WO2019078711A1 - Moyens et méthodes de traitement d'une dégénérescence musculaire - Google Patents

Moyens et méthodes de traitement d'une dégénérescence musculaire Download PDF

Info

Publication number
WO2019078711A1
WO2019078711A1 PCT/NL2018/050678 NL2018050678W WO2019078711A1 WO 2019078711 A1 WO2019078711 A1 WO 2019078711A1 NL 2018050678 W NL2018050678 W NL 2018050678W WO 2019078711 A1 WO2019078711 A1 WO 2019078711A1
Authority
WO
WIPO (PCT)
Prior art keywords
poh1
pabpn1
cell
protein
muscle
Prior art date
Application number
PCT/NL2018/050678
Other languages
English (en)
Inventor
Vered RAZ
Silvère Maria VAN DER MAAREL
Original Assignee
ACADEMISCH ZIEKENHUIS LEIDEN (h.o.d.n.LUMC)
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by ACADEMISCH ZIEKENHUIS LEIDEN (h.o.d.n.LUMC) filed Critical ACADEMISCH ZIEKENHUIS LEIDEN (h.o.d.n.LUMC)
Publication of WO2019078711A1 publication Critical patent/WO2019078711A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6489—Metalloendopeptidases (3.4.24)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/19—Omega peptidases (3.4.19)
    • C12Y304/19012—Ubiquitinyl hydrolase 1 (3.4.19.12)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin

