe scoliosis, grayish sclera, and DI. Types IV and V are moderate to severe forms. Patients with Type IV have moderate short stature, varying degrees of scoliosis, grayish or white sclera, and some have DI. Type V is characterized by mild to moderate short stature, metaphyseal dysplasia at birth, radial head dislocation, interosseous calcification of the forearm and lower leg, hyperplastic callus formation (especially during rapid growth periods such as infancy or adolescence), and white sclera, but without DI. Type V also presents with varying degrees of scoliosis. Diagnosis is based on skeletal and extraskeletal clinical findings. Radiographic examination reveals the presence of osteoporosis and Worm's bone. Bone density measurement confirms low bone mass.

[0007] As used herein, the terms "treatment" or "treating" refer to both prophylactic or preventive treatment and curative or disease-modifying treatment, and include treatment of patients at risk of developing a disease, patients suspected of developing a disease, and patients suffering from or diagnosed as suffering from a disease or condition, including suppression of clinical recurrence. Treatment may be performed for a patient having a medical disorder or a patient who may ultimately acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of the disorder or recurrent disorder, or to extend the patient's survival beyond the expected survival in the absence of such treatment. "Therapeutic regimen" means a treatment pattern for a disease, for example, an administration pattern used during treatment. The therapeutic regimen may include an induction regimen and a maintenance regimen. The phrases "induction regimen" or "induction regimen" refer to a therapeutic regimen (or part of a therapeutic regimen) used for the initial treatment of a disease. The general purpose of the induction regimen is to provide the patient with a high level of drug during the initial phase of the treatment regimen. The induction regimen may (in part or in whole) utilize a "loading regimen". A loading regimen may include administering a higher dose of the drug than a physician would utilize during a maintenance regimen, administering the drug at a higher frequency than a physician would administer during a maintenance regimen, or both. The phrases "maintenance regimen" or "maintenance period" refer to a therapeutic regimen (or part of a therapeutic regimen) used to maintain a patient during the treatment of a disease, for example, to keep the patient in a state of remission over an extended period (months or years). The maintenance regimen may utilize continuous treatment (e.g., administering the drug at regular intervals, such as once a week, once a month, once a year, etc.) or intermittent treatment (e.g., interrupted treatment, intermittent treatment, treatment upon recurrence, or treatment upon achievement of certain predetermined criteria [e.g., onset of disease symptoms, etc.]).

[0008] As used herein, the terms “microRNA” or “miRNA” refer to RNA molecules with a length of approximately 21 to 23 nucleotides (nt). miRNAs may range in length from 18 to 26 nucleotides. Typically, miRNAs are single-stranded. However, in some embodiments, miRNAs may be at least partially double-stranded. In some embodiments, miRNAs may contain an RNA double helix (referred to herein as a “double-stranded region”), which may further contain one or two single-stranded overhangs. In some embodiments, miRNAs may contain a double-stranded region ranging in length from 15 to 29 nucleotides, which may further contain one or two single-stranded overhangs. miRNAs may be formed from two RNA molecules that hybridize with each other, or alternatively, from a single RNA molecule containing a self-hybridization region. Generally, the free 5' end of a miRNA molecule has a phosphate group, and the free 3' end has a hydroxyl group. The double-stranded portion of a miRNA typically contains one or more bulges consisting of one or more unpaired nucleotides, but these are not always necessary. One strand of the miRNA contains the region that hybridizes with the target RNA. In some embodiments, one strand of the miRNA is not strictly complementary to the region of the target RNA. This means that the miRNA hybridizes with the target RNA with one or more mismatches. In some embodiments, one strand of the miRNA is strictly complementary to the region of the target RNA. This means that the miRNA hybridizes with the target RNA without mismatches. Typically, miRNAs are thought to mediate the repression of gene expression by inhibiting the translation of the target transcript. However, in some embodiments, miRNAs may mediate the inhibition of gene expression by causing the degradation of the target transcript.

[0009] As used herein, the term "miR-106b" has its ordinary meaning in the art and refers to the miRNA available under miRBase accession number MI0000734 (hsa-mir-106b) in the database http: / / mirbase.org. This term encompasses the mature sequences hsa-miR-106b-5p (MIMAT0000680, SEQ ID NO: 1) and hsa-miR-106b-3p (MIMAT0004672, SEQ ID NO: 2).

[0010]

Chemical Formula

[0011] As used herein, the term "miR-93" has its ordinary meaning in the art and refers to the miRNA available under miRBase accession number MI0000095 (hsa-mir-93) in the database http: / / mirbase.org. This term encompasses the mature sequences hsa-miR-93-5p (MIMAT0000093, SEQ ID NO: 3) and hsa-miR-93-3p (MIMAT0004509, SEQ ID NO: 4).

[0012]

Chemical Formula

[0013] As used herein, the term "miR-25" has its ordinary meaning in the art and refers to the miRNA available under miRBase accession number MI0000082 (hsa-mir-25) in the database http: / / mirbase.org. This term encompasses the mature sequences hsa-miR-25-5p (MIMAT0004498, SEQ ID NO: 5) and hsa-miR-25-3p (MIMAT0000081, SEQ ID NO: 6).

[0014]

Chemical Formula

[0015] As used herein, the term "miR-182" has its general meaning in the art and refers to the miRNA available in the database http: / / mirbase.org under miRBase accession number MI0000272 (hsa-mir-182). This term encompasses the mature sequences hsa-miR-182-5p (MIMAT0000259, SEQ ID NO: 7) and hsa-miR-182-3p (MIMAT0000260, SEQ ID NO: 8).

[0016] [ka]

[0017] As used herein, the term “miRNA inhibitor” refers to any compound that blocks the expression, processing, and / or function of miRNA. The miRNA inhibitors of the present invention are compounds that inhibit or reduce the activity of miR-106b, miR-93, miR-25, or miR-182. Alternatively, reducing and / or reducing the activity of miR-106b, miR-93, miR-25, or miR-182 may also be achieved by inhibiting the expression of miR-106b, miR-93, miR-25, or miR-182. The term “inhibiting the expression of miR-106b, miR-93, miR-25, or miR-182” means that in treated target cells, the production of miR-106b, miR-93, miR-25, or miR-182 is less than the amount produced before treatment, or the existing level of activity is neutralized. Those skilled in the art can easily determine, for example, whether the expression of miR-106b, miR-93, miR-25, or miR-182 is inhibited in target cells by using techniques for measuring miRNA transcription levels.

[0018] In some embodiments, the miRNA inhibitor of the present invention is a compound such as a nucleic acid that hybridizes with miR-106b, miR-93, miR-25, or miR-182, or has a sequence complementary to the sequence of miR-106b, miR-93, miR-25, or miR-182. In some embodiments, the miRNA inhibitor of the present invention is a compound such as a nucleic acid that has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence complementarity with the sequence of miR-106b, miR-93, miR-25, or miR-182.

[0019] Appropriate miRNA inhibitors include double-stranded RNA (e.g., short or small interfering RNA or "siRNA"), antagonists, antisense nucleic acids, and enzymatic RNA molecules, such as ribozymes. Each of these compounds can target a specific miRNA and disrupt or induce the disruption of the target miRNA. For example, the expression of a specific miRNA can be inhibited by inducing RNA interference of the miRNA with an isolated double-stranded RNA ("dsRNA") molecule having at least 90%, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to at least a portion of the miRNA.

[0020] In some embodiments, the dsRNA molecule is a “short or small interfering RNA” or “siRNA.” siRNA useful in the methods of the present invention comprises a short double-stranded RNA having a length of about 17 to about 29 nucleotides, preferably about 19 to about 25 nucleotides. siRNA comprises a sense RNA strand and a complementary antisense RNA strand, annealed to each other by a standard Watson-Crick base-pairing interaction (hereinafter, “base-pairing”). The sense strand contains a nucleic acid sequence substantially identical to the nucleic acid sequence contained within the target miRNA. As used herein, a nucleic acid sequence in siRNA that is “substantially identical” to the target sequence contained within the target miRNA is either identical to the target sequence or differs from the target sequence by one or two nucleotides. The sense and antisense strands of siRNA may contain two complementary single-stranded RNA molecules or a single molecule in which two complementary portions form base pairs and are covalently linked by a single-stranded “hairpin” region. Furthermore, siRNA can be a modified RNA that differs from native RNA by the addition, deletion, substitution, and / or modification of one or more nucleotides. Such modifications may include adding non-nucleotide substances to the ends of the siRNA or to one or more internal nucleotides of the siRNA, modifications that make the siRNA resistant to nuclease digestion, or substitution of one or more nucleotides in the siRNA with deoxyribonucleotides. Also, one or both strands of the siRNA may contain a 3' overhang. As used herein, "3' overhang" refers to at least one non-base-pairing nucleotide extending from the 3' end of a double-stranded RNA strand. For this reason, in some embodiments, the siRNA contains at least one 3' overhang of 1 to about 6 nucleotides in length (including ribonucleotides or deoxyribonucleotides), preferably 1 to about 5 nucleotides in length, more preferably 1 to about 4 nucleotides in length, and particularly preferably about 2 to about 4 nucleotides in length. In some embodiments, the 3' overhang is present on both strands of the siRNA and is 2 nucleotides in length.For example, each chain of an siRNA may contain a 3' overhang of dithymidylic acid ("TT") or diuridylic acid ("uu").

