Evidence of epigenetic landscape shifts in mucopolysaccharidosis IIIB and IVA.

Lysosomal storage diseases (LSDs) are a group of monogenic diseases characterized by mutations in genes coding for proteins associated with the lysosomal function. Despite the monogenic nature, LSDs patients exhibit variable and heterogeneous clinical manifestations, prompting investigations into epigenetic factors underlying this phenotypic diversity. In this study, we focused on the potential role of epigenetic mechanisms in the pathogenesis of mucopolysaccharidosis IIIB (MPS IIIB) and mucopolysaccharidosis IVA (MPS IVA). We analyzed DNA methylation (5mC) and histone modifications (H3K14 acetylation and H3K9 trimethylation) in MPS IIIB and MPS IVA patients' fibroblasts and healthy controls. The findings revealed that global DNA hypomethylation is present in cell lines for both diseases. At the same time, histone acetylation was increased in MPS IIIB and MPS IVA cells in a donor-dependent way, further indicating a shift towards relaxed open chromatin in these MPS. Finally, the constitutive heterochromatin marker, histone H3K9 trimethylation, only showed reduced clustering in MPS IIIB cells, suggesting limited alterations in heterochromatin organization. These findings collectively emphasize the significance of epigenetic mechanisms in modulating the phenotypic variations observed in LSDs. While global DNA hypomethylation could contribute to the MPS pathogenesis, the study also highlights individual-specific epigenetic responses that might contribute to phenotypic heterogeneity. Further research into the specific genes and pathways affected by these epigenetic changes could provide insights into potential therapeutic interventions for these MPS and other LSDs.

Genome Editing Tools for Lysosomal Storage Disorders.

Genome editing has multiple applications in the biomedical field. They can be used to modify genomes at specific locations, being able to either delete, reduce, or even enhance gene transcription and protein expression. Here, we summarize applications of genome editing used in the field of lysosomal disorders. We focus on the development of cell lines for study of disease pathogenesis, drug discovery, and pathogenicity of specific variants. Furthermore, we highlight the main studies that use gene editing as a gene therapy platform for these disorders, both in preclinical and clinical studies. We conclude that gene editing has been able to change quickly the scenario of these disorders, allowing the development of new therapies and improving the knowledge on disease pathogenesis. Should they confirm their hype, the first gene editing-based products for lysosomal disorders could be available in the next years.

CRISPR-Cas9 approach confirms Calcineurin-responsive zinc finger 1 (Crz1) transcription factor as a promising therapeutic target in echinocandin-resistant Candida glabrata.

Invasive fungal infections, which kill more than 1.6 million patients each year worldwide, are difficult to treat due to the limited number of antifungal drugs (azoles, echinocandins, and polyenes) and the emergence of antifungal resistance. The transcription factor Crz1, a key regulator of cellular stress responses and virulence, is an attractive therapeutic target because this protein is absent in human cells. Here, we used a CRISPR-Cas9 approach to generate isogenic crz1Δ strains in two clinical isolates of caspofungin-resistant C. glabrata to analyze the role of this transcription factor in susceptibility to echinocandins, stress tolerance, biofilm formation, and pathogenicity in both non-vertebrate (Galleria mellonella) and vertebrate (mice) models of candidiasis. In these clinical isolates, CRZ1 disruption restores the susceptibility to echinocandins in both in vitro and in vivo models, and affects their oxidative stress response, biofilm formation, cell size, and pathogenicity. These results strongly suggest that Crz1 inhibitors may play an important role in the development of novel therapeutic agents against fungal infections considering the emergence of antifungal resistance and the low number of available antifungal drugs.

Engineering a heterologously expressed fructosyltransferase from Aspergillus oryzae N74 in Komagataella phaffii (Pichia pastoris) for kestose production.