Definitions

  • the invention relates to the field of treating muscle wasting and other neuromuscular diseases (NMDs), such as oculopharyngeal muscular dystrophy (OPMD), in subjects in need thereof, particularly adults.
  • NMDs neuromuscular diseases
  • OPMD oculopharyngeal muscular dystrophy
  • the invention also relates to the use of compounds for the treatment of muscle degeneration/wasting.
  • the invention also relates to methods for upregulating PABPN1 in a cell, in particular a muscle cell, comprising contacting the cell with an agent that inhibits POH1 gene or POH1 protein.
  • the invention also relates to screening methods to identify putative agents to treat muscle degeneration/wasting (via POH1 inhibition).
  • the invention also relates to methods for selecting patients suffering from or likely to suffer from muscle deterioration for treatment with a POH1 inhibitor.
  • the methods and medical uses are particularly suited to the treatment of sarcopenia and adult muscular dystrophies such as OPMD.
  • Proper protein turnover and protein homeostasis are crucial for maintaining cellular integrity. Imbalance of protein homeostasis leading to an increase in protein aggregation is among the cellular signatures of aging (Ben-Zvi et aL Hum Mol Genet. 13: 829-838, 2009; Lindner & Demarez Biochimica et Biophysics Acta - General Subjects. 1790: 980-996, 2009; and David et al. PLoS biology. 8:e 1000450, 2010).
  • Protein aggregation characterizes a large spectrum of late-onset neuromuscular degenerative disorders, such as Alzheimer's disease, Huntington's, Parkinson's disease and OPMD, In these disorders, misfolded proteins accumulate in nuclear inclusions or insoluble inclusions (Bingol & Sheng. Neuron. 69:22-32, 2011). Aging associated muscle wasting, such as sarcopenia, affects mobility, stability and metabolic homeostasis.
  • Muscle wasting is characterized by multiple pathological features including: myofiber atrophy, alterations In myofiber type, thickening of the extracellular matrix (ECM), central nucleation and fatty infiltration, which are regulated by multiple network regulatory pathways (Lopez-Otin et al., Cell 153:1194-1217, 2013).
  • Muscle atrophy referring to a disease in muscle mass together with a net loss of protein homeostasis, is an important characteristic of aging-associated muscle wasting and muscular dystrophies, like OPMD (Lexell, J. Gerontology Ser. A Biol. Sci. Med. Sci. 6:11-16, 1995).
  • PABPN1 poly(A)-binding protein 1
  • PABPN1 is a multifunctional regulator of mRNA processing; PABPN1 regulates poly(A) tail length (Benoit et al. Developmental Cell 9 (4): 511-522, 2005), alternative polyadenylation site (APA) in the 3'-UTR of the transcripts (de Klerk et al., Nucleic acids research 40:9089-9101, 2012; Jenal et al., Cell 149:538-553, 2012), internal APA (Abbassi-Daloii, T. et al., Aging Mech. Dis. 1-8, 2017; Li et al., PLoS Genet 11, el005166, 2015) and mRNA decay (Bergeron et al., Molecular and Cellular Biology 55:2503-2517, 2015).
  • APA alternative polyadenylation site
  • Muscular dystrophy is a group of muscle disorders which result in the weakening and breakdown of skeletal muscles over time. These disorders are caused by mutations in genes involved in making muscle proteins and the mutations are either inherited from one or both parents or they arise in early development. In certain disorders the onset of disease is late in life (e.g. after age 20, sometimes after 40 or 60), and so may be termed age-related MD or adult MD. Despite a germline mutation and ubiquitous expression of the mutant gene, muscle weakness and fibrosis start only during adulthood. Examples of age-related MD include: Oculopharyngeal muscular dystrophy (OPMD), Myotonic dystrophy (MD), Facioscapulohumeral muscular dystrophy (FSHD) and Becker muscular dystrophy (Becker).
  • OPMD Oculopharyngeal muscular dystrophy
  • MD Myotonic dystrophy
  • FSHD Facioscapulohumeral muscular dystrophy
  • Becker Becker muscular dystrophy
  • Muscle mass loss can be a consequence of autoimmune diseases like AchR and MuSK myasthenia gravis, muscle denervation or neuronal dysfunction (inherited [SMA] as acquired [e.g. paraplegia]) or muscle disuse (e.g. cast). (Shieh. Neurol Clin. 31(4):1009-10029, 2013).
  • DM Myotonic dystrophies
  • DM type 1 is caused by expansion of a CTG triplet repeat in DMPK
  • DM type 2 is caused by expansion of a CCTG tetramer repeat in CNBP.
  • the DM mutations lead to expression of dominant-acting RNAs (see Thornton et al.. Current Opinion in Genetics and Development.44:135-140, 2017).
  • Facioscapulohumeral muscular dystrophy is a clinically recognizable and relatively common muscular dystrophy. It is inherited mostly as an autosomal dominant disease or in a minority of cases, in a digenic pattern (see Wang and Tawil. Current
  • OPMD is a late-onset autosomal dominant myopathy characterized by muscle weakness, initially specific skeletal muscles are affected, including the eyelids, pharyngeal muscles and limb muscles (Brais et al., Nat Genet 18:164-167, 1998).
  • the OPMD is a late-onset autosomal dominant myopathy characterized by muscle weakness, initially specific skeletal muscles are affected, including the eyelids, pharyngeal muscles and limb muscles (Brais et al., Nat Genet 18:164-167, 1998).
  • OPMD is a paradigm for accelerated sarcopenia (Raz and Raz, Front Aging Neurosci 6:317, 2014).
  • OPMD is characterised by ptosis, dysphagia and proximal limb muscle weakness that typically appears in the 5 th decade (Brais et al., Current Neurology and Neuroscience Reports. 9:76-82, 2009).
  • PABPN1 Poly A Binding Nuclear 1
  • the expansion mutation in PABPN1 results in an extended polyalanine tract, from 10 alanines to 12-18 alanines, in the N-terminus of the PABPN1 protein (expPABPN1) and is an insoluble form of PABPN1 which forms intranuclear aggregates, resulting in reduced levels of functionally available PABPN1. These aggregates are found in muscles from both OPMD patients and presymptomatic carriers of the PABPN1 mutation, and therefore in affected and nonaffected disease stages.
  • E1 and E2 prepare ubiquitin for conjugation
  • E3 Is the component of the ubiquitination process that brings protein specificity to the system (Stein TP, et al., Curr Opin Clin Nutr Metab Care.9:395-402, 2006).
  • DUBs deubiquitinating enzymes
  • Some specific DUBs are part of the 19S proteasome cup, which is connected to the 20S core catalytic particle of the proteasome, where proteolysis takes place.
  • Ubiquitin-mediated protein degradation is a major mechanism for regulation of essentially every cellular function.
  • the deubiquitinating enzymes (DUBs), which cleave ubiquitin-protein bonds, constitute the largest class of enzyme in the deubiquitin-mediated proteolysis pathway.
  • the DUB POH1 (and its yeast homolog RPN11) is a metalloprotease and is thought to facilitate proteolysis by removing ubiquitin from targeted substrates prior to proteasome entry.
  • PQH1 is also known as PSMD14.
  • the deubiquitinase POH1 is a regulator of muscle cell biology and it regulates PABPN1 levels and that PABPN1 regulates POH1 mRNA.
  • the expression of both proteins is maintained via interplay between both mRNA stability and protein turnover regulatory machineries operating in a feedforward regulatory loop.
  • POH1 affects proteosome activity in muscle cells. Reduced proteasome activity leads to increased protein accumulation. In muscles with reduced PABPN1 levels POH1 levels increase and proteasome activity decreases.
  • the inventor has surprisingly found that inhibition of POH1 leads to PABPN1 upregulation and an
  • a compound which inhibits POH1 is useful jn the treatment of a disease or disorder or condition associated with reduced PABPN1 in muscle cells.
  • a mouse model with muscle-specific reduction of PABPN1 the inventor found histological features of muscle regeneration and atrophy.
  • features of myogenesis were observed in muscle tissue.
  • the causative link between a decrease in POH1 and an increase in PABPN1 opens the opportunity to increase the amount of PABPN1 in a cell in need thereof, and to treat diseases, disorders or conditions mediated by PABPN1, such as muscle degeneration, by targeting POH1.
  • the invention provides a method for increasing the level of PABPN1 protein in a cell, in particular in the nucleus of the cell, said method comprising inhibiting POH1 in said cell.
  • the invention further provides a method for inhibiting a molecular effect of aging in a cell, said method comprising decreasing the level of POH1 protein or activity of POH1 protein in said cell.
  • the invention also provides an isolated oligonucleotide having 12-40 bases, wherein the oligonucleotide comprises a continuous stretch of at least 7 bases that is complementary to and capable of hybridizing to a continuous stretch of at least 7 bases that is complementary to and capable of hybridizing to POH1 mRNA.
  • the invention further provides a compound for increasing the level of PABPN1 protein in a cell for use in the treatment of an individual suffering from muscle
  • the individual is suffering from sarcopenia.
  • Oculopharyngeal muscular dystrophy OPMD
  • Myotonic dystrophy DM
  • Facioscapulohumeral muscular dystrophy FSHD
  • Becker muscular dystrophy Becker
  • Alzheimer's Parkinson's, autoimmune diseases like AchR and MuSK myasthenia gravis, muscle denervation or neuronal dysfunction
  • the individual is suffering from sarcopenia
  • the invention further provides a method for the treatment of an individual suffering from an age-related degenerative disease or condition comprising administering to the individual in need thereof a compound that decrease the level of POH1 protein In a cell or the activity of POH1 protein in the celL
  • the age related degenerative disease is a neurodegenerative disease.
  • the disease is oculopharyngeal muscular dystrophy (OPMD), myotonic dystrophy (DM), facioscapulohumeral muscular dystrophy (FSHD), Becker muscular dystrophy (Becker), Alzheimer's, Parkinson's, autoimmune diseases like AchR and MuSK myasthenia gravis, muscle denervation or neuronal dysfunction.
  • the condition is sarcopenia.
  • the compound is selected from an antibody or an RNAi molecule capable of hybridizing to POH1 mRNA produced by said ceil.
  • a method for increasing the amount of PABPN1 protein in a cell comprising contacting the cell with, an agent that inhibits POH1.
  • the cell is a muscle cell.
  • muscle cells are cardiomyocytes, smooth muscle cells, vascular muscle cells, skeletal muscles or myotubes.
  • the method is for increasing the amount of PABPN1 in the nucleus of the cell.
  • an agent that inhibits POH1 is also referred to as a POH1 inhibitor.
  • the compound or agent is a de-ubiquitination inhibitor.
  • the term "inhibit" refer to reducing or stopping the amount of or activity of the protein, e.g. POH1.
  • an inhibitor of PQH1 may affect the amount of POH1 protein produced by the cell, e.g. by targeting the transcription or translation of the protein in the cell.
  • it may affect the activity of the protein, e.g. by preventing the protein from forming a complex or catalysing a reaction e.g. deubiquitination, phosphorylation etc., such that the aforementioned amount of protein or level pf activity of the protein is less than that observed in the absence of the inhibiting compound (e.g.
  • Inhibition may be reversible or irreversible.
  • a method for increasing myotubule formation in a muscle cell comprising contacting the cell with an agent that inhibits POH1.
  • the method is performed in vitro.