[0021] siRNA can be produced chemically or biologically as described above, or expressed from recombinant plasmids or viral vectors. Exemplary methods for producing and testing dsRNA or siRNA molecules are described in US2002 / 0173478 (Gewirtz) and US2004 / 0018176 (Reich et al). These disclosures are incorporated herein by reference in their entirety.

[0022] Furthermore, the expression of a given miRNA may also be inhibited by antisense nucleic acids. As used herein, "antisense nucleic acid" refers to a nucleic acid molecule that binds to a target RNA via RNA-RNA, RNA-DNA, or RNA-peptide nucleic acid interactions and alters the activity of the target RNA. Suitable antisense nucleic acids for use in the methods of the present invention are generally single-stranded nucleic acids (e.g., RNA, DNA, RNA-DNA chimeras, PNAs) containing a nucleic acid sequence complementary to the continuous nucleic acid sequence in the miRNA. Preferably, the antisense nucleic acid contains a nucleic acid sequence that is 50-100% complementary, more preferably 75-100%, and most preferably 95-100% complementary to the continuous nucleic acid sequence in the miRNA. While we do not wish to be bound by theory, it is thought that antisense nucleic acids activate RNase H or some other intracellular nucleases that digest the miRNA / antisense nucleic acid double helix.

[0023] Furthermore, antisense nucleic acids may contain modifications to the nucleic acid backbone or the sugar and base moieties (or equivalents thereof) to enhance target specificity, nuclease resistance, delivery, or other properties related to molecular efficacy. Such modifications include the inclusion of cholesterol moieties, double-strand intercalators, such as acridine or one or more nuclease-resistant groups.

[0024] Antisense nucleic acids can be produced chemically or biologically, or expressed from recombinant plasmids or viral vectors, as described below. Exemplary methods for production and testing are within the scope of the art; see, for example, Stein and Cheng (1993), Science 261:1004 and US5,849,902 (Woolf et al). The entirety of these disclosures is incorporated herein by reference.

[0025] In some embodiments, the miRNA inhibitor is antagomir. As used herein, the term “antagomir” refers to a chemically engineered small RNA used to silence miR-106b, miR-93, miR-25, or miR-182. Antagomir is complementary to a particular miRNA target, having either incorrect base pairing or some base modification. Antagomir may also include some modification to make it more resistant to degradation. In some embodiments, antagomir is a chemically engineered cholesterol-conjugate single-stranded RNA analog.

[0026] Furthermore, inhibition of miR-106b, miR-93, miR-25, or miR-182 can also be achieved by using antisense 2'-O-methyl (2'-O-Me) oligoribonucleotides, 2'-O-methoxyethyl (2'-O-MOE), phosphorothioate, locked nucleic acid (LNA), mololino-oligomers, or lentiviral or adenovirus-expressed antagonists (Stenvang and Kauppinen (2008), Expert Opin. Biol. Ther. 8(1):59-81). In addition, miRNA activity can be inhibited using MOE (2'-O-methoxyethyl phosphorothioate) or LNA (locked nucleic acid (LNA) phosphorothioate chemical) modification of single-stranded RNA analogs.

[0027] Furthermore, the expression of a specific miRNA can also be inhibited by an enzymatic nucleic acid. As used herein, "enzymatic nucleic acid" refers to a nucleic acid that includes a substrate-binding region complementary to the continuous nucleic acid sequence of the miRNA and is capable of specifically cleaving the miRNA. Preferably, the substrate-binding region of the enzymatic nucleic acid is 50-100% complementary to the continuous nucleic acid sequence in the miRNA, more preferably 75-100%, and most preferably 95-100% complementary. The enzymatic nucleic acid may also include modifications at the base, sugar, and / or phosphate group. An exemplary enzymatic nucleic acid for use in the method of the present invention is a ribozyme. Enzymatic nucleic acids can be produced chemically or biologically, or expressed from recombinant plasmids or viral vectors as described below. Exemplary methods for producing and testing dsRNA or siRNA molecules are described in Werner and Uhlenbeck (1995), Nucl. Acids Res. 23:2092-96; Hammann et al. (1999), Antisense and Nucleic Acid Drug Dev. 9:25-31 and US4,987,071 (Cech et al). These disclosures are incorporated herein by reference in their entirety.

[0028] The miRNA inhibitors of the present invention can be obtained using many standard techniques. For example, the miRNA inhibitors of the present invention can be chemically synthesized or recombinantly produced using methods known in the art. Typically, the miRNA inhibitors of the present invention are chemically synthesized using appropriately protected ribonucleoside phosphoramidites and conventional DNA / RNA synthesizers. Commercial suppliers of synthetic RNA molecules or synthetic reagents include, for example, Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colo., USA), Pierce Chemical (part of Perbio Science, Rockford, 111., USA), Glen Research (Sterling, Va., USA), ChemGenes (Ashland, Mass., USA), and Cruachem (Glasgow, UK).

[0029] In some embodiments, the synthetic miRNA inhibitors of the present invention contain one or more design elements. These design elements include, but are not limited to, (i) substituents for the phosphate or hydroxyl group of a nucleotide at the 5' end of the complementary region; and (ii) one or more sugar modifications. In some embodiments, the synthetic miRNA inhibitors of the present invention have a nucleotide at its 5' end of the complementary region in which the phosphate group and / or hydroxyl group is replaced by another chemical group (referred to as a “substitution design”). In some cases, the phosphate group is replaced, and in other cases, the hydroxyl group is replaced. In some embodiments, the substituents are biotin, amine groups, lower alkylamine groups, acetyl groups, 2'O-Me (2'-oxygen-methyl), DMTO (oxygen-containing 4,4'-dimethoxytrityl), fluorescein, thiols, or acridine, but other substituents are well known to those skilled in the art and can also be used. In some embodiments, the sugar modification is a 2'O-Me modification. In some embodiments, one or more sugar modifications are present in the first or last 2-4 residues or the first or last 4-6 residues of the complementary region.

[0030] In some embodiments, the miRNA inhibitors of the present invention are resistant to degradation by nucleases. Those skilled in the art can easily synthesize nuclease-resistant nucleic acids, for example, by incorporating one or more ribonucleotides modified at the 2'-position into the miRNA. Suitable 2'-modified ribonucleotides include those modified at the 2'-position with fluoro, amino, alkyl, alkoxy, and O-allyl groups.

[0031] Alternatively, the miRNA inhibitors of the present invention can be expressed from recombinant linear or circular DNA plasmids using any suitable promoter. Suitable promoters for expressing RNA from plasmids include, for example, the U6 promoter sequence or the cytomegalovirus promoter. The selection of other suitable promoters is within the scope of the art. The recombinant plasmids of the present invention may also contain inducible or regulating promoters for expressing the miRNA inhibitors of the present invention in target cells.

[0032] The miRNA inhibitor of the present invention, expressed from a recombinant plasmid, can be isolated from a cultured cell expression system using standard techniques. Furthermore, the miRNA inhibitor of the present invention, expressed from a recombinant plasmid, can be delivered to target cells and directly expressed within those target cells. The use of recombinant plasmids for delivering the miRNA inhibitor of the present invention to target cells is discussed in more detail below.

[0033] The miRNA inhibitors of the present invention may be expressed from a separate recombinant plasmid or from a proprietary recombinant plasmid. Preferably, the miRNA inhibitors of the present invention are expressed as nucleic acid precursor molecules from a single plasmid, and these precursor molecules are processed into functional miRNA inhibitors by an appropriate processing system, including a processing system present in the target cell. Other appropriate processing systems include, for example, the in vitro Drosophila cell lysate system described in US2002 / 0086356 (Tuschl et al) and the E. coli RNAse III system described in US2004 / 0014113 (Yang et al). These disclosures are incorporated herein by reference in their entirety.

[0034] Selection of a plasmid suitable for expressing the miRNA inhibitor of the present invention, a method for inserting a nucleic acid sequence into a plasmid to express a gene product, and a method for delivering a recombinant plasmid to target cells are within the scope of the art. See, for example, Zeng et al. (2002), Molecular Cell 9:1327-1333; Tuschl (2002), Nat. Biotechnol, 20:446-448; Brummelkamp et al. (2002), Science 296:550-553; Miyagishi et al. (2002), Nat. Biotechnol. 20:497-500; Paddison et al. (2002), Genes Dev. 16:948-958; Lee et al. (2002), Nat. Biotechnol. 20:500-505 and Paul et al. (2002), Nat. Biotechnol. 20:505-508. These disclosures are incorporated herein by reference in their entirety.