Fructo-oligosaccharides (FOS) are one of the most well-studied and commercialized prebiotics. FOS can be obtained either by controlled hydrolysis of inulin or by sucrose transfructosylation. FOS produced from sucrose are typically classified as short-chain FOS (scFOS), of which the best known are 1-kestotriose (GF2), 1,1-kestotetraose (GF3), and 1,1,1-kestopentaose (GF4), produced by fructosyltransferases (FTases) or ß-fructofuranosidases. In previous work, FOS production was studied using the Aspergillus oryzae N74 strain, its ftase gene was heterologously expressed in Komagataella phaffii (Pichia pastoris), and the enzyme's tertiary structure modeled. More recently, residues that may be involved in protein-substrate interactions were predicted. In this study, the aim was to experimentally validate previous in silico results by independently producing recombinant wild-type A. oryzae N74 FTase and three single-point mutations in Komagataella phaffii (Pichia pastoris). The R163A mutation virtually abolished the transfructosylating activity, indicating a requirement for the positively charged arginine residue in the catalytic domain D. In contrast, transfructosylating activity was improved by introducing the mutations V242E or F254H, with V242E resulting in higher production of GF2 without affecting that of GF3. Interestingly, initial sucrose concentration, reaction temperature and the presence of metal cofactors did not affect the enhanced activity of mutant V242E. Overall, these results shed light on the mechanism of transfructosylation of the FTase from A. oryzae and expand considerations regarding the design of biotechnological processes for specific FOS production.

Molecular characterization of mucopolysaccharidosis type IVA patients in the Andean region of Colombia.

Colombia has a high prevalence of mucopolysaccharidosis (MPS) type IVA. Nevertheless, data regarding the mutation spectrum for MPS IVA in this population have not been completely characterized. Forty-seven families and 53 patients from seven different Colombian regions were tested for MPS IVA mutations. We compared the sequences with the N-acetylgalactosamine-6-sulfatase (GALNS) reference sequence NM_000512.4, and gene variants were reported. Bioinformatics analysis was performed using SWISS-MODEL. The mutant proteins were generated by homology from the wild-type GALNS 4FDJ template obtained from the PDB database, and visualization was performed using Swiss-PDBViewer and UCSF Chimera. The predictive analysis was run using different bioinformatic tools, and the deleterious annotation of genetic variants was performed using a neural network. We found that 79% and 21% of the cohort was homozygous and compound heterozygous, respectively. The most frequent mutation observed was p.Gly301Cys (78.3% of alleles), followed by p.Arg386Cys (10.4% of alleles). A novel mutation (p.Phe72Ile) was described and classified in silico as a pathogenic variant. This study reveals the mutation spectrum of MPS IVA in Colombia. The high prevalence of the p.Gly301Cys mutation suggests a founder effect of this variant in the Colombian population that causes diseases in the Andean region (via migration). These data can facilitate genetic counseling, prenatal diagnosis, and the design of therapeutic interventions.

Use of a neuron-glia genome-scale metabolic reconstruction to model the metabolic consequences of the Arylsulphatase a deficiency through a systems biology approach.

Metachromatic leukodystrophy (MLD) is a human neurodegenerative disorder characterized by progressive damage on the myelin band in the nervous system. MLD is caused by the impaired function of the lysosomal enzyme Arylsulphatase A (ARSA). The physiopathology mechanisms and the biochemical consequences in the brain of ARSA deficiency are not entirely understood. In recent years, the use of genome-scale metabolic (GEM) models has been explored as a tool for the study of the biochemical alterations in MLD. Previously, we modeled the metabolic consequences of different lysosomal storage diseases using single GEMs. In the case of MLD, using a glia GEM, we previously predicted that the metabolism of glycosphingolipids and neurotransmitters was altered. The results also suggested that mitochondrial metabolism and amino acid transport were the main reactions affected. In this study, we extended the modeling of the metabolic consequences of ARSA deficiency through the integration of neuron and glial cell metabolic models. Cell-specific models were generated from Recon2, and these were used to create a neuron-glial bi-cellular model. We propose a workflow for the integration of this type of model and its subsequent study. The results predicted the impairment pathways involved in the transport of amino acids, lipids metabolism, and catabolism of purines and pyrimidines. The use of this neuron-glial GEM metabolic reconstruction allowed to improve the prediction capacity of the metabolic consequences of ARSA deficiency, which might pave the way for the modeling of the biochemical alterations of other inborn errors of metabolism with central nervous system involvement.

Evaluation of HIV-1 derived lentiviral vectors as transductors of Mucopolysaccharidosis type IV a fibroblasts.