  • the cell is a cell of an animal, preferably of a mammal or a bird.
  • the cell is a primate cell, preferably a human cell.
  • the POH1 inhibitor causes an increase in the amount of PABPN1 protein in the nucleus of the cell.
  • the method is performed in vitro.
  • the agent that inhibits POH1 can be a small molecule compound or a large molecule biologic.
  • the agent that inhibits POH1 is specific for POH1.
  • the agent that inhibits POH1 is a nucleic acid molecule capable of inhibiting mRNA of POH1.
  • the agent that inhibits POH1 is an antibody molecule that binds to POH1 protein.
  • the human and mouse gene and protein sequences for PABPN1 and POH1/psmdl4 sequences are known and available from various gene sequence databases such as Ensembl, GenBank, and UniProt.
  • a representative human gene sequence is found in Ensembl under accession number: NM_0Q4643 (SEQ ID NO: 6).
  • the mouse PABPN1 sequences are available from UniProtKB under identifier Q8CCS6.
  • a representative mouse gene sequence is found in Ensembl under accession number: NM_019402.2 (SEQ ID NO: 9).
  • the human POH1/PSMD14 sequences are available from UniProtKB under identifier: 000487 (SEQ ID NO: 5).
  • a representative human gene sequence is found in Genbank under accession number: NM_005805 (SEQ ID NO:7).
  • the mouse POH1/PSMD14 sequences are available from UniProtKB under identifier 035593.
  • a representative mouse gene sequence is found in Ensemble under accession number NM_021526.2 (SEQ ID NO:8).
  • antibody and antibody molecule includes a derivative of an antibody, such as scFV, HCAbs, Fab and VHH molecules, and the like.
  • the antibody molecule is preferably one that neutralizes the activity of POH1.
  • the antibody molecule is an intracellular antibody.
  • the antibody is a monoclonal antibody.
  • the antibody is an antibody-derived molecule, such as one selected from a Fab, ScFv, HcABs or VHH antibody.
  • the antibody molecule may be provided to the cell by means of a nucleic acid delivery vehicle comprising one or more nudeic acids encoding said antibody molecule. Intracellular antibody molecules thereof may be provided to the cell by means of said gene delivery vehicle.
  • said POH1 inhibitor is an RNA inhibitor.
  • RNA inhibitor is preferably an RNAi molecule specific for POH1; shRNA molecule, specific for POH1 mRNA; or an antisense oligonucleotide (AON) specific for POH1 mRNA.
  • the agent that inhibits POH1 is a nucleic acid or peptide- based aptamer.
  • Antibody and RNAi technologies are suitably advanced that the person skilled in the art would be able to make an antibody or antibody-derived molecule or an RNAi molecule that could inhibit POH1.
  • the agent that inhibits POH1 is selected from the group consisting of small interfering RNAs (siRNAs), nucleic acid aptamers, small molecules, inorganic compounds, peptide aptamers, antibodies, such as Fab, scFV, VHH, natural single domain antibodies, nanpbodies, affibodies, affibody-antibody chimeras, heavy-chain only antibodies (HCAbs) and non-immunoglohulins. Certain of these agents are discussed in Muyldermans (Ann Rev Biochem. 82:775-797, 2013).
  • Functional inhibitor agents can be identified on the basis of their ability to induce a down-regulation or inhibition of gene expression and/or down- regulation or inhibition of the activity of a transcriptional or translational product thereof (i.e. POH1/POH1).
  • the expression is for example reduced or down-regulated to less than 90%, such as less than 80% such as less than 70% for example less than 60%, for example less than 50%, such as less than 40%, such as less than 30% such as less than 20% for example less than 10%, for example less than 5%, such as completely inhibited (0%) relative to the expression or activity in the absence of the agent that inhibits POH1.
  • a method of treating a patient suffering from or at risk of developing muscle degeneration comprising administering to the patient an effective amount of an agent. that inhibits POH1.
  • Muscle degeneration also referred to herein as muscle wasting, muscle atrophy or muscle fibrosis, is characterized by multiple pathological features including: atrophy, alterations in myofiber type, thickening of the extracellular matrix (ECM), central nudeation, split myofibers and fatty infiltration, and is in effect a decrease in the mass and strength of the muscle.
  • Muscular dystrophy is a group of inherited diseases that are characterized by weakness and wasting away of muscle tissue, with or without the breakdown of nerve tissue.
  • a method of treating a patient suffering from or at risk of developing muscle degeneration comprising administering to the patient an effective amount of an agent that indirectly increases the amount of PABPN1 in the cell, wherein the agent is one that inhibits POH1.
  • a method of treating a patient suffering from or at risk of developing muscle degeneration comprising administering to the patient an effective amount of an agent that increases the amount of PABPN1 in the cell, wherein said agent is one that inhibits POH1.
  • a method of treating a patient whose muscle cells have reduced levels of PABPN1 relative to control or normal cells comprising administering to the patient an effective amount of an agent that inhibits POH1.
  • administration of the agent results in an increase in PABPN1 in the nucleus of the cell, in particular in a muscle cell.
  • the muscle degeneration may be due to a muscular dystrophy or sarcopenia.
  • the muscle degeneration may be associated with other neurodegenerative diseases such as Parkinson's and Alzheimer's.
  • the muscle degeneration is age- related, by which is meant arises later in life, such as after childhood.
  • This type of muscle degeneration is termed age-related muscle degeneration and includes muscle degeneration seen in old age (typically after age 70), like sarcopenia, or in muscular dystrophies like OPMD, Becker, DM and FSHD where the muscle degeneration occurs later in life, typically during adulthood.
  • the inventor has found that inhibition of POH1 leads to an increase in the amount of PABPN1, particularly in the nucleus, and an increase in myogenesis in muscle cells. This increase in PABPN1 may be due to reduced breakdown of PABPN1 protein by the proteasome due to there being less or no POH1.
  • a method of reducing the amount of PABPN1 breakdown/degradation in a cell comprising contacting the cell with an agent that inhibits POH1.
  • the myogenesis is increased muscle cell fusion or myotubule formation.
  • PABPN1 protein levels Aberrant PABPN1 protein levels, particularly in the muscle cell nucleus, are known to be involved in muscle degeneration and reduced myogenesis. Thus, therapeutic or prophylactic agents that can regulate PABPN1 protein levels could be useful in the treatment of diseases or disorders or conditions that are mediated by PABPN1.
  • a method of treating a patient suffering from a PABPN1-mediated disease, disorder or condition comprising administering to the patient an effective amount of an agent that inhibits POH1.
  • the PABPN1-mediated disease is a muscular dystrophy, such as one selected from OPMD, Becker, DM and FSHD.
  • the PABPN1-mediated condition is sarcopenia.
  • the POH1 inhibitor reduces the amount of PABPN1 protein in the nucleus of the cell.
  • the agent that inhibits POH1 is a small molecule compound or a large molecule biologic.
  • the agent that inhibits POH1 is specific for POH1.
  • the agent that inhibits POH1 is a nucleic acid molecule capable of inhibiting mRNA of POH1.
  • the agent that inhibits POH1 is an antibody molecule that binds to POH1 protein.
  • the antibody is preferably an antibody that neutralizes the activity of POH1.
  • said antibody is an intracellular antibody.
  • said antibody is a monoclonal antibody.
  • said antibody is an antibody- derived molecule such as a Fab, ScFv or VHH antibody.
  • the antibody may be provided to the cell by means of a nucleic acid delivery vehicle comprising one or more nucleic acids encoding said antibody, or derivative thereof. Intracellular antibodies may be provided to the cell by means of said gene delivery vehicle.
  • said POH1 inhibitor is an RNA inhibitor.
  • RNA inhibitor is preferably an RNAi molecule specific for POH1; shRNA molecule specific for POH1 mRNA; or an antisense oligonucleotide (AON) specific for POH1 mRNA.
  • AON antisense oligonucleotide
  • specific means that the molecule does not significantly binds to any other mRNA. Examples of two RNAi molecules that can inhibit POH1 mRNA are disclosed in
  • RNAi molecule for use in one of the aspects of the present invention consists of or comprises SEQ, ID NO: 2 or SEQ ID NO: 3.
  • antibody refers to a polypeptide or group of polypeptides that are comprised of at least one binding domain that is formed from the folding of polypeptide chains having three-dimensional binding spaces with internal surface shapes and charge distributions complementary to the features of an antigenic determinant of an antigen, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies formed from at least two different epitope binding fragments (e.g., bispecific antibodies), human antibodies, humanized antibodies, camelid antibodies, chimeric antibodies, single-chain Fvs (scFv), single-chain antibodies, single domain antibodies, domain antibodies * VHH, Fab fragments, F(ab')2 fragments, antibody fragments that exhibit the desired biological activity (e.g, the antigen binding portion), disulfide-linked Fvs (dsFv), and anti-idiotypic (anti-Id) antibodies (including, e.
  • monoclonal antibodies including full-length monoclonal antibodies
  • polyclonal antibodies multi
  • monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma (Kohler et al., Nature, 256:495 (1975); Harlow et aL, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, etal, in: Monodonaf Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N,Y., 1981), recombinant, and phage display technologies, or a
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous or isolated antibodies, e.g., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site or multiple antigenic sites in the case of multi-specific engineered antibodies.
  • Nucleic acid oligomeric compounds are another class of therapeutic compounds that can be used to inhibit POH1, by targeting the POH1 gene product, especially POH1 mRNA.
  • Oligomeric compounds that include nucleotide sequences at least partially complementary to a target nucleic acid have been shown to alter the function and activity of the target, both in vitro and in vivo. When delivered to a cell containing a target nucleic acid (such as mRNA), oligomeric compounds have been shown to modulate the expression of the target resulting in altered transcription or translation of the target nucleic acid.
  • the oligomeric compound can reduce the expression of the gene by inhibiting the nucleic acid target and/or triggering the degradation of the target nucleic acid.
  • An example of such nucleic acid oligomeric compounds is the aptamer, a class of high- affinity nucleic acid ligands that specifically bind a desired target molecule (see U.S. Pat. No. 5,475,096).
  • Aptamer technology started in the early 1990s and has advanced to the stage where therapeutics have now been approved (e.g. FDA approved pegaptanib for age- related macular degeneration in 2004). The person skilled in the art would be able to design a nucleic acid or peptide-hased aptamer against PQH1.