[0035] In some embodiments, the plasmid expressing the miRNA inhibitor of the present invention includes a sequence encoding a miRNA inhibitor precursor under the control of the CMV intermediate early promoter. As used herein, “under the control of the promoter” means that the nucleic acid sequence is located 3' to the promoter, and as a result the promoter can initiate transcription of the miRNA inhibitor encoding sequence.

[0036] Furthermore, the miRNA inhibitors of the present invention can also be expressed from recombinant viral vectors. It is intended that the miRNA inhibitors of the present invention can be expressed from separate recombinant viral vectors or from proprietary viral vectors. The miRNA inhibitors expressed from recombinant viral vectors can be isolated from cultured cell expression systems using standard techniques, or they can be directly expressed in target cells. The use of recombinant viral vectors to deliver miRNA inhibitors to target cells is discussed in more detail below. The recombinant viral vectors of the present invention comprise a sequence encoding the miR-106b, miR-93, miR-25, or miR-182 inhibitor compound of the present invention, and any promoter suitable for expressing the miRNA inhibitor sequence. Suitable promoters include, for example, the U6 or HI RNA pol III promoter sequence or the cytomegalovirus promoter. Selection of other suitable promoters is within the scope of the art. The recombinant viral vectors of the present invention may also comprise an inducible or regulating promoter for the expression of the miRNA inhibitor in target cells. Any viral vector capable of accepting the coding sequence of the miRNA inhibitor of the present invention can be used, such as vectors derived from adenovirus (AV), adeno-associated virus (t), retroviruses (e.g., lentivirus (LV), rhabdovirus, mouse leukemia virus), herpesvirus, etc. The tropism of the viral vector can be modified as needed by pseudotyping the vector with envelope proteins or other surface antigens derived from other viruses, or by substitution with different viral capsid proteins. For example, the lentiviral vector of the present invention can be pseudotyped with surface proteins of vesicular stomatitis virus (VSV), rabies, Ebola, Mocola, etc. The AAV vector of the present invention can target various cells by manipulating the vector to express different capsid protein serotypes. For example, an AAV vector expressing serotype 2 capsid on a serotype 2 genome is called AAV 2 / 2.The AAV 2 / 5 vector can be produced by replacing the serotype 2 capsid gene in this AAV 2 / 2 vector with the serotype 5 capsid gene. Techniques for constructing AAV vectors expressing various capsid protein serotypes are within the realm of the art. See, for example, Rabinowitz JE et al. (2002), J Virol 76:791801. This disclosure is incorporated herein by reference in its entirety.

[0037] miRNA inhibitors can be administered to a patient by any means suitable for delivering these compounds to target cells. For example, miRNA inhibitors can be administered by a method suitable for transfecting the patient's cells with these compounds or nucleic acids containing sequences encoding these compounds. Preferably, the cells are transfected with a plasmid or viral vector containing a sequence encoding at least one miRNA inhibitor. miRNA inhibitors can be administered to a patient by any suitable enteral or parenteral administration route. Suitable enteral administration routes for the methods of the present invention include, for example, oral, rectal, or intranasal delivery. Suitable parenteral administration routes include, for example, intravascular administration (e.g., intravenous bolus injection, intravenous infusion, intra-arterial bolus injection, intra-arterial infusion, and intravascular catheter infusion); peritissue and intratissue injection (e.g., intraretinal or subretinal injection); subcutaneous injection or deposition including (e.g., by an osmotic pump); and direct application to the tissue of interest by catheter or other placement devices (e.g., implants containing porous, non-porous, or gel-like materials) and inhalation. The preferred routes of administration are injection, infusion, and direct injection into tumor tissue.

[0038] In the methods of the present invention, the miRNA inhibitor can be administered to the patient as naked RNA, in combination with a delivery reagent, or as a nucleic acid (e.g., recombinant plasmid or viral vector) containing a sequence expressing the miRNA inhibitor. Suitable delivery reagents include, for example, Minis Transit TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations (e.g., polyethyleneimine, which becomes nanoparticles (less than 50 nm) when mixed with glucose solution; or, for example, polylysine) and liposomes. In some embodiments, liposomes are used to deliver the miRNA inhibitor (or nucleic acid containing a sequence encoding it) to the patient. Liposomes can also extend the blood half-life of the gene product or nucleic acid. Liposomes suitable for use in the present invention can be formed from standard vesicle-forming lipids. These vesicle-forming lipids generally include neutral or negatively charged phospholipids and sterols, such as cholesterol. The selection of lipids is generally guided by considering factors such as the desired liposome size and the liposome half-life in the bloodstream. Various methods for preparing liposomes are known, for example, as described in Szoka et al. (1980), Ann. Rev. Biophys. Bioeng. 9:467 and US4,235,871, US4,501,728, US4,837,028 and US5,019,369. These disclosures are incorporated herein by reference in their entirety. Liposomes for use in the methods of the present invention may contain ligand molecules that target the liposomes to target cells. Ligands that bind to receptors widely present on target cells, such as monoclonal antibodies that bind to cancer cell antigens, are preferred. Liposomes for use in the methods of the present invention may also be modified to avoid removal by the mononuclear macrophage system ("MMS") and the reticuloendothelial system ("RES"). Such modified liposomes have an opsonization inhibitory moiety on their surface or incorporated into the liposome structure. In particular, the liposomes of the present invention may contain both an opsonization inhibitory moiety and a ligand.The opsonization inhibitory moieties used for preparing liposomes in the present invention are typically large hydrophilic polymers bound to the liposome membrane. As used herein, "bound" to the liposome membrane refers to a state in which the opsonization inhibitory moiety is chemically or physically attached to the membrane, for example, by intercalation of lipid-soluble anchors to the membrane itself or by direct binding to active groups of membrane lipids. These opsonization inhibitory hydrophilic polymers form a protective surface layer that significantly reduces liposome uptake by MMS and RES, as described, for example, in US4,920,016 (the entirety of which is incorporated herein by reference). Opsonization inhibitory moieties suitable for modifying liposomes are preferably water-soluble polymers with a number-average molecular weight of about 500 to about 40,000 daltons, more preferably about 2,000 to about 20,000 daltons. Such polymers include polyethylene glycol (PEG) or polypropylene glycol (PPG) derivatives; for example, methoxyPEG or PPG and PEG or PPG stealth; synthetic polymers, for example, polyacrylamide or poly-N-vinylpyrrolidone; linear, branched, or dendrimeric polyamidoamines; polyacrylic acid; polyalcohols, for example, polyvinyl alcohol and polyxylitol with chemically bonded carboxylic acid or amino groups, and gangliosides, for example, ganglioside GM1. Copolymers of PEG, methoxyPEG, or methoxyPPG or their derivatives are also suitable. In addition, opsonization-inhibiting polymers may be block copolymers of PEG with any of the following: polyamino acids, polysaccharides, polyamidoamines, polyethyleneamines, or polynucleotides. Furthermore, opsonization-inhibiting polymers may be natural polysaccharides containing amino acids or carboxylic acids, such as galacturonic acid, glucuronic acid, mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginic acid, carrageenan; active polysaccharides or oligosaccharides (linear or branched); or carboxylated polysaccharides or oligosaccharides, such as carboxylic acid derivatives, to which carboxyl groups are bonded. Preferably, the opsonization-inhibiting moiety is PEG, PPG, or derivatives thereof.Liposomes modified with PEG or PEG derivatives are sometimes called "PEGylated liposomes." The opsonization inhibitory moiety can be attached to the liposome membrane by one of numerous known techniques. For example, the N-hydroxysuccinimide ester of PEG can be attached to a phosphatidylethanolamine lipid-soluble anchor and then to the membrane. Similarly, dextran polymers can be derivatized with a stearylamine lipid-soluble anchor via reductive activation using Na(CN)BH3 and a solvent mixture, such as tetrahydrofuran and water in a 30:12 ratio, at 60°C. Liposomes modified with opsonization inhibitory moieties remain in circulation much longer than unmodified liposomes. For this reason, such liposomes are sometimes called "stealth" liposomes. Stealth liposomes are known to accumulate in tissues nourished by porous or "leaky" microvascular networks. Therefore, tissues characterized by defects in such microvascular networks will efficiently accumulate these liposomes. See Gabizon, et al. (1988), Proc. Natl. Acad. Sci., USA, 18:6949-53. In addition, reduced uptake by RES prevents significant accumulation of liposomes in the liver and spleen, thereby reducing the toxicity of stealth liposomes. For this reason, liposomes modified with opsonization inhibitory moieties are particularly suitable for delivering miRNA inhibitors (or nucleic acids containing encoding sequences) to target cells.

[0039] Those skilled in the art can easily determine the therapeutically effective dose of a miRNA inhibitor to be administered to a given patient by considering factors such as the patient's size and weight; the stage of the disease; the patient's age, health condition, and sex; the route of administration, and whether the administration is local or systemic. The effective dose of the compound may be based on the approximate or estimated weight of the patient being treated. Preferably, such an effective dose is administered parenterally or enterally, as described herein. For example, the effective dose of the compound administered to a patient may be in the range of about 5 to 10,000 micrograms / kg body weight, preferably about 5 to 3,000 micrograms / kg body weight, preferably about 700 to 1,000 micrograms / kg body weight, and more preferably about 1,000 micrograms / kg body weight. Furthermore, those skilled in the art can easily determine a suitable administration regimen for administering the compound to a given patient. For example, the compound may be administered to the patient once (e.g., as a single injection or deposition).