Mucopolysaccharidosis type IVA (MPS IVA) is a lysosomal storage disease produced by the deficiency of the N-acetylgalactosamine-6-sulfate sulfatase (GALNS) enzyme, leading to glycosaminoglycans (GAGs) accumulation. Since currently available treatments remain limited and unspecific, novel therapeutic approaches are essential for the disease treatment. In an attempt to reduce treatment limitations, gene therapy rises as a more effective and specific alternative. We present in this study the delivery assessment of GALNS and sulfatase-modifying factor 1 (SUMF1) genes via HIV-1 derived lentiviral vectors into fibroblasts from MPS IVA patients. After transduction, we determined GALNS enzymatic activity, lysosomal mass change, and autophagy pathway impairment. Additionally, we computationally assessed the effect of mutations over the enzyme-substrate interaction and phenotypic effects. The results showed that the co-transduction of MPS IVA fibroblasts with GALNS and SUMF1 cDNAs led to a significant increase in GALNS enzyme activity and a reduction of lysosomal mass. We show that patient-specific differences in cellular response are directly associated with the set of mutations on each patient. Lastly, we present new evidence supporting autophagy impairment in MPS IVA due to the presence and changes in autophagy proteins in treated MPS IVA fibroblasts. Our results offer new evidence that demonstrate the potential of lentiviral vectors as a strategy to correct GALNS deficiency.

Human recombinant lysosomal ß-Hexosaminidases produced in Pichia pastoris efficiently reduced lipid accumulation in Tay-Sachs fibroblasts.

GM2 gangliosidosis, Tay-Sachs and Sandhoff diseases, are lysosomal storage disorders characterized by the lysosomal accumulation of GM2 gangliosides. This accumulation is due to deficiency in the activity of the ß-hexosaminidases Hex-A or Hex-B, which are dimeric hydrolases formed by αß or ßß subunits, respectively. These disorders show similar clinical manifestations that range from mild systemic symptoms to neurological damage and premature death. There is still no effective therapy for GM2 gangliosidoses, but some therapeutic alternatives, as enzyme replacement therapy, have being evaluated. Previously, we reported the production of active human recombinant ß-hexosaminidases (rhHex-A and rhHex-B) in the methylotrophic yeast Pichia pastoris. In this study, we evaluated in vitro the cellular uptake, intracellular delivery to lysosome, and reduction of stored substrates. Both enzymes were taken-up via endocytic pathway mediated by mannose and mannose-6-phosphate receptors and delivered to lysosomes. Noteworthy, rhHex-A diminished the levels of stored lipids and lysosome mass in fibroblasts from Tay-Sachs patients. Overall, these results confirm the potential of P. pastoris as host to produce recombinant ß-hexosaminidases intended to be used in the treatment of GM2 gangliosidosis.

Bone-Specific Drug Delivery for Osteoporosis and Rare Skeletal Disorders.

PURPOSE OF REVIEW: The skeletal system provides an important role to support body structure and protect organs. The complexity of its architecture and components makes it challenging to deliver the right amount of the drug into bone regions, particularly avascular cartilage lesions. In this review, we describe the recent advance of bone-targeting methods using bisphosphonates, polymeric oligopeptides, and nanoparticles on osteoporosis and rare skeletal diseases. RECENT FINDINGS: Hydroxyapatite (HA), a calcium phosphate with the formula Ca10(PO4)6(OH)2, is a primary matrix of bone mineral that includes a high concentration of positively charged calcium ion and is found only in the bone. This unique feature makes HA a general targeting moiety to the entire skeletal system. We have applied bone-targeting strategy using acidic amino acid oligopeptides into lysosomal enzymes, demonstrating the effects of bone-targeting enzyme replacement therapy and gene therapy on bone and cartilage lesions in inherited skeletal disorders. Virus or no-virus gene therapy using techniques of engineered capsid or nanomedicine has been studied preclinically for skeletal diseases. Efficient drug delivery into bone lesions remains an unmet challenge in clinical practice. Bone-targeting therapies based on gene transfer can be potential as new candidates for skeletal diseases.

Bromocriptine as a Novel Pharmacological Chaperone for Mucopolysaccharidosis IV A.

Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disease caused by mutations in the gene encoding for the enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS), leading to lysosomal accumulation of keratan sulfate (KS) and chondroitin-6-sulfate. In this study, we identified and characterized bromocriptine (BC) as a novel PC for MPS IVA. BC was identified through virtual screening and predicted to be docked within the active cavity of GALNS in a similar conformation to that observed for KS. BC interacted with similar residues to those predicted for natural GALNS substrates. In vitro inhibitory assay showed that BC at 50 µM reduced GALNS activity up to 30%. However, the activity of hrGALNS produced in HEK293 cells was increased up to 1.48-fold. BC increased GALNS activity and reduced lysosomal mass in MPS IVA fibroblasts in a mutation-dependent manner. Overall, these results show the potential of BC as a novel PC for MPS IVA and contribute to the consolidation of PCs as a potential therapy for this disease.

A perspective on research, diagnosis, and management of lysosomal storage disorders in Colombia.

Lysosomal storage diseases (LSDs) are a group of about 50 inborn errors of metabolism characterized by the lysosomal accumulation of partially or non-degraded molecules due to mutations in proteins involved in the degradation of macromolecules, transport, lysosomal biogenesis or modulators of lysosomal environment. Significant advances have been achieved in the diagnosis, management, and treatment of LSDs patients. In terms of approved therapies, these include enzyme replacement therapy (ERT), substrate reduction therapy, hematopoietic stem cell transplantation, and pharmacological chaperone therapy. In this review, we summarize the Colombian experience in LSDs thorough the evidence published. We identified 113 articles published between 1995 and 2019 that included Colombian researchers or physicians, and which were mainly focused in Mucopolysaccharidoses, Pompe disease, Gaucher disease, Fabry disease, and Tay-Sachs and Sandhoff diseases. Most of these articles focused on basic research, clinical cases, and mutation reports. Noteworthy, implementation of the enzyme assay in dried blood samples, led to a 5-fold increase in the identification of LSD patients, suggesting that these disorders still remain undiagnosed in the country. We consider that the information presented in this review will contribute to the knowledge of a broad spectrum of LSDs in Colombia and will also contribute to the development of public policies and the identification of research opportunities.

Design and applications of gene therapy vectors for mucopolysaccharidosis in Colombia.

The authors briefly describe their work in the construction of viral derived vectors for the use in gene therapy of muchopolysaccharide storage diseases (MPS), especially in Morquio A syndrome. The motivations to undertake that line of research about twenty years ago was the belief that gene therapy was the most plausible treatment for monogenic diseases due to the transient effect and its difficulty to reach bone tissue of the only effective treatment in use, the enzyme replacement therapy. The strategy used to increase the bone targeting was to include in the vectors an aspartic acid octapeptide that increases their affinity for the oppositely charged hydroxyapatite molecule of bone. It is also discussed the difficulties to do front line research in many developing countries, due to the extended belief that their research money should be mainly devoted to projects that render solutions in a very short time. However, the authors argue in favor of doing research in gene therapy, because it is proving to be the solution for many monogenic diseases, and therefore there is a need of people with good command of GT all over the world, in order to make good use of that therapy especially for ex-vivo treatments.

Media improvement for 10 L bioreactor production of rPOXA 1B laccase by P. pastoris.

In this work, we statistically improved culture media for rPOXA 1B laccase production, expressed in Pichia pastoris containing pGAPZαA-LaccPost-Stop construct and assayed at 10 L bioreactor production scale (6 L effective work volume). The concentrated enzyme was evaluated for temperature and pH stability and kinetic parameter, characterized by monitoring oxidation of different ABTS [2, 20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] substrate concentrations. Plackett-Burman experimental design (PBED) implementation improved previous work results by 3.05-fold, obtaining a laccase activity of 1373.72 ± 0.37 U L-1 at 168 h of culture in a 500 mL shake flask. In contrast, one factor experimental design (OFED) applied after PBED improved by threefold the previous study, additionally increasing the C/N ratio. Employing OFED media at 10 L bioreactor scale was capable of producing 3159.93 ± 498.90 U L-1 at 192 h, representing a 2.4-fold increase. rPOXA 1B concentrate remained stable between 10 and 50 °C and retained over 70% residual enzymatic activity at 60 °C and 50% at 70 °C. Concerning pH stability, the enzyme was stable at pH 4.0 ± 0.2 with a residual activity greater than 90%. The lowest residual activity (60%) was obtained at pH 10.0 ± 0.2. Furthermore, the apparent kinetic parameters were V max of 3.163 × 10-2 mM min-1 and K m of 1.716 mM. Collectively, regarding enzyme stability our data provide possibilities for applications involving a wide range of pH and temperatures.