  • RNA interference refers to the process of sequence-specific post- transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs) (e.g. Zamore et al.. Cell, 101:25-33, 2000; Fire et al., Nature. 391:806, 1998; Hamilton et al., Science.286:950-951, 1999; Lin et al.. Nature.402:128-129,1999; and Elbashir et al., Nature. 411:494-98, 2000).
  • siRNAs short interfering RNAs
  • the types of synthetic RNA or RNA-like molecules that can trigger the RNAi response mechanism may be comprised of modified nucleotides and/or one or more non-phosphodiester linkages.
  • single-stranded RNA and RNA-like molecules which can also include modified nucleotides and have one or more non-phosphodiester linkages, can also alter the expression of a target nucleic acid, such as a target mRNA.
  • Agents that decrease expression of POH1 include non-enzymatic nucleic acid molecules that binds to target RNA by means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Eghoim et al., Nature. 365:566, 1993) interactions and alters the activity of the target RNA (for a review see Stein and Cheng, Science. 261:1004, 1993) as well as enzymatic nudeic acid agents.
  • RNA-RNA or RNA-DNA or RNA-PNA protein nucleic acid
  • a suitable embodiment of an agent that decreases expression of POH1 may be selected from the group consisting of: agents for use in RNA interference (RNAi), including small interfering RNA (siRNA) molecules that inhibit POM expression; antisense oligonucleotides that inhibit POM expression; and ribozymes that inhihit POM expression.
  • RNAi RNA interference
  • siRNA small interfering RNA
  • Suitable antisense oligonucleotides may be designed with reference to the previously published sequences of nucleic acids, such as mRNA, that encode POH1.
  • Suitable examples of siRNA molecules that may be used as agents that decrease expression of POH1, and which may be employed in the methods or uses of the invention may be commercially available siRNA molecules that inhibit POH1 expression.
  • suitable siRNA molecules include those in Example 5 herein (AON_14.1 and AON_14.2; see Table 1), which comprise sequences disclosed in SEQ ID NO: 2 or 3.
  • An enzymatic nucleic add molecule is one that has complementarity in a substrate binding region to a specified gene target (e.g. POM), and also has an enzymatic activity which is active to spedfically cleave target RNA. Such that the enzymatic nucleic add molecule is able to intermolecularly cleave RNA and thereby inactivate a target RNA molecule.
  • the complementary regions allow sufficient hybridization of the enzymatic nudeic acid molecule to the target RNA and thus permit cleavage.
  • One hundred percent complementarity is preferred, but complementarity as low as 50-75% may also be useful in this invention.
  • the nucleic acids may be modified at the base, sugar, and/or phosphate groups.
  • enzymatic nucleic acid molecule is used interchangeably with phrases such as enzymatic nucleic add, ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, aptazyme or aptamer-binding ribozyme, regulatable ribozyme, catalytic oligonucleotides, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease, endonuclease, minizyme, leadzyme, oligozyme or DNA enzyme. All of these terminologies describe nucleic acid molecules with enzymatic activity.
  • RNAi and other nucleic acid based technologies are suitably advanced that the person skilled in the art would be able to make a molecule that could inhibit pohl.
  • Compounds that inhibit rpn11/POH1 are known.
  • Thioiutin (THL) and other dithiopyrrolones are reported to inhibit rpnll as well as a number of other JAMM
  • the agent that inhibits POH1 is selected from quinoline-8- thiol (STQ) or its derivative capzimin. These compounds are disclosed in Jing U et al., (Nature Chemical Biology. 13:486-496, 2017).
  • age-related muscular dystrophies such as one selected from OPMD, Becker, DM and FSHD arise in later life. Without being bound by theory it is believed that a secondary trigger brings about the onset of the disease. It has been observed that reduced PABPN1 levels are associated with the onset of muscle atrophy and so PABPN1 may be a second trigger.
  • the effect that the decline of the levels of PABPN1 has in a cell can be decreased and/or reversed by inhibiting the level of POH1 mRNA and/or protein in the cell.
  • Decreasing the level of POH1 mRNA and/or protein in a cell has the effect of elevating the level of PABPN1 mRNA and/or protein in the cell, particularly the amount of protein in the nucleus.
  • elevating or increasing the level of PABPN1 is meant a higher level when compared to the same circumstances in the absence of POH1 manipulation. This increase may be attributed to there being a reduced amount of PABPN1 protein removal or breakdown by the prateasame by virtue of the inhibition of POH1.
  • the term also encompasses a stabilization or slower decrease of the level of PABPN1 over time.
  • the level of PABPN1 preferably refers to the level (amount) of PABPN1 in the nucleus.
  • inhibitor is meant that the expression of the gene, or level of RNA or equivalent RNA encoding POH1 protein, or the amount of protein (e.g. POH1), or the activity of the protein, is reduced below that observed in the absence of the agent. Such reduction can be complete (100%), or a lesser amount, such as reduced by 99%, 90%, 75%, 50%, 35%, 25%, 15%, 15%, 5% or less.
  • agents that can inhibit POH1 can result in an increase in the amount of PABPN1 protein in muscle cells and induce myogenesis opens the opportunity to treat patients as well as means for identifying or selecting patients for a suitable treatment.
  • a method for selecting an individual suffering from, or at risk of developing, muscle degeneration for treatment with an agent that inhibits POH1 comprising, determining whether the patient's cells express reduced PABPN1 protein levels relative to a control or normal cell, wherein if PABPN1 protein levels in the individual's cells are reduced relative to control or normal cell the individual is selected for treatment with an agent that inhibits POH1.
  • the patient having selected the patient for treatment with an agent that inhibits POH1 the patient is administered an effective amount of the agent that inhibits POH1.
  • a method for determining whether an individual suffering from, or at risk of developing, muscle degeneration is likely to be responsive to treatment with an agent that inhibits POH1 comprising determining whether the patient's cells express reduced PABPN1 protein levels relative to a control or normal cell, wherein if PABPN1 protein levels are reduced in the cells the patient is likely to be responsive to treatment with an agent that inhibits POH1.
  • the patient is administered an effective amount of the agent that inhibits POH1.
  • the patient is administered an effective amount of the agent that inhibits POH1.
  • prophylactic treatment we mean treating patients before the actual symptoms of muscle deterioration are detected so as to endeavour to prevent or delay the onset in those patients likely to develop the disease, disorder or condition.
  • a method for selecting an individual at risk of developing age-related muscle deterioration for treatment with an agent that inhibits POH1 comprising, determining whether the patient's cells express reduced PABPN1 protein levels relative to a control or normal cell, wherein if PABPN1 protein levels in the individual's cells are reduced relative to control or normal cell the individual is selected for treatment with an agent that inhibits POH1.
  • a patient at risk of developing age-related muscle deterioration includes an individual who carries one or more germ-line mutations making them susceptible to develop muscular dystrophies such as oculopharyngeal muscular dystrophy (OPMD), myotonic dystrophy (DM1),
  • FSHD facioscapulohumeral muscular dystrophy
  • Becker Becker muscular dystrophy
  • the reference level of PABPN1 can, for example, be determined from normal muscles or could be a pre-determined/recognised value. Thus for example, using a simple scale of 0-10, if from a population based assessment normal muscle cells typically exhibit PABPN1 protein levels between 7-10 and those with clinical muscle
  • deterioration/degeneration have levels of 5 and below
  • the physician or health authority could determine that a value of 4 constituted the threshold for determining whether or not a subject should be treated with the POH1 inhibitor, such that if the detected PABPN1 levels in the subject's muscle cell was 0-4 they would be selected for treatment with the POH1 inhibitor.
  • a method of treating a patient with or at risk of developing age-related muscle degeneration comprising determining the amount of PABPN1 protein present in muscle cells of the patient and if this level is lower than a reference level indicative of risk of muscle degeneration selecting the patient for treatment with a POH 1 inhibitor.
  • a method of treating a patient with or at risk of developing age-related muscle degeneration comprising determining the amount of PABPN1 protein present in muscle cells of the patient and if this level is lower than a reference level indicative of risk of age-related muscle degeneration administering to the patient an effective amount of a POH1 inhibitor.
  • the age-related muscle degeneration is sarcopenia.
  • the age-related muscle degeneration is a muscular dystrophy, such as one selected from: OPMD, Becker, DM and FSHD.
  • the patient at risk of age-related muscle degeneration is one that has been identified as possessing a germ line mutation that makes them likely to develop OPMD, Becker, DM or FSHD.
  • a reference level indicative of muscle cells that are likely to degenerate would be determined from population studies; such that for example if a patient had a genetic predisposition to developing an age-related muscular dystrophy, e.g. OPMD, they would have the PABPN1 levels measured at periodic stages in adulthood and if the levels dropped to a level (e.g.
  • the patient would be selected for treatment with the POH1 inhibitor so as to endeavour to delay or prevent the onset of muscle degeneration.
  • muscle wasting, muscle degeneration, muscle atrophy and muscle deterioration are used interchangeably.
  • the amount of PABPN1 in the patient's cells can be determined in a sample of the patient's cells that have been previously Isolated from the patient.
  • the amount of PABPN1 protein in the cells can be determined using suitably labelled molecules capable of specifically binding to PABPN1 protein, such as a labelled antibody or probe.
  • a label can be any molecule that produces or can be induced to produce a signal, including but not limited to fluoresced, radiolabels, enzymes, chemiluminescers or photosensitizers.
  • binding may be detected and/or measured by detecting, for example, by fluorescence or luminescence, radioactivity, enzyme activity or light
  • Suitable labels include, by way of illustration and not limitation, enzymes such as alkaline phosphatase, glucose-6-phosphate dehydrogenase ("G6PDH”) and horseradish peroxidase; dyes; fluoresces, such as fluorescein, rhodamine compounds, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, fluorescamirte, fluorophores such, as lanthanide cryptates and chelates (Perkin Elmer and Cis Blointernational); chemiluminescers such as isofuminol; sensitizers; coenzymes; enzyme substrates; radiolabels including but not limited to 125l, 131l, 35S, 32 P, 14C and 3H and other radiolabels known to the person skilled in the art.
  • enzymes such as alkaline phosphatase, glucose-6-phosphate dehydrogenase (“G6PDH”) and horseradish peroxida