[0040] In some embodiments, the miRNA inhibitors of the present invention are administered to a patient in combination with one or more bisphosphonates. As used herein, the term "bisphosphonate" means a compound characterized by two C-PO3 2-bonds. When the two bonds are located on the same carbon atom, the compound is called a geminal bisphosphonate. As used herein, the term "bisphosphonate" also means diphosphonates, biphosphonic acids, and diphosphonic acids, as well as salts and derivatives of these substances.

[0041] In some embodiments, the miRNA inhibitors of the present invention are administered to patients in combination with one or more sclerostin inhibitors. As used herein, the term "sclerostin" has its general meaning in the art and refers to the protein encoded by the SOST gene in humans (Balemans, Wendy, et al. "Increased bone density in sclerosteosis is due to the deficiency of a novel secreted protein (SOST)." Human molecular genetics 10.5 (2001): 537-544). Sclerostin is a secreted glycoprotein having a cysteine ​​knot-like (CTCK) domain at its C-terminus and exhibiting sequence similarity to the DAN (differential screening selection gene disorder in neuroblastoma) family, which are bone morphogenetic protein (BMP) antagonists. As used herein, the term “sclerostin inhibitor” means any molecule that inhibits the bioactivity of sclerostin in bone, as measured by changes in bone mineralization, bone density, bone height, effects on osteoblasts and / or osteoclasts, bone formation markers, bone resorption markers, osteoblast activity markers and / or osteoclast activity markers. Such inhibitors may act by binding to sclerostin or its receptor or binding partner. Inhibitors in this category include “sclerostin conjugates,” such as antibody or peptide-based molecules. “Sclerostin inhibitors” also refer to low-molecular-weight organic compounds that bind to sclerostin and inhibit its activity, and which may have a molecular weight of less than approximately 1000 daltons. Alternatively, inhibitors may act by inhibiting sclerostin expression. Inhibitors in this category include polynucleotides or oligonucleotides that bind to sclerostin DNA or mRNA and inhibit sclerostin expression, and these include, but are not limited to, antisense oligonucleotides, inhibitory RNA, DNA enzymes (deoxyribozymes), ribozymes, aptamers, or pharmaceutically acceptable salts thereof that inhibit sclerostin expression.Examples of sclerostin inhibitors include romosozumab, brosozumab, and setorusumab.

[0042] In some embodiments, the miRNA inhibitors of the present invention are administered to a patient in combination with one or more TGF-beta inhibitors. As used herein, the term "TGF-beta" has its general meaning in the art and refers to one or more members of the transforming growth factor beta family proteins, e.g., TGF-beta 1, TGF-beta 2, and TGF-beta 3. As used herein, the term "TGF-beta inhibitor" refers to a drug that has the ability to directly or indirectly inhibit the biological function of TGF-beta. Therefore, TGF-beta inhibitors include, but are not limited to, TGF-beta-specific inhibitors (e.g., blocking (neutralizing) antibodies), soluble TGF-beta receptors (which would competitively inhibit TGF-beta), membrane-bound TGF-beta receptors, protease inhibitors that inactivate proteases responsible for the activation of precursor TGF-beta into mature TGF-beta, inhibitors specific to TGF-beta receptors (type I, type II, or type III) that prevent TGF-beta from binding to the receptor (e.g., antibodies or small molecules), and siRNA or antisense RNA that block TGF-beta expression, or combinations thereof. In some embodiments, the TGF-beta inhibitor is fresolimmab.

[0043] The miRNA inhibitor of the present invention is preferably formulated as a pharmaceutical composition according to techniques known in the art before administration to a patient. The pharmaceutical composition of the present invention is characterized by being at least sterile and free of pyrogens. As used herein, “pharmaceutical formulation” includes formulations for human and veterinary use. Methods for preparing the pharmaceutical composition of the present invention are within the realm of those skilled in the art, for example, as described in Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company, Easton, Pa. (1985) (this disclosure is incorporated herein by reference in its entirety). The pharmaceutical formulation of the present invention comprises a miRNA inhibitor (e.g., 0.1 to 90% by weight) or a physiologically acceptable salt thereof, mixed with a pharmaceutically acceptable carrier. The pharmaceutical formulation of the present invention may also comprise a miRNA inhibitor encapsulated in liposomes and a pharmaceutically acceptable carrier. Preferred pharmaceutically acceptable carriers include water, buffer water, ordinary saline, 0.4% saline, 0.3% glycine, hyaluronic acid, etc. Furthermore, the pharmaceutical compositions of the present invention may also contain conventional pharmaceutical excipients and / or additives. Suitable pharmaceutical excipients include stabilizers, antioxidants, osmotic regulators, buffers, and pH adjusters. Suitable additives include, for example, the addition of physiologically biocompatible buffers (e.g., tromethamine hydrochloride), chelating agents (e.g., DTPA or DTPA-bisamide, etc.), or calcium chelate complexes (e.g., calcium DTPA, CaNaDTPA-bisamide, etc.), or optionally, calcium or sodium salts (e.g., calcium chloride, calcium ascorbate, calcium gluconate, or calcium lactate). The pharmaceutical compositions of the present invention may be packaged for use in liquid form or may be lyophilized.

[0044] The present invention will be further illustrated by the following drawings and embodiments. However, these embodiments and drawings should not be construed as limiting the scope of the present invention. [Brief explanation of the drawing]

[0045] [Figure 1] Figure 1: Flowchart of the miROI study. In the initial screening phase, potential miRNAs were identified, these were quantified in a larger control group in the validation phase, and in the clinical phase, the potential association between circulating miRNAs and disease phenotypes was investigated. [Figure 2] Figure 2: Volcano plot showing significant differences in miRNA expression between the control group and the OI group. The miRNA expression levels were compared between groups using the tag ratio per million, and relative data was obtained by calculating the log2 change. The -log10 change in FDR-corrected p-value was plotted against the log2 change. Continuous lines represent an FDR-corrected p-value threshold of 0.05, and discontinuous lines represent 0.0005. MiRNAs with p<0.0005 are labeled. [Figure 3] Figure 3: Relative levels of eight miRNAs that showed significant differences in expression between OI patients (n=48) and matched control subjects (n=48). Data are shown as the mean log2 change and 95% confidence interval for the OI group and the control group. Relative levels were measured by RT-qPCR on a pre-spotted Taqman low-density array card. [Figure 4A] Figure 4A: Expression levels of COL1A1 by qPCR 5 days after treatment with Mimic-CTRL, Mimic miR-93-3p, Mimic miR-106b-3p, Antago-CTRL, Antago miR-93-3p, and Antago miR-106b-3p in MC3T3E1 osteoblasts. Expression levels are shown as the ratio of 2^-delta Ct to each control. Results are shown as mean + / - SEM (n=5). *P<0.05 compared to the control group. [Figure 4B]Figure 4B: Expression levels of COL1A2 by qPCR 5 days after treatment with Mimic-CTRL, Mimic miR-93-3p, Mimic miR-106b-3p, Antago-CTRL, Antago miR-93-3p, and Antago miR-106b-3p in MC3T3E1 osteoblasts. Expression levels are shown as the ratio of 2^-delta Ct to each control. Results are shown as mean + / - SEM (n=5). *P<0.05 compared to the control group. [Figure 5A] Figure 5A: Expression levels of COL1A1 by qPCR 5 days after treatment with Mimic-CTRL, Mimic miR-93-3p, Mimic miR-106b-3p, Antago-CTRL, Antago miR-93-3p, and Antago miR-106b-3p in osteoblasts derived from long bones of 12-week-old adult C57BL / 6 mice. Expression levels are shown as the ratio of 2^-delta Ct to each control. Results are shown as mean + / - SEM (n=3). *P<0.05 compared to the control group. [Figure 5B] Figure 5B: Expression levels of COL1A2 by qPCR 5 days after treatment with Mimic-CTRL, Mimic miR-93-3p, Mimic miR-106b-3p, Antago-CTRL, Antago miR-93-3p, and Antago miR-106b-3p in osteoblasts derived from long bones of 12-week-old adult C57BL / 6 mice. Expression levels are shown as the ratio of 2^-delta Ct to each control. Results are shown as mean + / - SEM (n=3). *P<0.05 compared to the control group. [Figure 6]Figure 6: Modulation of miR-93-3p affects type I collagen expression in MC3T3 and primary osteoblasts: Western blot analysis and quantification. Representative Western blot images show type I collagen and beta-actin expression in osteoblasts 5 days after treatment with Antago-CTRL and Antago-miR-93-3p (A) or Mimic-CTRL and Mimic-miR-93-3p (B) in the MC3T3E1 line. Relative quantification of type I collagen normalized to beta-actin in MC3T3E1 cells (C) or primary osteoblasts (E) 5 days after treatment with Antago-miR-93-3p compared to Antago-CTRL. Relative quantification of type I collagen normalized to beta-actin in MC3T3E1 cells (D) or primary osteoblasts (F) 5 days after treatment with Mimic-miR-93-3p compared to Mimic-CTRL. P-value (T-test) < 0.05 = * [Figure 7] Figure 7: Immunofluorescence analysis of type I collagen expression in MC3T3E1 cells treated with Antago-CTRL and Antago miR-93-3p. (A) Confocal microscopy of MC3T3E1 cells stained for type I collagen after treatment with Antago-CTRL (Zeiss 880, ×20). (B) Confocal microscopy of MC3T3E1 cells treated with Antago miR-93-3p. The same parameters were applied (A-B). (C) Automated quantification of type I collagen expression normalized to cell number across the entire slide (n=3). P-value T-test <0.05:* [Figure 8] Figure 8: Immunofluorescence analysis of type I collagen expression in MC3T3E1 cells treated with Antago-CTRL and Antago miR-93-3p. (A) Confocal microscopy of MC3T3E1 cells stained for type I collagen after treatment with Mimic-CTRL (Zeiss 880, ×40). (B) Confocal microscopy of MC3T3E1 cells treated with Mimic miR-93-3p. The same parameters were applied (A-B). (C) Automated quantification of type I collagen expression normalized to cell number across the entire slide (n=3). P-value T-test <0.05:*