Identification of the iduronate-2-sulfatase proteome in wild-type mouse brain.

Iduronate-2-sulfatase (IDS) is a lysosomal enzyme involved in the metabolism of the glycosaminoglycans heparan (HS) and dermatan (DS) sulfate. Mutations on IDS gene produce mucopolysaccharidosis II (MPS II), characterized by the lysosomal accumulation of HS and DS, leading to severe damage of the central nervous system (CNS) and other tissues. In this study, we used a neurochemistry and proteomic approaches to identify the brain distribution of IDS and its interacting proteins on wild-type mouse brain. IDS immunoreactivity showed a robust staining throughout the entire brain, suggesting an intracellular reactivity in nerve cells and astrocytes. By using affinity purification and mass spectrometry we identified 187 putative IDS partners-proteins, mainly hydrolases, cytoskeletal proteins, transporters, transferases, oxidoreductases, nucleic acid binding proteins, membrane traffic proteins, chaperons and enzyme modulators, among others. The interactions with some of these proteins were predicted by using bioinformatics tools and confirmed by co-immunoprecipitation analysis and Blue Native PAGE. In addition, we identified cytosolic IDS-complexes containing proteins from predicted highly connected nodes (hubs), with molecular functions including catalytic activity, redox balance, binding, transport, receptor activity and structural molecule activity. The proteins identified in this study would provide new insights about IDS physiological role into the CNS and its potential role in the brain-specific protein networks.

Identification of Ezetimibe and Pranlukast as Pharmacological Chaperones for the Treatment of the Rare Disease Mucopolysaccharidosis Type IVA.

Mucopolysaccharidosis type IVA (MPS IVA) is a rare disease caused by mutations in the gene encoding the lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS). We report here two GALNS pharmacological chaperones, ezetimibe and pranlukast, identified by molecular docking-based virtual screening. These compounds bound to the active cavity of GALNS and increased its thermal stability as well as the production of recombinant GALNS in bacteria, yeast, and HEK293 cells. MPS IVA fibroblasts treated with these chaperones exhibited increases in GALNS protein and enzyme activity and reduced the size of enlarged lysosomes. Abnormalities in autophagy markers p62 and LC3B-II were alleviated by ezetimibe and pranlukast. Combined treatment of recombinant GALNS with ezetimibe or pranlukast produced an additive effect. Altogether, the results demonstrate that ezetimibe and pranlukast can increase the yield of recombinant GALNS and be used as a monotherapy or combination therapy to improve the therapeutic efficacy of MPS IVA enzyme replacement therapy.

Therapeutic Options for Mucopolysaccharidoses: Current and Emerging Treatments.

Mucopolysaccharidoses (MPS) are inborn errors of metabolism produced by a deficiency of one of the enzymes involved in the degradation of glycosaminoglycans (GAGs). Although taken separately, each type is rare. As a group, MPS are relatively frequent, with an overall estimated incidence of around 1 in 20,000-25,000 births. Development of therapeutic options for MPS, including hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT), has modified the natural history of many MPS types. In spite of the improvement in some tissues and organs, significant challenges remain unsolved, including blood-brain barrier (BBB) penetration and treatment of lesions in avascular cartilage, heart valves, and corneas. Newer approaches, such as intrathecal ERT, ERT with fusion proteins to cross the BBB, gene therapy, substrate reduction therapy (SRT), chaperone therapy, and some combination of these strategies may provide better outcomes for MPS patients in the near future. As early diagnosis and early treatment are imperative to improve therapeutic efficacy, the inclusion of MPS in newborn screening programs should enhance the potential impact of treatment in reducing the morbidity associated with MPS diseases. In this review, we evaluate available treatments, including ERT and HSCT, and future treatments, such as gene therapy, SRT, and chaperone therapy, and describe the advantages and disadvantages. We also assess the current clinical endpoints and biomarkers used in clinical trials.