  • an POH1 inhibitor for use as a medicament.
  • an POH1 inhibitor for use in a method of treating a patient.
  • an POH1 inhibitor for use in a method of treating a patient suffering from muscle degeneration.
  • an POH1 inhibitor for use in a method of treating a patient at risk of developing age-related muscle
  • an POH1 inhibitor for use in a method of treating a patient suffering from, or at risk of developing, muscle degeneration by enhancing PABPN1 protein in the muscle cells.
  • a pharmaceutical composition comprising a POH1 inhibitor and at least one pharmaceutically acceptable excipient for use in a method of treating a patient suffering from, or at risk of developing, muscle degeneration.
  • a pharmaceutical composition comprising a POH1 inhibitor and at least one pharmaceutically acceptable excipient for use in a method of treating a patient suffering from muscle, degeneration.
  • a pharmaceutical composition comprising a POH1 inhibitor and at least one pharmaceutically acceptable excipient for use in a method of treating a patient at risk of developing age-related muscle degeneration.
  • a pharmaceutical composition comprising a POH1 inhibitor and at least one pharmaceutically acceptable excipient for use in a method of treating a patient suffering from muscle degeneration by enhancing PABPN1 protein in the muscle cells.
  • a POH1 Inhibitor in the manufacture of a medicament for use in a method of treating a patient suffering from, or at risk of developing, muscle degeneration.
  • an POH1 Inhibitor in the manufacture of a medicament for use in a method of treating a patient suffering from, or at risk of developing, muscle degeneration.
  • POH1 inhibitor in the manufacture of a medicament for use in a method of treating a patient at risk of developing age-related muscle degeneration.
  • an POH1 inhibitor in the manufacture of a medicament for use in a method of treating a patient suffering from muscle degeneration by enhancing PABPN1 protein in the muscle cells.
  • an POH1 inhibitor in the manufacture of a medicament for use in a method of treating a patient at risk of developing muscle degeneration by enhancing PABPN1 protein in the muscle cells.
  • an POH1 Inhibitor in the manufacture of a medicament for use in a method of treating a patient at risk of developing muscle degeneration by inhibiting the breakdown of PABPN1 protein in the muscle cells.
  • the patient at risk of developing muscle degeneration is one that is predicted to develop a muscular dystrophy (MD) selected from: OPMD, Becker, DM and FSHD.
  • MD muscular dystrophy
  • an individual may be predicted to develop a muscular dystrophy (such as OPMD, Becker, DM and FSHD) If they have particular germ line mutations that are associated with the specific MD.
  • the mutations that associate with a particular muscular dystrophy are well known (e.g. see Thornton et al., Current Opinion in Genetics and Development.44:135-140, 2017; Wang and Tawil. Current Neurology and Neuroscience Reports.
  • an POH1 inhibitor for use in a method of treating a patient suffering. from PABPN1-mediated disease, disorder or condition.
  • the PABPN1-mediated disease or disorder is selected from OPMD, Becker, DM and FSHD.
  • the PABPN1-mediated condition is sarcopenia.
  • an POH1 inhibitor for use in a method of increasing myogenesis in a patient in need thereof.
  • compositions suitable for administration typically comprise the compound and a pharmaceutically acceptable carrier.
  • pharmaceutically- acceptable carrier means one or more compatible solid or liquid fillers, diluents or encapsulating substances that are suitable for administration into a human.
  • carrier denotes an organic or Inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • the invention relates to a pharmaceutical composition comprising an inhibitor of POH1, for use in the prevention and/or treatment of muscle degeneration.
  • the muscle degeneration is associated with the presence of reduced levels of PABPN1 protein in the muscle cells.
  • compositions can take the form of solutions, suspensions, emulsion, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained- release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use.
  • compositions of the present invention are administered in pharmaceutically acceptable preparations.
  • Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents or compounds.
  • Administration may be topical, i.e., substance is applied directly where its action is desired, enteral or oral, i.e., substance is given via the digestive tract, parenteral, i.e., substance is given by other routes than the digestive tract such as by injection.
  • the active agent and optionally another therapeutic or prophylactic agent are formulated in accordance with routine procedures as pharmaceutical compositions adapted for intravenous administration to human beings.
  • the active compounds for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the compositions can also include a solubilizing agent.
  • Compositions for intravenous administration can optionally include a local anaesthetic such as lignocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule.
  • the active compound is to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.
  • compositions for oral delivery can be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs, for example.
  • Orally administered compositions can contain one or more optional agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavouring agents such as peppermint, oil of wintergreen, or cherry; colouring agents; and preserving agents, to provide a pharmaceutically palatable preparation.
  • sweetening agents such as fructose, aspartame or saccharin
  • flavouring agents such as peppermint, oil of wintergreen, or cherry
  • colouring agents such as peppermint, oil of wintergreen, or cherry
  • preserving agents to provide a pharmaceutically palatable preparation.
  • a time delay material such as glycerol monostearate or glycerol stearate can also be used.
  • compositions can include standard vehicles such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
  • Compositions for use in accordance with the present invention can be formulated in conventional manner using one or more physiologically acceptable carriers or excipients.
  • the active agent (POH1 inhibitor) and optionally another therapeutic or prophylactic agent and their physiologically acceptable salts and solvates can be formulated into pharmaceutical compositions for administration by inhalation or insufflation (either through the mouth or the nose) or oral, parenteral or mucosal (such as buccal, vaginal, rectal, sublingual) administration.
  • parenteral or mucosal such as buccal, vaginal, rectal, sublingual
  • local or systemic parenteral administration is used.
  • compositions can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).
  • binding agents e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
  • fillers e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate
  • lubricants e.g., magnesium stearate, talc or silica
  • disintegrants e.g., potato starch or sodium
  • Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • the preparations can also contain buffer salts, flavouring, colouring and sweetening agents as appropriate.
  • the pharmaceutical compositions of the invention are for administration in an effective amount.
  • An "effective amount” is the amount of a composition that alone, or together with further doses, produces the desired response.
  • the agent that inhibits P0H1 can be administered as a pharmaceutical composition in which the pharmaceutical composition comprises between 0.1-lmg, 1-10 mg, 10-50mg 50-100mg, 100-500mg, or 500mg to 5 g of the active agent. If the amount of PABPN1 in a patient's cells is to be measured, this can be done on a sample of cells (e.g. tissue biopsy) from the patient. For example, a muscle cell or muscle-cell containing sample obtained or obtainable from the patient/individual.
  • a sample of cells e.g. tissue biopsy
  • the diagnostic/determining methods of the invention can be undertaken using a sample previously taken from the individual or patient. Such, samples may be preserved, by freezing or fixed and embedded in formalin-paraffin or other media. Alternatively, a fresh musde cell containing sample may be obtained and used.
  • a method of screening an agent for its ability to prevent or treat muscle degeneration in which the ability of the agent to inhibit POH1 activity is assessed.
  • the agent is able to inhibit POH1 activity, then this indicates that the agent has potential as a drug to prevent or treat musde degeneration in a patient in need thereof.
  • an in vitro method for determining whether a test compound has potential as an agent to treat muscle degeneration comprising determining the effect that the test compound has on the amount of POH1 protein expressed in a cell that has been contacted with the test compound, wherein if the test compound causes a reduction in the amount of POH1 protein expressed in the contacted cell then the test compound has potential as an agent to treat muscle degeneration.
  • an in vitro method for determining whether a POH1 inhibitor test compound has potential as an agent to treat muscle degeneration comprising determining the effect that the test compound has on the amount of PABPN1 protein expressed in a cell that has been contacted with the test compound, wherein if the test compound causes an increase in PABPN1 protein levels in the contacted cell then the test compound has potential as an agent to treat muscle degeneration.
  • the method determines the amount or level of PABPN1 protein in the nucleus of the cell.
  • an in vitro method for determining whether a test compound has potential as an agent to treat muscle degeneration comprising determining the effect that the test compound has on the amount of POH1 protein and the amount of PABPN1 protein expressed in a cell that has been contacted with the test compound, wherein if the test compound causes a reduction in the amount of POH1 protein levels and an increase in PABPN1 protein levels in the contacted cell then the test compound has potential as an agent to treat muscle degeneration.
  • the method determines the amount or PABPN1 protein in the nucleus of the cell.
  • the invention is also directed at screening methods to identify agents that inhibit POH1. These candidates constitute leads for development of therapeutic agents suitable for treating muscle degeneration by impacting PABPN1 protein levels in the cell.
  • an in vitro method for determining whether a test compound has potential as an agent to treat muscle degeneration comprising determining the effect that the test compound has on the fold change (FC) in amount of PABPN1 protein expressed in a cell that has been contacted with the test compound, wherein if the test compound causes a FC increase to above 1.5, such as >1.5, >1.6, >1.7, >1.8,>1.9, >2.0, >2.1, >2.2, >2.5 or >3.0, in the contacted cell then the test compound has potential as an agent to treat muscle degeneration.
  • FC fold change
  • a suitable screening method could use a standardised ubiquitinated or neddylated protein or peptide substrate for POH1 enzyme.
  • the substrate is tagged with a detectable moiety, such as a fluorescent or radioisotope label, that will enable differentiation between the substrate having the ubiquitin or nedd moiety attached or one that has been cleaved.
  • a detectable moiety such as a fluorescent or radioisotope label