[0046] Example 1 Materials and methods This study was conducted in three stages. First, the inventors performed a screening stage using next-generation sequencing (NGS) to measure the differences in expression levels of all miRNAs in the serum of 10 OI patients and 10 control subjects. Next, the inventors performed a validation stage using RT-qPCR analysis of the miRNAs selected in the screening stage in a larger serum collection. Finally, the inventors investigated the potential association between differences in miRNA expression levels and clinicopathological characteristics in their OI patient group.

[0047] Recruitment OI patients were recruited for the mirOI study from October 2019 to October 2021. Recruitment took place either during hospitalization or during consultations at the bone disease reference center at Edouard Herriot University Hospital (Lyon, France) (OSCAR network). Patients were selected based on the following criteria: male or female, 18 years of age or older; having a COL1A1 or COL1A2 mutation; and having a pre-established Sillence classification by a bone disease specialist rheumatologist. (23) Participants had to meet the criteria of having been diagnosed with type I or type III osteoporosis (OI) based on the following criteria. Exclusion criteria included long-term glucocorticoid treatment (more than 3 months), ongoing anti-osteoporosis treatment, presence of inflammatory disease, uncontrolled hypothyroidism / hyperthyroidism or hypoparathyroidism / hyperparathyroidism, presence of active cancer, malabsorption, benign bone tumors and other bone diseases within the past 2 years, pregnancy or lactation, insufficient French language proficiency that could hinder informed consent, and judicial protection. Written informed consent was obtained from each participant. In the screening phase, the inventors recruited 10 OI patients, and in the validation phase, 48 patients were included. The control group was defined by age (in 5-year increments), sex, and body mass index (BMI, 2 kg / m²). 2These are healthy individuals (matched in the group) recruited from the OFELY, MODAM, and STRAMBO population cohorts at INSERM UMR 1033 (Lyon, France). (24~27) OFELY (Os des Femmes de Lyon) is a prospective cohort of 1089 healthy women aged 31–89 years, recruited in 1992 and 1993 to study determinants of bone fragility. MODAM (Mother Daughter Microarchitecture) is a case-control study conducted from May 2010 to February 2012. STRAMBO (the bone structure of aging men) is a cohort of 1169 healthy men aged 20–85 years, recruited in Lyon between 2006 and 2016. The participant distribution and study design are shown in Figure 1. This study was approved by the French Ethics Committee (CPP Ile-de-France VI, registration No. 2019-A00521-56) and conducted in accordance with the ethical standards of the French National Ethics Guidelines and the Revised Declaration of Helsinki. This study is registered on ClinicalTrials.gov (NCT T04009733).

[0048] Blood sample collection Blood samples were preferentially collected in the morning after fasting from the previous night. For serum preparation, the samples were centrifuged at 2000g at 4°C within one hour of collection, divided into equal portions, and stored at -80°C until analysis. To assess serum quality, blood samples were visually analyzed (including the presence of hemolysis and fibrin coagulation).

[0049] Collection of clinical and biological data Clinical information was collected using both clinical questionnaires submitted by physicians and patients, and the SF-36 QOL questionnaire completed by the patients themselves. (28)Patient clinical data was obtained from standardized electronic medical records (DPI Easily®, from 2009 to the present). Sillence classification and disease severity assessment were performed by rheumatologists at a reference center for rare bone diseases.

[0050] OI MiRNome Analysis using Next-Generation Sequencing The screening stage was performed to compare serum miRNA profiles (miRNomes) between OI patients and control subjects. For this purpose, the inventors performed miRNome analysis in the serum of 10 OI patients and 10 matched controls by total RNA extraction, library preparation and quantification followed by NGS and miRNA sequencing (Illumina platform; QIAGEN Genomic Services, Germantown, MD, USA). Total RNA (5 μL) extracted from serum using the miRNeasy serum / plasma kit (Qiagen®) was used to prepare the miRNA-NGS library using the QIAseq miRNA library kit (Qiagen®). First, ligation was performed on mature miRNAs, adding an adapter poly(A) sequence to the 3'-OH end, and then the adapter sequence was ligated to the 5'-PO4 end using ATP ligase. Mature single-stranded miRNAs containing a specific nucleotide sequence, which is the molecular barcode (UMI), were obtained. Reverse transcription (RT) was performed to generate cDNA containing the UMI. This enabled library identification after sequencing. After 22 cycles of PCR amplification, cDNA was incubated with QMN beads (QIAseq miRNA NGS beads), washed, and eluted from the beads with RNase / DNase-free water. Then, each library was amplified using the Qiaseq miRNA index kit. Prior to this, the miRNA-enriched fraction was isolated by agarose gel electrophoresis and subjected to quality control using a Bioanalyzer 2100 (Agilent®, Santa Clara, CA, USA). After final quantification by PCR, libraries obtained from various samples were pooled at equimolar concentrations, clusters were formed on the flow cell surface, and then sequencing was performed using a NextSeq500 sequencer (Illumina). The collected "read" format data was subjected to read quality control to correct for library amplification bias, and then mature miRNA sequences and GRCh38 human genomes referenced to miRBase-20 were analyzed. (29~32)Alignment was performed. After demultiplexing the unprocessed reads, the data was expressed as counts. This data was then calculated by dividing the count of a specific miRNA by the total count in the sample, and 10 6 The data was multiplied by a factor and then normalized using the Trimmed Mean of M-values ​​(TMM) method. miRNA expression levels were expressed in TPM (number of tags per million reads), and then compared using the TPM ratio between groups. This ratio was then converted into relative data by calculating Log2(log-free change) (LogFC).

[0051] MiRNA analysis by RT-qPCR Total RNA was extracted from 200 μL of serum using the Nucleospin miRNA plasma kit (Macherey-Nagel, Duren, Germany) according to the manufacturer's recommendations. The samples were thawed on ice and centrifuged at 3000 g for 5 minutes. A lysis buffer containing 1 μL of glycogen as an RNA carrier and synthetic spike-in control RNA (cel-miR-39-3p) as an exogenous control was added to the serum (4 μL of a 0.1 nmol / L dilution). After cell lysis, protein precipitation, and washing, the RNA was eluted from a silica microcolumn with 40 μL of RNAse / DNAse-free water and stored at -80°C. miRNA was quantified using TaqMan Advanced miRNA technology (Applied Biosystems, Thermo-Fisher Scientific, Waltham, MA, USA). Quantification of miRNAs expressed at low copy numbers was improved by 14 cycles of cDNA amplification in a 2× TaqMan PreAmp master mix containing Megaplex PreAmp primers. miRNAs were subjected to RT (Restorative Synthesis) to prepare cDNA from 2 μL of total RNA using the TaqMan Advanced miRNA Synthesis Kit. qPCR amplification was performed on 5 μL of the RT sample using a 2× Fast Advanced master mix and a 20× TaqMan Advanced miRNA assay, at a 1:20 dilution of the cDNA obtained from the miRNA amplification reaction. Subsequently, the miRNAs were amplified and quantified using a TaqMan Low Density MicroRNA Array (TLDA) card and TaqMan Fast Advanced master mix (Applied Biosystems, ThermoFisher Scientific). The TLDA was designed for real-time PCR quantification of 23 miRNAs (19 candidate miRNAs, 3 endogenous controls (let-7a-5p, miR-222-3p, and miR-361-5p), and the exogenous control cel-miR-39-3p) using QuantStudio® 7 flex (Applied Biosystems).Threshold cycle (CT) values ​​were recorded as the number of cycles in which the fluorescence generated within the reaction exceeded the fluorescence threshold (a fluorescence signal significantly higher than the ROX® fluorescence background recorded in each sample). The inventors used Expression Suite software (Applied Biosystems) to express miRNA levels as relative quantitative values ​​(RQ). The CT values ​​of each miRNA were normalized by the average expression level of three endogenous miRNAs (let7a-5p, miR-222-3p, and miR-361-5p) that are universally expressed and not associated with bone and joint tissue metabolism. RQ was calculated as 2-ΔΔCT (where ΔCT = (CT of miRNA - average CT of the three endogenous targets) and ΔΔCT = (ΔCT of miRNA - average ΔCT of miRNA across all samples)), and converted to double change (FC) = log2(2-ΔΔΔCT). Exogenous spike-in cel-miR-39-3p was used as quality control for qPCR. (33) In addition, to control the quality of these processes and ensure the quality of the analysis, the inventors used a 96-well plate (Taqman® advanced miRNA assay) with primers specific to the tested miRNA after each extraction, RT, and pre-amplification. For this purpose, prior to each TLDA analysis, the inventors performed 96-well plate analysis for seven miRNAs. The seven miRNAs included two endogenous control miRNAs, an exogenous control miRNA, and four target miRNAs at different expression levels.