Characterization of Human Recombinant N-Acetylgalactosamine-6-Sulfate Sulfatase Produced in Pichia pastoris as Potential Enzyme for Mucopolysaccharidosis IVA Treatment.

Mucopolysaccharidosis IVA (MPS IVA or Morquio A syndrome) is a lysosomal storage disease caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), leading to lysosomal storage of keratan sulfate and chondroitin-6-sulfate. Currently, enzyme replacement therapy using an enzyme produced in CHO cells represents the main treatment option for MPS IVA patients. As an alternative, we reported the production of an active GALNS enzyme produced in the yeast Pichia pastoris (prGALNS), which showed internalization by cultured cells through a potential receptor-mediated process and similar post-translational processing as human enzyme. In this study, we further studied the therapeutic potential of prGALNS through the characterization of the N-glycosylation structure, in vitro cell uptake and keratan sulfate reduction, and in vivo biodistribution and generation of anti-prGALNS antibodies. Taken together, these results represent an important step in the development of a P. pastoris-based platform for production of a therapeutic GALNS for MPS IVA enzyme replacement therapy.

Generation of an induced pluripotent stem cell line (TRNDi005-A) from a Mucopolysaccharidosis Type IVA (MPS IVA) patient carrying compound heterozygous p.R61W and p.WT405del mutations in the GALNS gene.

Mucopolysaccharidosis type IVA (MPS IVA) is a rare genetic disease caused by mutations in the GALNS gene and is inherited in an autosomal recessive manner. GALNS encodes N-acetylgalactosamine-6-sulfatase that breaks down certain complex carbohydrates known as glycosaminoglycans (GAGs). Deficiency in this enzyme causes accumulation of GAGs in lysosomes of body tissues. A human induced pluripotent stem cell (iPSC) line was generated from dermal fibroblasts of a MPS IVA patient that has compound heterozygous mutations (p.R61W and p.WT405del) in the GALNS gene. This iPSC line offers a useful resource to study the disease pathophysiology and a cell-based model for drug development.

Advances in the Development of Pharmacological Chaperones for the Mucopolysaccharidoses.

The mucopolysaccharidoses (MPS) are a group of 11 lysosomal storage diseases (LSDs) produced by mutations in the enzymes involved in the lysosomal catabolism of glycosaminoglycans. Most of the mutations affecting these enzymes may lead to changes in processing, folding, glycosylation, pH stability, protein aggregation, and defective transport to the lysosomes. It this sense, it has been proposed that the use of small molecules, called pharmacological chaperones (PCs), can restore the folding, trafficking, and biological activity of mutated enzymes. PCs have the advantages of wide tissue distribution, potential oral administration, lower production cost, and fewer issues of immunogenicity than enzyme replacement therapy. In this paper, we will review the advances in the identification and characterization of PCs for the MPS. These molecules have been described for MPS II, IVA, and IVB, showing a mutation-dependent enhancement of the mutated enzymes. Although the results show the potential of this strategy, further studies should focus in the development of disease-specific cellular models that allow a proper screening and evaluation of PCs. In addition, in vivo evaluation, both pre-clinical and clinical, should be performed, before they can become a real therapeutic strategy for the treatment of MPS patients.

Tailoring the AAV2 capsid vector for bone-targeting.

BACKGROUND: Targeting specific tissues remains a major challenge to the promise of gene therapy. For example, several strategies have failed to target adeno-associated virus 2 (AAV2) vectors, to bone. We have evaluated in vitro and in vivo the affinity of an AAV2 vector to bone matrix, hydroxyapatite (HA) to treat Mucopolysacccharidosis IVA. METHODS: To increase vector affinity to HA, an aspartic acid octapeptide (D8) was inserted immediately after the N-terminal region of the VP2 capsid protein. The modified vector had physical titers and transduction efficiencies comparable to the unmodified vector. RESULTS: The bone-targeting vector had significantly higher HA affinity and vector genome copies in bone than the unmodified vector. The modified vector was also released from HA, and its enzyme activity in bone, 3 months post infusion, was 4.7-fold higher than the unmodified vector. CONCLUSION: Inserting a bone-targeting peptide into the vector capsid increases gene delivery and expression in the bone without decreasing enzyme expression. This approach could be a novel strategy to treat systemic bone diseases.