  • an in vitro method for determining whether a test compound has potential as an agent to treat muscle degeneration comprising determining the effect that the test compound has on the enzyme activity of the POH1 protein comprising contacted the test compound with a labelled POH1 substrate, adding POH1 enzyme and determining the rate of enzyme activity of the POH1 wherein if the test compound causes a reduction in the activity of POH1 protein the test compound has potential as an agent to treat muscle degeneration.
  • screening assays can also be employed that determine the effect that the candidate molecule has on the production of POH1 protein.
  • an in vitro method for determining whether a test compound has potential as an agent to treat muscle degeneration comprising determining the effect that the test compound has on the amount of POH1 protein expressed in a cell that has been contacted with the test compound, wherein if the test compound causes a reduction in the amount of POH1 protein expressed in the contacted cell then the test compound has potential as an agent to treat muscle degeneration.
  • An in vitro method for determining whether a POH1 inhibitor test compound has potential as an agent to treat muscle degeneration comprising determining the effect that the test compound has on the amount of PABPN1 protein present in a cell that has been contacted with the test compound, wherein if the test compound causes an increase in PABPN1 protein levels in the contacted cell then the test compound has potential as an agent to treat muscle degeneration.
  • An increase in PABPN1 in the cell(s) can be determined using, for example, a labelled probe or antibody.
  • the method of treating muscle degeneration diseases, disorders or conditions relies on the ability of POH1 to regulate PABPN1 protein.
  • Models of DM1 are disclosed in EMBO Mol Med. 2013 Dec;5(12):1887-900. doi: 10.1002/emmm.201303275 Models of FSHD are disclosed in Krom et al. PLOS Genet 9(4) el003415, 2013.
  • nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art. The invention will be described further by the following non-limiting examples and Figures.
  • M-1 mouse one (high PABPN1 FC)
  • M-2 mouse 2 (low PABPN1) FC
  • a representative western blot shows PABPN1 protein accumulation in scram and shPab muscles. UBE2N was used as loading control. Dot chart shows PABPN1 protein levels after normalization to UBE2n. FC for each muscle pair is depicted above the connecting line.
  • C. Dot chart shows a paired analysis of the mean fraction central nuclei in myofibers. Whiskers represent SD from six image frames (D-E). D.
  • Dot chart show a paired analysis of the mean number of myofibers per image frame. Whiskers represent SD from six image frames.
  • E. Dot plot shows a paired analysis of the fraction of Pax7 positive nuclei. The mouse with the highest PABPN1 FC (M-l) is depicted in the middle line and the mouse with the lowest PABPN1 FC (M-2) is depicted in the upper line. Quantification of the muscle pathology was carried out over the entire muscle section. P-values were calculated with ratio-paired Student's t-test.
  • C Bar chart indicates the percentage of the mRN A and proteins (FC> l 1.5 l ) that are deregulated in similar or opposite direction.
  • Fig 4. DUBs are up-regulated in shPab muscles.
  • M-l mouse one (high PABPN1 FC)
  • M-2 mouse 2 (low PABPN1) FC
  • A. Western blot with anti-ubiquitin antibodies shows accumulation of ubiquitinated proteins in the mouse with the highest PABPN1 fold change. UBE2n shows equal loading.
  • C. Western blots show the OTUB1, USP14, UCH-L1, UCH-L3 and POH1 levels in the mouse with the highest PABPN1 FC. UBE2n shows equal loading.
  • A-C Characterization of down-regulated cell lines in C2C12.
  • A UCH-L1 (UL1),
  • B USP14 (U14) and
  • C POH1 (P14).
  • Bar chart shows mRNA or protein levels in each cell line. mRNA fold change was normalized to Hprt housekeeping genes and Scram (S) cell culture. Averages and standard deviation are from three experiments. Image of a representative Western blot show protein levels and tubulin loading control and fold change was calculated after normalization to tubulin and Scram.
  • D Bar chart shows the proteasomal activity in the down-regulated cell lines. Black tors indicate the ⁇ 1/5 subunits and the grey bars indicate the ⁇ 2 subunits.
  • the subunits activities in the cell lines were normalized to scram. Averages and standard deviation are from three experiments.
  • E. A representative Western blot shows ⁇ 5 and ⁇ 2 expression across the cell lines. Tubulin was used as loading control,
  • F. Representative images show the myotube formation in the DUB- DR cell cultures. The upper row shows the overlay of DAPI and MH20. The lower row shows the segmentation of the overlapping used for quantitation. The scale bar Is 20 um. The adjacent bar chart shows the fusion index.
  • G. A Western blot shows the PABPN1 protein expression in the different shDub cell lines. Tubulin is used as a loading control. The bar chart shows the normalized PABPN 1 fold change. Averages and standard deviation are from four experiments.
  • PABPN1 immunofluorescence in the down-regulated ceil lines.
  • the upper row shows PABPN1 signal (grey).
  • the lower row shows PABPN1 and nuclei counterstaining.
  • Scale bar is 10 um.
  • Bar chart shows LWA300 mean florescence intensity.
  • G Bar chart shows PABPN1 protein levels after normalization to GAPDH and Scram.
  • H Bar chart shows PABPN1 mRNA levels, values were normalized to HPRT and Scram control. Averages and standard variations are from three biological experiments. The Student's Mest was carried out to assess statistical significance, P ⁇ 0J05 is assigned by *.
  • FIG. 8 IU1 and Capzimin have a differential effect in human muscle cells.
  • Human muscle cell cultures were treated with DMSO (mock), Capzimin (CAPZ), a proteasome inhibitor (MG132) or IU1 (an inhibitor of USP14) for 18 hours.
  • C Representative immunofluorescence images of ubiquitin and poly ubiquitin (TUBEs) in muscle cells.
  • DAPI Immunofluorescence with anti-Ubiquitin or polyubiquitin (TUBEs), DAPI is used as a nuclear counter staining.
  • D-E Cell-based analysis of Ubiquitin (D) or poly-ubiquitin (E) florescence signal in the cytoplasm. Foci were measured from over 1500 cells in muscle cells that were treated with increasing inhibitor concentration. Averages and standard deviations are from three experiments.
  • F-G The effect of inhibitor treatment on proteasome activity.
  • Proteasomal activity was measured with LWA300 (Raz V, Raz Y, Paniagua-Soriano G et al: Proteasomal activity-based probes mark protein homeostasis in muscles. J Cachexia Sarcopenia Muscle 2017; 8: 798-807) using activity gels.
  • F A representative LWA300 activity gel.
  • G Quantification of LWA300 in beta2 and betal/5 subunits. Averages and standard deviations are from three experiments. Statistical significance (Students t-test ,p ⁇ 0.05) is depicted with an asterisk *.
  • Figure 9 Capzimin, but not IU1, restores ATP concentration and protein synthesis in muscle cell culture with reduced PABPN1 levels.
  • shPAB muscle cell cultures. Muscle cell cultures were treated with DMSO (mock), Capzimin (CAPZ), or IU1 for 6 hours.
  • MG132 is used as control, and cycloheximide (CHX) treatment as negative control. Per condition >1000 cells were analyzed. Averages and standard deviations are from three experiments. Statistical significance (Students t-test ,p ⁇ 0.05) is depicted with an asterisk *.
  • Figure 10 Capzimin, hut not IU1, reduces PABPN1 aggregation in COS-7 ceil culture with reduced PABPN1 levels.
  • the Alal7-GFP or GFP alone expression plasmids were transfected into stable scramble shRNA (scram) or shRNAto PABPN1 (shPAB) COS-7 cell cultures, prior or after treatments with Capzimin (CAPZ), or IU1 for 3 hours, as detailed in the figure. Transfection was carried out for 18 hours. Mock was left untreated for 21 hours. PABPN1 aggregation in the nucleus was measured after KCI treatment using a cell-based assay. Transfection with GFP alone did not result in nuclear aggregates. Per condition >1000 cells were analyzed. Averages and standard deviations are from three replicated. Each bar represents an independent experiment. Statistical significance (Students t-test ,p ⁇ 0.05) is depicted with an asterisk *.
  • Example 1 Muscle pathology in shPab musdes correlates with Pabpnl levels
  • TA muscle in four male mice of seven weeks old were injected contralateral with AAV9 particle expressing shRNAto Pabpnl (shPab) or scramble shRNA (Scram) and muscles were harvested 5 weeks post injection for ex-vivo analyses.
  • Transduction efficiency was assessed in living mice by GFP expression and muscles.
  • GFP mean fluorescence intensity (MFI) did not differ between Scram and shPab muscles. Every harvested muscle was cross-sectional cryo- sectioned and sections across the entire muscle were collected for RNA, protein, or histology. GFP in cryosections confirmed that there was no difference in GFP MFI between shPab and. scram. This indicated that molecular differences between paired muscles could not cause by differences in transduction efficiency.
  • Pabpnl affects a broad spectrum of muscle histology including regeneration, atrophy and ECM thickening, and that shPab histology severity seems to correlate with Pabpnl fold change in muscles.
  • PABPN1 causes a switch from distal to proximal polyadenylation site (PAS) in the 3'-UTR (de Klerk et al., Nucleic Acids Research 40:9089- 9101, 2012; Jenal et al., Cell 149:538-553, 2012).
  • PAS proximal polyadenylation site
  • FC direction correlation we focused on the molecules with an average FC> l 1.5 l , and found only 67 mRNAs and 254 proteins that were affected by PABPN1.
  • the FC> l 1.5 l cut-off was selected since PABPN1 1.4 FC had a resilient effect on muscle pathology (Fig. 2B). From these 67 transcripts, nearly 50% were up regulated, but from the 254 proteins, 88% had higher expression in shPab muscles (Fig. 2B).
  • Fig. 2C the vast majority of genes (87%) showed a positive FC (Fig. 2C).
  • the fraction of down-regulated genes was only ⁇ 13%.
  • the fraction of the up-regulated genes (positive average FC of the protein and negative average FC of RNA) was 91.5%, and only 9 genes showed a negative protein FC and positive RNA FC (Fig. 2C). Together, this suggests that in shPab muscles protein accumulation surpasses mRNA alterations.
  • Example 3 Proteomic changes in skeletal muscles correlate with Pabpnl levels
  • the GO term with most genes was the mitochondria (one third) and about 8-12% of the genes were mapped to cytoskeleton, ribosome, focal adhesion and sarcomere GO terms.
  • One third of the proteins were not clustered to significant GO terms.
  • DAVID analysis was carried out on the 407 proteins (FC >
  • the UPS, ECM and nucleolus were found in addition to the GO terms that were found with the significant proteins.
  • the mitochondrion was found in the down-regulated cluster, and in the up-regulated cluster the ribosome, cytoskeleton, focal adhesion, ECM, nucleolus, and the UPS were found.
  • the sarcomere was not assigned with a specific dysregulation direction. Up-regulation of the ECM proteins is consistent with ECM thickening in shPab muscles.
  • dysregulation of the UPS could relate to reduced proteasomal activity in shPab muscles (Riaz et al., PLoS Genet 12,
  • Example 4 Deubiquftinating enzymes are predominantly up-regulated in shPab muscles
  • Fig.4A protein ubiquitination using WB and found higher levels ubiquitinated proteins in shPab muscles