[0052] statistical analysis Controls were matched to patients for age, sex, and BMI. NGS processes and circulating miRNA expression difference analysis were performed using Qiagen with the EdgeR software package. An exact test similar to Fisher's exact test was used for expression difference analysis, but adapted for overvariance data. (34)Each gene was evaluated using [a specific method / tool]. Relative quantitative values ​​for RT-qPCR were calculated using Expression-Suite software. Patient and control characteristics were expressed as mean ± SD for continuous variables and as numbers (%) for qualitative variables. Significant differences in characteristics between the two groups were investigated using the Wilcoxon-Mann-Whitney test. P values ​​were corrected for false detection rate (FDR) using the Benjamin-Hochberg method. In the second stage, with N>30, the Anderson-Darling test, Shapiro test, and compatible distribution curves indicated that the approximation to a normal distribution was correct. Therefore, Student's t-test was used to analyze significant differences in serum miRNA levels between patients and controls. P values ​​were corrected for false detection rate (FDR) using the Benjamin-Hochberg method, and values ​​less than 0.05 were considered significant. Subgroup analysis by molecular species was performed using the non-parametric Mann-Whitney test due to the small sample size. The association between miRNA levels and disease severity was evaluated using the Mann-Whitney rank-sum test for qualitative data. The inventors investigated the correlation between miRNA expression levels and clinical characteristics using Pearson's r for continuous data and Spearman's r for ordinal data. All analysis, graphing, and charting were performed using GraphPad Prism version 8.0.0 for Windows, GraphPad Software, San Diego, California, USA, http: / / www.graphpad.com.

[0053] result Screening stage: Identification of circulating miRNAs with differing expression levels in OI using NGS analysis. Characteristics of the group Table 1 shows the characteristics of 10 OI patients (5 males, 5 females) and 10 controls (5 males, 5 females). There was no significant difference in BMI, but the patients were significantly shorter and lighter than the controls.

[0054] Comparison of circulating miRNA expression levels in OI patients and control subjects using NGS. From a total of 1297 miRNAs quantified in 10 OI patients and 10 control patients, 79 miRNAs were identified by next-generation sequencing (NGS) that showed a significant difference in expression levels between the OI group and the control group, with a Benjamin-Hochberg false detection rate (FDR) adjusted probability of less than 0.05 (Table 2). Of these miRNAs, 19 were downregulated and 60 were overexpressed in the OI group compared to the control group. Of these 79 miRNAs, 27 had FDR-adjusted p-values ​​of 5 × 10⁻⁶. -4 It was less than (Figure 2).

[0055] In the validation stage, the inventors selected 19 miRNAs that exhibited the highest level of statistical significance and the highest expression level (RT-qPCR is technically less sensitive than NGS) (Table 3). The candidate miRNAs for validation are shown in Table 4.

[0056] Verification stage: Differences in circulating miRNA expression levels determined by RT-qPCR Characteristics of the group In the validation phase of the candidate miRNAs, an additional 48 patients with OI and 48 age-, sex-, and BMI-matched controls were included. Of these, 45 had type I OI and 3 had type III OI. The mean age was 43 years. The inventors confirmed that OI patients were lighter in weight and shorter in height compared to controls. This is consistent with the classic morphological characteristics of OI patients. OI patients had a median of 17 fractures in the past, with wrist, elbow, tibia, and vertebral fractures being prominent (approximately 30% prevalence for each site), and 61% had previously received bisphosphonates. Interestingly, according to French government figures... (35)In 2014, 55% of patients had a secondary education level or higher, compared to 34% of the French population as a whole. The characteristics of all patients are shown in Table 5. To better understand the potential epigenetic regulation based on the molecular subtype of OI, the inventors classified OI patients into molecular types I and molecular types III-IV. Patients suspected of having structural collagen deficiencies resulting from mutations causing early stop codons were considered type I OI. Patients suspected of having qualitative collagen deficiencies associated with mutations involving amino acid substitutions were considered type III and type IV OI (12 out of 15 patients included glycine substitutions) (Table 6). For mutations involving splicing sites, the inventors used the translation tool from http: / / www.expasy.org and splicing site prediction algorithms including SpliceSiteFinder, MaxEntScan, NNSPLICE, and GeneSplicer. The results were reviewed by a geneticist (CC) to determine stop codon occurrence, frameshift, exon skipping, or intron translation. Based on these outcomes, splicing site mutations were classified as either quantitative or structural abnormalities (Table 7). (36~40) Please note that one patient was excluded from this analysis because the type of mutation was not recorded.

[0057] Comparison of serum circulating miRNA expression levels in patients and controls during the validation phase. The inventors compared the levels of 19 pre-selected circulating miRNAs in 96 individuals (48 OIs and 48 controls) by performing RT-qPCR on pre-spotted TaqMan Low Density Array (TLDA) cards. Of these 19 miRNAs, the inventors identified 10 that showed significant differences in expression levels between the two groups. Eight of these miRNAs maintained significant differences even after correction using the Benjamin-Hochberg method (miR-106b-3p, miR-191-5p, miR-363-3p, miR-451a, miR-93-3p, miR-93-5p, let-7g-5p, miR-15b-5p) (Figure 3). Only miR-15b-5p showed decreased expression in the OI group compared to healthy controls. All other miRNAs were overexpressed in OIs (Table 8). Considering the comparison of expression levels according to the molecular type of OI, among the 19 selected miRNAs, six (miR-106b-3p, miR-191-5p, miR-363-3p, miR-451a, miR-19b-3p, miR-93-5p) that were already disregulated in the overall OI population remained significantly increased in OI patients with quantitative collagen deficiencies. In the group with structural collagen deficiencies, two miRNAs (miR-191-5p, miR-451a) remained significantly upregulated, and miR-93-5p and miR-93-3p showed a strong trend, although not reaching statistical significance (p=0.06). The expression level of let-7g-5p (p<0.043), which was elevated in the entire population, did not maintain a significant difference in either the quantitative or structural group after population classification (Table 9).

[0058] Clinical stage: Association between miRNA deregulation and phenotype and severity of osteogenesis imperfecta Some phenotypic characteristics are shown in Table 5. The inventors investigated the relationship between validated phenotypic characteristics and circulating miRNA levels in OI patients. Specifically, the inventors investigated whether differences in miRNA expression levels were associated with the number of frequent severe fractures, hearing impairment, hyperflaccidity, practitioner classification of OI severity, Silence classification, and dentinogenesis imperfecta. The inventors found that only the latter was significantly associated with the circulating levels of miR-363-3p and miR-451a (Table 10). The inventors found no significant correlation between validated miRNA expression levels and the number of severe fractures. Adjusting for age in a multiple regression model did not change the results for fractures or hearing impairment. No additional significant correlations were found when molecular subgroups were refined to investigate the relationship between OI phenotype and miRNA deregulation. However, there is a weak correlation between the severity of scoliosis as assessed by X-ray measurement of the Cobb angle and miR-96-5p (r=-0.442, p=0.045) and miR-93-5p (r=-0.457, p=0.037).

[0059] The relationship between miRNA deregulation and the pathophysiological mechanisms of osteogenesis imperfecta. To address questions regarding the biological regulation of pathogenic mutations in OI, the inventors investigated the relationship between differences in miRNA expression levels in OI and changes in molecular mechanisms in this disease. For this purpose, the inventors used the miRwalk3® algorithm to predict potential interactions between given miRNAs and corresponding genes. Classically, it has been reported that miRNAs can destabilize mRNA and partially inhibit translation initiation. (41、42) Therefore, the inventors investigated the interaction between the validated miRNA and COL1A1 or COL1A2. Here, the inventors found an estimated 100% interaction between hsa-miR-106b-3p and COL1A1 and COL1A2, and between hsa-miR-93-3p and COL1A2.