  • Fig.4A the highest effect was found in the mouse with highest PABPN1 FC, whereas the smallest difference between shPab and scram was found in the mouse with the lowest PABPN1 FC.
  • DUBs deubiquitinating enzymes
  • UCH-L1, USP14 and POH1 play a role in myogenesis using C2C12 muscle cell lines that stably express shRNA to Uch-Ll, Uspl4 or Pohl in C2C12 muscle cell culture (named here UL1, U14 and P14, respectively).
  • UL1, U14 and P14 C2C12 muscle cell lines that stably express shRNA to Uch-Ll, Uspl4 or Pohl in C2C12 muscle cell culture (named here UL1, U14 and P14, respectively).
  • UL1, U14 and P14 were selected these three genes for functional genomic studies because UCH-L1 and POH1 are regulated by PABPN1, and USP14 plays a regulatory role in the 19S proteasome cap (Lee et al., Nature 532, 398-401, 2016).
  • PAS utilization is altered by PABPN1 expression.
  • A17.1 mouse model which expressed Alanine expanded PABPN1
  • the proximal PAS is utilized, but in control mice the distal PAS is utilized (Fig. 7A).
  • AON antisense oligo nucleotides flanking Proximal PAS in m_Psmdl43'-UTR.
  • AONs were designed using mFOLD online tool and two AONs (AON_14.1 and AON_14.2; Table 1) masking PAS utilization was assayed in transfected shPab muscle cell culture. Scramble AON (sAON) (Table 1) was used as control. PAS utilization was determined in qRT-PCR using proximal or distal primer sets (Fig. 7A). The distal primer set measures the long transcript from distal PAS, and the proximal primer set measures long and short transcripts. A change in the ratio between proximal and distal PCR products indicates a change in PAS utilization. In shPab the ratio decreases indicating proximal PAS utilization (Fig. 7B).
  • the ratio was elevated in shPab transfected with AON.14.2 (named AON_14) indicating masking of proximal PAS and reversion of PAS utilization (Fig. 8B).
  • AON_14 named AON_14
  • Psmdl4 mRNA levels were elevated in shPab cell culture after transection with AON_14 (Fig. 7C).
  • AON_14.2 transfection resulted in a more pronounced affect and further studies were carried out only with AON_14.
  • PSMD14/POH1 protein levels were determined using Western blot. In agreement with reduced mRNA levels, in shPab culture POH1 protein levels were lower compared with Scram, but protein levels were higher after AON_14 transfection (Fig. 7D-E).
  • POH1 is part of the 19S cup, and altered reduced activity is associated with reduced proteasomal activity.
  • PABPN1 protein levels but not mRNA are higher in shPab culture AON_14-transfected (Fig. 7G-H).
  • FIG. 8 shows that CAPZ but not IU1 affects the bulk of protein ubiquitination in human muscle cell culture (Fig. 8A-B). Moreover, CAPZ but not IU1 affects polyubiquitination and accumulation of polyubiquitinated proteins in cytosolic foci (Fig. 8C-E). In contrast, IU1 but not CAPZ affects proteasomal activity of the catalytic beta subunits (Fig. 8F-G). IU1 was shown to enhance proteasomal activity in other cells. These results suggest that in muscle cells PSMD14 and USP14 have a non-redundant function, and PSMD14 affects targets polyubiquitinated proteins to the 19S cup, whereas USP14 is in contact with the catalytic subunits. We show that ATP concentration and translational activity are specifically restored after capzimin treatment in muscle cell cultures with a stable PABPNl-knockdown (Fig. 9).
  • PABPN1 levels are elevated by IU1 treatment (not shown), Overexpressjon of PABPN1 leads to its aggregation. Since PABPN1 and the proteasome co-localize in the aggregates, it is possible that the aggregated PABPN1 block the proteasome leading to reduced activity of the catalytic subunit. In contrast, inhibition of PSMD14, using CAPZ treatment, prevents the entrance of ⁇ 1 to the proteasome and hereby prevents aggregation.
  • Injections were carried out under general anaesthesia of 2% isoflurane (Pharmachemie BV, Haarlem, The Netherlands). Mice were housed in ventilated cages with sterile bedding, water, rodent food and air at DM-III containment level. Mice were imaged for GFP expression at the third week post-injection using the MaestroTM In-vivo fluorescence imaging system (Xenogen product from Caliper Life Sciences, Hopkinton, Massachusetts, USA) as detailed previously (Riaz et al., PLoS Genet 12, el006031, 2016). Harvested TA muscles were immersed immediately in isopentane chilled with liquid nitrogen (30-45 seconds) and stored at -80°C prior to ex-vlvo analyses. Muscles were divided for proteomic, histology and RNA extraction.
  • Muscle cell culture Knockdown in immortalized mouse myoblasts were generated by Lipofectamine* 2000 reagent (ThermoFisher) transfection of shRNA DNA constructs. Stable cells were selected by cell growth in the presence of puromycin Sigma- Aldrich). Per gene two shRNA were analysed, and the clone with the highest DR was selected for functional analysis. Muscle cell fused was carried out in 48 well plate, cells were seeded to ⁇ 90% confluence (100,000 cells per well) and fusion was carried out in 2% horse- serum for 10 days.
  • RNA was isolated from soleus and quadriceps muscles (N 6 for each muscle) using Qjagen miRNeasy Mini kit (Qjagen BV, Venlo, The Netherlands) as described in Riaz et al., (PLoS Genet 12, el006031, 2016). Briefly, RNA was extracted using miRNeasy kit. Following DNAse treatment and phenol-chloroform extraction RNA was participated with ethanol. The quality of bulk RNA including small RNA was determined using bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). cDNA library for mRNAseq was prepared from 500ng RNA using oligo-dT Dynabeads (UfeTech 61002)(Parkhomchuk et al., 2009). The NEB Next generation sequencing kit was used to prepare the samples for strand-specific sequencing without a size selection step, according to the manufacturer instructions with following modifications: the library was treated with USER enzyme (NEB M5505L, New
  • TMM trimmed mean of M values
  • Raw counts were normalized in EdgeR version 3.14.0using TMM, and differential expression was determined after fitting a generalized linear model with two fixed effects, donor (mouse) and type (muscle type), using the glmFit function and using tag-wise dispersion estimates. Differential expression was determined using the paired log-ratio likelihood (LRT) carried out on gene level. P-values were corrected for multiple testing using Benjamini and Hochberg's false discovery rate.
  • LRT log-ratio likelihood
  • RNA analyses were used for protein analysis. 20 mg muscle tissue was homogenised with plastic beads using bead beater (Precellys Stretton Scientific). Proteins were extracted using RIPA buffer (50mM Tris, pH 7.4, 150mM NaCI, 1% SDS, 0.5% sodium deoxycholate, 1% Igepal CA-630 and protease inhibitors cocktail (Sigma-Aldrich), and protein concentration was determined with a BCA protein assay kit (Pierce, Thermofisher Scientific).
  • Mass spectrometry and proteomics analysis Peptides were obtained by a tryptic digestion according to the filter-aided sample preparation (FASP) method as previously described (Wisniewski et al., Nat Meth 6, 359-362, 2009).
  • FASP filter-aided sample preparation
  • 50 ⁇ ig protein was pretreated with DTT (20mM), IAA (1M) and washed with urea (8M) in between.
  • Protein digestion was carried out with 1.7 ug trypsin at 37oC overnight. Trypsin was deactivated with 5% formic acid (FA) and the peptides were then washed with NaCI (0.5M) and H 2 O.
  • FASP filter-aided sample preparation
  • C Carbamidomethylation
  • N deamidation
  • M oxidation
  • FDR false discovery rate
  • NP-40 buffer 50 itiM Tris (pH 7.4), 5 mM MgCI 2 , 250 mM sucrose, 1 mM DTT, 0.5mM EDTA and 0.5% NP-40
  • Active DUBs of 15ug protein lysate were labelled with HAUbBr2 for 1 hourat 37 oC (Borodovsky et al.. Chemistry & Biology 9, 1149-1159, 2002).
  • the labelled DUBs were subsequently blotted and probed with anti-HA tag monoclonal antibody 12CA5, In Vitro analysis ofproteasomal activity
  • Proteasomal activity was determined after cell labelling with LWA300 activity probe as previously described (Raz et al., J Cachexia, Sarcopenia and Muscle. 2017 Jul 3. doi: 10.1002/jcsm.12211).
  • C2C12 cells were incubated with 0.5 ⁇ LWA300 for 40 min at 37'C and proteins were directly extracted in lysis buffer (50 mM Tris-HCI [pH 7.5], 250 mM sucrose, 5 mM MgCl 2 , 1 mM DTT, 0.025% digitonin, Proteins were separated using 12.5% SDS-PA6E.
  • proteasome ⁇ -subunit 2 (PSB2) (1:1000, anti-mouse, sc-58410, Santa Cruz, CA, USA), proteasome ⁇ -subunit 5 (PSB5) (1: 1000; anti-rabbit, #09-278, Millipore, MA, USA), rabbit anti-OTUBl (1:800, 10573-1-ap, Proteintech, MAN, UK), mouse anti- Ubiquitin (1:1000, BML-PW0930-0100, Enzolife, EXE, UK), rabbit anti-SUMO-2/3 (1:1000, 4974, Cell Signaling, MA, USA) rabbit anti-CSRP3 (1:1000; GTX110536, GeneTex, CA. USA); rabbit anti-MURC (1:1000, HPA021021, Atlas antibodies), rabbit anti-UBE2n (1:1000;
  • Cryosections (10 ⁇ m) were made from the middle of TA muscles with a cryostat CM3050S (Leica Microsystems) and pasted on Super Frost Plus glass slides (Menzel-Glaser; Thermofisher Scientific). Cryosections were stained with Gomori-Trichrome staining (Gomori, American journal of clinical pathology 20, 661-664, 1950). GFP distribution in myofibers was directly visualised in non-fixed sections after mounting with Aqua Polymount (Polyscience) containing DAPi. Muscle immunohistochemistry was carried out in non-fixed sections as previously described Riaz et al., (PLoS Genet 12, el006031, 2016). Primary antibodies employed here is: Pax-7 (1:200; DSHB). Sections were mounted with Aqua Polymount (Polyscience) containing DAPI.
  • Fused cells in cell culture were visualized using the MF20 antibody, described in (Anvar et al., Aging (Albany NY) 5, 412-426, 2013).
  • the fraction of nuclei within MF20 structures was determined using co-localization bio-application V4 in the Cellomics software.
  • Nuclear PABPN1 was determined in myoblast cell cultures using anti-PABPN1 antibody. The nucleus was segmented using DAPI staining, and nuclear PABPN1 signal was determined using compartment bio-application V4 in the Cellomics software.
  • Fluorescent and Nomarski interference imaging were carried out with DM5500 (Leica Microsystems). Images were captured with the LAS AF software versions: 2.3.6 for the DM5500. Gomori-Trichome stained images were captured using a LAS software version: 4.5.0 for the DM-LB light microscope. High throughput imaging in cell culture was carried out with Arrayscan VTI HCA, Cellomics (Thermo Scientific). Statistical analyses were carried out in Graphpad Prism version 6.0.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Virology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Plant Pathology (AREA)
  • Toxicology (AREA)
  • Neurology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne l'utilisation d'un agent qui inhibe POH1 en vue de traiter un patient souffrant ou à risque de développement d'une dégénérescence musculaire. L'invention provient de la découverte selon laquelle POH1 régule la protéine PABPN1 et offre ainsi la possibilité de traiter des maladies ou des états pathologiques médiés par des taux de PABPN1 anormaux ou réduits, tels que la dégénérescence musculaire. L'invention concerne en outre des méthodes destinées à sélectionner un patient destiné à être traité par un inhibiteur de POH1.
PCT/NL2018/050678 2017-10-16 2018-10-16 Moyens et méthodes de traitement d'une dégénérescence musculaire WO2019078711A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
NL2019739A NL2019739B1 (en) 2017-10-16 2017-10-16 Means and methods for treating muscle degeneration
NL2019739 2017-10-16