[0060] Consideration In our case-control study of 48 patients with OI, we observed differences in the expression levels of eight circulating miRNAs compared to healthy controls. After classifying the OI population according to collagen mutations, two miRNAs (miR-191 and miR-451a) remained significantly upregulated in the structural group. On the other hand, six miRNAs showed significant differences in expression levels in the quantitative group. These data suggest that miRNA-induced modulation of biological function differs according to the pathological mutation of OI. Two of these miRNAs (miR-106b-3p and miR-93-3p) may be directly involved in the pathophysiology of this disease by inducing further enhancement of collagen deficiency through predicted interactions with COL1A1 and COL1A2. This regulation of collagen levels may have been mediated by miR-106b-3p in OI patients with quantitative collagen deficiency, as the increase in miR-106b-3p in the structural group was not significant. On the other hand, the near-significant increase in miR-93-3p in the structural group suggests that it may play the same role in structural mutations. By further examining the roles of these disregulated miRNAs in OI, we observed that some of them have been consistently reported as regulators of osteogenic genes. This report points to possible changes in pathophysiological pathways induced by OI in osteocytes, bone mineralization, and remodeling. (13、15、20、21) Therefore, the inventors investigated the relationship between the circulating levels of significantly disregulated miRNAs and their phenotypic characteristics in OI patients. Overexpression of miR-363-3p and miR-451a was significantly correlated with the state of dentinogenesis imperfecta, but the inventors were unable to demonstrate further correlations between the expression levels of these miRNAs and other severity criteria (i.e., number of fractures, classification severity). The pathophysiology of OI is currently unknown. Specifically, considering the possibility of extraskeletal symptoms, (43)The phenotypic diversity of OI remains unclear. This suggests the existence of other pathways that regulate gene expression downstream of mutations, such as miRNAs acting as epigenetic regulators. In our research, we further support this hypothesis by reporting not only the possibility of direct interaction with mutant genes but also the possibility of direct interaction with other pathways in bone pathophysiology. Due to somewhat incomplete complementarity and small seed size, each miRNA influences the regulation of many mRNAs with varying efficiencies. Some miRNAs have multiple binding sites within 3'-UTR sequences that strongly downregulate target genes, and each miRNA can interact with hundreds of different 3'-UTR regions. (12) Furthermore, this makes it difficult to saturate the miRNA target site, thus inducing weak to moderate downstream regulation. (44) Therefore, overexpression or inhibition of these miRNAs is likely to be involved in multiple unexplained or unknown pathophysiological pathways in OI, and this discovery represents a new approach to the disease. The inventors conducted a literature review and investigated the interactions of eight experimentally validated miRNAs with target genes already reported to be involved in bone homeostasis. The inventors focused on the miR-25 / 93 / 106 cluster in MCM7 intron 13 located at chromosome 7q.22.1. These three miRNAs were significantly upregulated in the serum of OI patients in NGS analysis, with only miR-93-5p, miR-93-3p, and miR-106b-3p being significantly increased in the larger OI population at the validation stage. Furthermore, miR-93-5p also interacts with Osx and is a well-known regulator of osteoblastogenesis. (45、46) As has already been widely reported, Osx plays an essential role in bone homeostasis by regulating the differentiation and maturation of osteoblasts. (47~49)This interaction may induce inhibition of Osx transcription, and the increase in miR-93 in OI patients may be involved in reduced osteoblast maturation via a maladaptive response in the miR-93 / Osx regulatory loop. The presence of strong regulation of Osx is consistent with literature data suggesting that mutations in the latter cause OI formation in a recessive inheritance pattern. (50) Furthermore, both miR-93-5p and miR-106b-3p are thought to inhibit osteoblast formation by inhibiting the expression of osteogenic protein 2 (BMP2) through repression of bone formation. (51) A study by Zhang et al. demonstrated an interaction between miR-93 and BMP2 in vitro. Stimulation of miR-93 leads to a decrease in BMP-2 expression. BMP-2 plays an essential role through its interaction with Runx-2 and Osterix, and as a member of the TGF-β superfamily, but it is also directly involved in bone homeostasis. (52) Overexpression of miR-93 in the OI cohort can lead to repression of BMP-2 expression, potentially causing inhibition of osteoblast differentiation. miR-106b is known to be an inhibitor of Smad5 and BMP-2, and therefore inhibits bone formation and differentiation. (53) Furthermore, inhibition of miR-106b improves osteoblast formation and bone density in a mouse model of corticosteroid-induced osteoporosis. (54)Furthermore, our data suggest that in the quantitative group, the downregulation of Col1A1 / A2 is further amplified as a result of the discordance of the miR-25 / 93 / 106 cluster, and that the main effect is the upregulation of miR-106b-3p due to direct targeting of Col1A1 / 2. The molecular mechanism of this Col1A1 / 2 regulation will likely be limited to the quantitative group, as the upregulation of miR-106b-3p and miR-93-3p did not reach a significant level in the structural group. However, the increase in miR-93-3p approaching the significance level may suggest that miR-93-3p may have a regulatory role in the structural group, but we lack the power to support this. While no literature suggests a regulatory mechanism for this cluster in OI, we hypothesize the existence of direct or indirect negative feedback by type I collagen to the expression of miR-93 and miR-106b. This feedback would likely be released by silencing of affected alleles that are not present in the structural form. Further in vitro studies are needed to investigate this point. In summary, the regulation of miR-106 and miR-93 gene expression within the miR-106b-25 cluster in OI may affect their expression levels as -5p / -3p matures, which may then differentiate the targeting of osteogenic factors. Release into the bloodstream, reflecting changes within tissues, suggests that miR-106 and miR-93 are non-invasive biomarkers for this disease. We have previously reported that miR-25-3p may act as a negative regulator of osteoblast formation by downregulating the expression of genes involved in osteoblast differentiation, and that targeting RANKL may reduce osteoclast formation. (21)Therefore, the role of miR-25-3p in the regulation of osteocyte-related genes remains under investigation and may be patient-dependent. This explains the lack of significant differences observed during the validation phase. Expression changes in this cluster are involved in multiple regulatory pathways in bone and other tissues. This strengthens the hypothesis that these miRNAs are regulators of this disease and that their activity differs depending on the OI patient group. We also reported that miR-19b-3p is upregulated in patients with quantitative collagen deficiency but not in patients with structural collagen deficiency. Interestingly, the expression of miR-19b-3p, which belongs to the miR-17-92 cluster, is modulated by the PI3K / Akt / mTOR signaling pathway. (55) This upregulation increases the levels of osteocalcin, osteopontin, RunX2, and Col1A1 proteins. (56、57) Negative regulation of phosphate and tension homology (PTEN) gene expression promotes bone formation. miR-19b-3p is upregulated in the serum of patients with osteoporosis accompanied by vertebral fractures and low BMD. (58、59) Recent studies have demonstrated that inhibiting miR-19a / b in osteoporotic rats removes the inhibition of TG interaction factor 1 and reduces RANKL-mediated resorption activity, thereby significantly improving the parathyroid hormone effect on bone formation. (60) Since no significant difference was observed in the structural mutation group, it is possible that this mechanism is not effective and that it may only be triggered by a quantitative decrease in COL1A1. Positive overexpression of miR-191-5p has been reported as a response to mechanical stress acting on osteoblasts through mRNA regulation, which confirms the protein-level interaction of bone morphogenetic proteins (BMP-2 and BPM-4) by the present inventors. (61)miR-191 may reduce the migration of bone marrow mesenchymal stem cells. Under osteogenic culture conditions for these cells, inhibition of miR-191 induces osteogenicity and affects chondrogenesis by directly targeting CCAAT enhancer-binding protein. (62) Furthermore, miR-191 was strongly overexpressed in patients with structural mutations, but the inventors did not find such overexpression in the aforementioned clusters except for miR-93-5p. This suggests that bone formation is inhibited elsewhere in these patients. In the OI population as a whole, overexpression of miR-451a and miR-363-3p was associated with dentinogenesis imperfecta. This suggests that even among patients with quantitative mutations, overexpression of miR-363-3p and miR-451a may regulate signaling pathways in patients with dentinogenesis imperfecta, and that only miR-451a may function in structural dentinogenesis imperfecta. miR-363-3p promotes activation of the PTEN / PI3K / AKT pathway, thereby enhancing osteoclast formation and inhibiting osteoblast formation. (63) miR-451 inhibits BMP-6, thereby inhibiting osteoblast formation. (64) In a mouse model of osteoporosis induced by ovariectomy, inhibiting these miRNAs improves the qualitative and quantitative parameters of bone. Although there are no studies reporting a relationship between these two miRNAs and pulp formation itself, the inventors have found that PTEN It can be inferred that the respective effects on the BMP6 pathway may be causing the disorder of pulp formation. (65~67) The miRNAs let-7g-5p and miR-15b-5p are largely unknown in bone. Some studies suggest that let-7g-5p may be involved in osteoblast differentiation, but the mechanism is still not fully understood, and it has been suggested that it may play a more carcinogenic role in extraosseous bone. (68~71)In miRNome analysis, miR-15b-5p, which was overexpressed in the serum of OI patients, was weakly but significantly downregulated in OI patients at the validation stage (p<0.042), with no difference between the quantitative and structural groups. It is known that miR-15b-5p plays a dual role in carcinogenesis, acting as either an oncogene or a tumor suppressor, depending on its disregulation in various tissues and its numerous gene targets. (72) Although there are a few papers suggesting that it plays a role in promoting or inhibiting osteoblast formation, (72、73) However, it has not been strongly reported as an important regulator in bone homeostasis. On the other hand, miR-15b indirectly targets RUNX2, thereby promoting the differentiation of mesenchymal stem cells and bone progenitor cells into osteoblasts. (73) However, miR-15b indirectly targets USP1, inhibiting osteoblast differentiation and autophagy in mice, thus worsening osteoporosis. (74) The inventors report that there is inconsistency in the response of miR-15b-5p expression in OI serum, which may be due to differences in mutational states. Another interesting feature revealed in this study is the overexpression of miR-191-5p. miR-191-5p has often been reported as an endogenous control and has been used for data normalization. (75、76)Meanwhile, in the miROI study, miR-191-5p was shown to be significantly overexpressed in OI serum during the NGS screening phase. Since NGS is the only technique that allows for the detection of all miRNAs, including those previously unidentified, the normalization procedure expresses each miRNA level relative to the total amount of miRNAs in the sample. This provided the inventors with a basis for re-evaluating the stability of miR-191-5p, at least in OI. In the validation phase, miR-191-5p was found to be significantly overexpressed in OI serum when the raw data was normalized using three other endogenous controls. This was also confirmed when the raw data (CT) of miR-191-5p was normalized using the exogenous spike-in cel-miR-39-3p (data not shown). In the discovery phase 1, NGS showed that three members of the miR-182 / 96 / 183 cluster located on human chromosome 7q32.2 were overexpressed. This suggests a common regulation in this disease: polycistronic clusters contain two or more miRNAs transcribed from adjacent loci. 50% of mammalian miRNAs have loci that cluster near other miRNA loci. (12) These are classically regulated synchronously. After RT-qPCR analysis, only miR-182-5p showed a strong increase, but it did not reach statistical significance. This miR-182 / 96 / 183 cluster interacts with multiple targets, particularly FoxO1, heme oxygenase-1, or Osterix (Osx) (77~81) By stimulating osteoclast formation and inhibiting osteoblast formation, it is involved in the regulation of bone homeostasis and bone-related diseases. (21)However, no difference in expression levels was found in this miRNA cluster during the validation phase analysis. This mirOI study is the first to investigate the association between miRNAs and the severity of osteogenesis imperfecta in a significant number of patients with this rare bone disease. Recruitment was conducted at a bone disease reference center, and data quality was ensured through rigorous selection criteria. In addition, this study was constructed using a rigorous methodology, including analytical screening and validation phases with two different methods for analyzing circulating miRNA levels. This is the first study to report miRNA signatures related to the pathophysiological mechanisms of OI using molecular and clinical approaches. This opens up new avenues for exploring not only the pathophysiology of this disease but also treatment options. Currently, the management of osteogenesis imperfecta is based on surgical management of fractures and deformities, but there is a lack of effective drugs. Bisphosphonates remain the primary drug therapy in children. (82、83) Over 50% of these patients had already received bisphosphonates. However, despite the well-established effects on bone density and reduction of vertebral compression in growing children, it remains difficult to demonstrate true benefit in the management of osteogenesis imperfecta in adults. (84~87) Other approaches, such as anti-sclerostin treatments currently being evaluated in Phase III trials and the development of other approaches targeting TGF-beta, are triggering hope for new treatments. (88) Currently, no approved treatments exist. miRNAs represent an interesting pathway for targeted therapies and are expected to be increasingly explored in various research areas. This is particularly true in oncology, where miRNAs are emerging as tumor suppressors, with several promising clinical trials underway. (89、90) Therefore, it is reasonable to consider this approach in bone diseases, especially since some animal model studies of osteoporosis (miR-106b, miR-19a / b) are considered promising. (54、60) The inventors' results may form the basis for in vitro and in vivo research in this direction.

[0061] In conclusion, our research profiled circulating miRNAs in OI patients and identified eight disorder-associated miRNAs that can be used as potential biomarkers for this disease. These miRNAs are thought to be involved in pathophysiological mechanisms that directly affect COL1A1 and COL1A2 mutant genes or other pathways involved in bone homeostasis, particularly the regulation of osteoblastogenesis. This observation is supported by our cell-based research, which showed that COLA1A and COL1A2 expression is reduced in the presence of miR93 or miR-106b inhibitors (Figures 4A, 4B, and 5A, 5B). Therefore, the identified miRNAs are promising therapeutic targets.

[0062] Example 2 Materials and methods For Western blot analysis, osteoblasts were treated with either Antago-CTRL, Antago-miR-93-3p, Mimic-CTRL, or Mimic-miR-93-3p for 5 days. Proteins were extracted, separated by SDS-PAGE, and subsequently transferred to a PVDF membrane (Bio-Rad). The primary antibody used was rabbit polyclonal anti-type I collagen (Proteintech). Beta-actin was used as a loading control. The membranes were incubated with HRP-conjugated goat anti-rabbit secondary antibody and subsequently detected using an ECL substrate. Quantitative values ​​were normalized to beta-actin using ImageJ (FIJI). For immunofluorescence (IF), MC3T3E1 cells were treated under the same conditions. Cells were fixed, stained with anti-type I collagen rabbit polyclonal antibody (Proteintech), and visualized using CoraLite® Plus 488-goat anti-rabbit secondary antibody. Images were acquired using confocal microscopy (Zeiss 880). Quantification of type I collagen staining was automated across the entire slide using ImageJ and normalized to cell count.

[0063] result Western blotting results showed that treatment with Antago-miR-93-3p significantly increased type I collagen expression in both MC3T3 and primary osteoblasts compared to Antago-CTRL (p=0.040, n=3) (Figures 6A, 6B, 6C, 6D, 6E, 6F). Conversely, treatment with Mimic-miR-93-3p decreased collagen levels compared to Mimic-CTRL, although the difference was not statistically significant (p=0.055, n=3), confirming that miR-93-3p plays a role in collagen regulation. Immunofluorescence imaging supported these findings, showing increased collagen staining in cells treated with Antago-miR-93-3p and decreased staining after Mimic-miR-93-3p treatment. Quantification of fluorescence intensity further supported the Western blot data, revealing significant differences in collagen expression between antago-93-3p and antago-CTRL, and between mimic-93-3p and mimic-CTRL (Figures 7A, 7B, 7C, and 8A, 8B, 8C).

[0064] table: [Table 1]

[0065] [Table 2] TIFF2026534194000007.tif106165

[0066] [Table 3]

[0067] [Table 4]

[0068] [Table 5]

[0069] [Table 6]

[0070] [Table 7]

[0071] [Table 8]

[0072] [Table 9]

[0073] [Table 10] TIFF2026534194000016.tif250165

[0074] References Throughout this application, various references describe the state of the art related to the present invention. The disclosures of these references are incorporated by reference into this disclosure.

[0075] [Table 11] TIFF2026534194000018.tif241165 TIFF2026534194000019.tif241165 TIFF2026534194000020.tif249165 TIFF2026534194000021.tif248165 TIFF2026534194000022.tif249165 TIFF2026534194000023.tif249165 TIFF2026534194000024.tif226165

Claims

1. A method for treating osteogenesis imperfecta (OI) in patients requiring treatment for OI, The treatment involves administering to the patient a therapeutically effective dose of at least one miRNA inhibitor selected from the group consisting of miR-93 inhibitors, miR-106b inhibitors, miR-25 inhibitors, and miR-182 inhibitors. method.

2. The method according to claim 1, wherein the miRNA inhibitor is a nucleic acid or other compound that hybridizes with miR-106b, miR-93, miR-25, or miR-182, or has sequence complementarity with the sequence of miR-106b, miR-93, miR-25, or miR-182.

3. The method according to claim 1 or 2, wherein the miRNA inhibitor is selected from the group consisting of double-stranded RNA (e.g., short-chain or small interfering RNA or "siRNA"), antagonists, antisense nucleic acids, and enzymatic RNA molecules, such as ribozymes.

4. The method according to claim 1 or 2, wherein the miRNA inhibitor is an antisense oligonucleotide.

5. The method according to claim 1 or 2, wherein the miRNA inhibitor is antagonistomil.

6. The method according to any one of claims 1 to 5, wherein a miRNA inhibitor is administered to the patient in combination with one or more bisphosphonates.

7. The method according to any one of claims 1 to 5, wherein a miRNA inhibitor is administered to the patient in combination with one or more sclerostin inhibitors.

8. The method according to any one of claims 1 to 5, wherein a miRNA inhibitor is administered to the patient in combination with one or more TGF-beta inhibitors.

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