Publications (1)

Publication Number Publication Date
WO2019078711A1 true WO2019078711A1 (fr) 2019-04-25

Family

ID=61187777

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/NL2018/050678 WO2019078711A1 (fr) 2017-10-16 2018-10-16 Moyens et méthodes de traitement d'une dégénérescence musculaire

Country Status (2)

Country Link
NL (1) NL2019739B1 (fr)
WO (1) WO2019078711A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5475096A (en) 1990-06-11 1995-12-12 University Research Corporation Nucleic acid ligands
WO2012158435A1 (fr) 2011-05-17 2012-11-22 Han-Jie Zhou Composition et procédés pour l'inhibition des protéines jamm

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5475096A (en) 1990-06-11 1995-12-12 University Research Corporation Nucleic acid ligands
WO2012158435A1 (fr) 2011-05-17 2012-11-22 Han-Jie Zhou Composition et procédés pour l'inhibition des protéines jamm

Non-Patent Citations (82)

* Cited by examiner, † Cited by third party
Title
A. MALERBA ET AL: "PABPN1 gene therapy for oculopharyngeal muscular dystrophy", NATURE COMMUNICATIONS, vol. 8, 31 March 2017 (2017-03-31), pages 14848, XP055467318, DOI: 10.1038/ncomms14848 *
ABBASSI-DALOII ET AL., NPJ AGING AND MECHANISMS OF DISEASE, vol. 3, 2017, pages 6
ABBASSI-DALOII, T. ET AL., AGING MECH. DIS., 2017, pages 1 - 8
ANN BYRNE ET AL: "Knockdown of human deubiquitinase PSMD14 induces cell cycle arrest and senescence", EXPERIMENTAL CELL RESEARCH, vol. 316, no. 2, 15 January 2010 (2010-01-15), pages 258 - 271, XP055051181, ISSN: 0014-4827, DOI: 10.1016/j.yexcr.2009.08.018 *
ANVAR ET AL., AGING (ALBANY NY, vol. 5, 2013, pages 412 - 426
ANVAR ET AL., AGING., vol. 5, 2013, pages 412
ANVAR ET AL., SKELET MUSCLE, vol. 1, 2011, pages 15
ANVAR ET AL., SKELET MUSCLE, vol. 1, no. 1, 2011, pages 15
ANVAR, S. Y. ET AL., SKELET. MUSCLE, vol. 1, 2011, pages 15
ATTAIX, D. ET AL., BIOCHEM. SOC., vol. 41, 2005, pages 173 - 186
BELENGEANU V ET AL: "Ade novo2.3Mb deletion in 2q24.2q24.3 in a 20-month-old developmentally delayed girl", GENE, ELSEVIER, AMSTERDAM, NL, vol. 539, no. 1, 4 February 2014 (2014-02-04), pages 168 - 172, XP028661410, ISSN: 0378-1119, DOI: 10.1016/J.GENE.2014.01.060 *
BENOIT ET AL., DEVELOPMENTAL CELL, vol. 9, no. 4, 2005, pages 511 - 522
BEN-ZVI ET AL., HUM MOL GENET., vol. 13, 2009, pages 829 - 838
BERGERON ET AL., MOLECULAR AND CELLULAR BIOLOGY, vol. 35, 2015, pages 2503 - 2517
BINGOL & SHENG. NEURON., vol. 69, 2011, pages 22 - 32
BORODOVSKY ET AL., CHEMISTRY & BIOLOGY, vol. 9, 2002, pages 1149 - 1159
BRAIS ET AL., CURRENT NEUROLOGY AND NEUROSCIENCE REPORTS, vol. 9, 2009, pages 76 - 82
BRAIS ET AL., NAT GENET, vol. 18, 1998, pages 164 - 167
DAVID ET AL., PLOS BIOLOGY., vol. 8, 2010, pages e 1000450
DE KLERK ET AL., NUCLEIC ACIDS RESEARCH, vol. 40, 2012, pages 9089 - 9101
EGHOLM ET AL., NATURE, vol. 365, 1993, pages 566
ELBASHIR ET AL., NATURE, vol. 411, 2000, pages 494 - 98
EMBO MOL MED., vol. 5, no. 12, December 2013 (2013-12-01), pages 1887 - 900
FIRE ET AL., NATURE, vol. 391, 1998, pages 806
GOMORI, AMERICAN JOURNAL OF CLINICAL PATHOLOGY, vol. 20, 1950, pages 661 - 664
HALE; MARHAM: "The Harper Collins Dictionary of Biology", 1991, HARPER PERENNIAL
HAMILTON ET AL., SCIENCE, vol. 286, 1999, pages 950 - 951
HAMMERLING ET AL.: "Monoclonal Antibodies and T-Cell Hybridomas", 1981, ELSEVIER, pages: 563 - 681
HARLOW ET AL.: "Antibodies: A Laboratory Manual", 1988, COLD SPRING HARBOR LABORATORY PRESS
HUANG ET AL., NATURE PROTOC., vol. 4, no. 1, 2009, pages 44 - 57
JEANINE A. HARRIGAN ET AL: "Deubiquitylating enzymes and drug discovery: emerging opportunities", NATURE REVIEWS. DRUG DISCOVERY, vol. 17, no. 1, 29 September 2017 (2017-09-29), GB, pages 57 - 78, XP055467597, ISSN: 1474-1776, DOI: 10.1038/nrd.2017.152 *
JENAL ET AL., CELL, vol. 149, 2012, pages 538 - 553
JING LI ET AL., NATURE CHEMICAL BIOLOGY, vol. 13, 2017, pages 486 - 496
KOHLER ET AL., NATURE, vol. 256, 1975, pages 495
KROM ET AL., PLOS GENET, vol. 9, no. 4, 2013, pages el003415
LAUINGER ET AL., NATURE CHEMICAL BIOLOGY, vol. 13, 2017, pages 709 - 714
LEE B-H; LEE MJ; PARK S ET AL.: "Enhancement of Proteasome Activity by a Small-Molecule Inhibitor of Uspl4", NATURE, vol. 467, 2010, pages 179 - 184, XP055467744, DOI: doi:10.1038/nature09299
LEE ET AL., NATURE, vol. 467, 2010, pages 179 - 184
LEE ET AL., NATURE, vol. 532, 2016, pages 398 - 401
LEPPER ET AL., DEVELOPMENT, vol. 138, 2011, pages 3639 - 3646
LEXELL, J., GERONTOLOGY SER. A BIOL. SCI. MED. SCI., vol. 6, 1995, pages 11 - 16
LI ET AL., NAT PROTOC, vol. 8, no. 6, 2013, pages 1155 - 1168
LI ET AL., NATURE CHEMICAL BIOLOGY, vol. 13, 2017, pages 486 - 496
LI ET AL., PLOS GENET, vol. 11, 2015, pages e1005166
LI ET AL., PLOS GENETICS, vol. 11, no. 4, 2015, pages 1 - 28
LI J; YAKUSHI T; PARLATI F ET AL.: "Capzimin is a potent and specific inhibitor of proteasome isopeptidase Rpnll.", NATURE CHEMICAL BIOLOGY, vol. 13, 2017, pages 486 - 493
LIN ET AL., NATURE, vol. 402, 1999, pages 128 - 129
LINDNER; DEMAREZ, BIOCHIMICA ET BIOPHYSICA ACTA - GENERAL SUBJECTS, vol. 1790, 2009, pages 980 - 996
LLORIAN; SMITH, CURR OPIN GENET DEV, vol. 21, 2011, pages 380 - 387
LOPEZ-OTIN ET AL., CELL, vol. 153, 2013, pages 1194 - 1217
MALERBA, A. ET AL., NAT. COMMUN., vol. 8, 2017, pages 1 - 14
MUHAMMAD RIAZ ET AL: "PABPN1-Dependent mRNA Processing Induces Muscle Wasting", PLOS GENETICS, vol. 12, no. 5, 6 May 2016 (2016-05-06), pages e1006031, XP055467448, DOI: 10.1371/journal.pgen.1006031 *
MUYLDERMANS, ANN REV BIOCHEM., vol. 82, 2013, pages 775 - 797
NUCLEIC ACIDS RES., vol. 43, no. D447-D452, 28 January 2015 (2015-01-28)
PEREZ ET AL., J. MEDICINAL CHEMISTRY
PEREZ-RIVEROL ET AL., MOLECULAR & CELLULAR PROTEOMICS : MCP, vol. 15, 2016, pages 305 - 317
PHILIPPE A. BILODEAU ET AL: "The ubiquitin proteasome system in atrophying skeletal muscle: roles and regulation", AMERICAN JOURNAL OF PHYSIOLOGY. CELL PHYSIOLOGY., vol. 311, no. 3, 1 September 2016 (2016-09-01), US, pages C392 - C403, XP055467594, ISSN: 0363-6143, DOI: 10.1152/ajpcell.00125.2016 *
RANG CAO, P. ET AL., INT. J. BIOCHEM. CELL BIOL., vol. 37, 2005, pages 2088 - 2097
RAZ ET AL., AM J PATHOL., vol. 179, no. 4, 2011, pages 1988 - 2000
RAZ ET AL., AM J PATHOL., vol. 30, no. 14, 2014, pages 00016 - 00019
RAZ ET AL., J CACHEXIA, SARCOPENIA AND MUSCLE, 3 July 2017 (2017-07-03)
RAZ ET AL., J. CACHEXIA SARCOPENIA MUSCLE, 2017
RAZ V; RAZ Y; PANIAGUA-SORIANO G ET AL.: "Proteasomal activity-based probes mark protein homeostasis in muscles", J CACHEXIA SARCOPENIA MUSCLE, vol. 8, 2017, pages 798 - 807
RAZ V; STERRENBURG E; ROUTLEDGE S ET AL.: "Nuclear entrapment and extracellular depletion of PCOLCE is associated with muscle degeneration in oculopharyngeal muscular dystrophy", BMC NEUROLOGY, vol. 13, 2013, pages 70, XP021156358, DOI: doi:10.1186/1471-2377-13-70
RAZ; RAZ, FRONT, AGING NEUROSCI, vol. 6, 2014, pages 317
RIAZ ET AL., PLOS GENET, vol. 12, 2016, pages el006031
RIAZ ET AL., PLOS GENET., vol. 12, no. 5, 6 May 2016 (2016-05-06), pages el006031
RIAZ, M. ET AL., PLOS GENET., vol. 12, 2016, pages 1 - 19
SAMBASIVAN ET AL., DEVELOPMENT, vol. 138, 2011, pages 3647 - 3656
SANDRI, M., INT. J. BIOCHEM. CELL BIOL., vol. 45, 2013, pages 2121 - 2129
SHIEH, NEUROL CLIN, vol. 31, no. 4, 2013
SHIEH. NEUROL CLIN., vol. 31, no. 4, 2013, pages 1009 - 10029
SINGLETON; SAINSBURY: "Dictionary of Microbiology and Molecular Biology", JOHN WILEY AND SONS
STEIN TP ET AL., CURR OPIN CLIN NUTR METAB, vol. 9, 2006, pages 395 - 402
STEIN; CHENG, SCIENCE, vol. 261, 1993, pages 1004
THORNTON ET AL., CURRENT OPINION IN GENETICS AND DEVELOPMENT, vol. 44, 2017, pages 135 - 140
TROLLET ET AL., HUM MOL GENET., vol. 19, no. 11, 2010, pages 2191 - 2207
TROLLET ET AL., HUMAN MOLECULAR GENETICS, vol. 19, 2010, pages 2191 - 2207
WANG; TAWIL, CURRENT NEUROLOGY AND NEUROSCIENCE REPORTS, vol. 16, 2016, pages 66
WISNIEWSKI ET AL., NAT METH, vol. 6, 2009, pages 359 - 362
ZAMORE ET AL., CELL, vol. 101, 2000, pages 25 - 33
ZHENG ET AL., FRONTIERS IN AGING NEUROSCIENCE, vol. 8, 2016, pages 303

Also Published As

Publication number Publication date
NL2019739B1 (en) 2019-04-23

Similar Documents

Publication Publication Date Title
US9662314B2 (en) Compounds and methods for the treatment of muscular disease, and related screening methods
US9486521B2 (en) Therapeutic applications targeting SARM1
Zhong et al. Upstream open reading frame with NOTCH2NLC GGC expansion generates polyglycine aggregates and disrupts nucleocytoplasmic transport: implications for polyglycine diseases
Crabtree et al. Over‐expression of an inactive mutant cathepsin D increases endogenous alpha‐synuclein and cathepsin B activity in SH‐SY 5Y cells
US20080003570A1 (en) Translation enhancer elements of genes encoding human Tau protein and human alpha-synuclein protein
EP3655013B1 (fr) Ciblage de la synaptogyrine-3 dans le traitement de tauopathie
KR20100111282A (ko) 당쇄 관련 유전자, 및 그의 이용
WO2013181618A2 (fr) Méthodes pour traiter la maladie d'alzheimer à l'aide d'inhibiteurs d'apoe
CN104520439A (zh) Tdp-43细胞内存在量关联疾患的认定方法
WO2020150290A2 (fr) Procédés et compositions pour restaurer les taux de stmn2
US20230304088A1 (en) Compositions and methods of detection of pre-symptomatic als
US20220026444A1 (en) Methods for diagnosing and/or treating acute or chronic liver, kidney or lung disease
NL2019739B1 (en) Means and methods for treating muscle degeneration
Reynolds et al. Proteomic analysis after status epilepticus identifies UCHL1 as protective against hippocampal injury
WO2016009225A2 (fr) Méthode de prévention ou de traitement d'une maladie liée à l'agrégation des protéines
US11273161B2 (en) Methods of treating autism spectrum disorders
Kostyunina et al. Transcriptomics and proteomics revealed sex differences in human pulmonary microvascular endothelial cells
CN116744791A (zh) 用于抑制神经元细胞中的chmp7表达以治疗神经退行性紊乱的方法
Xie Characterization of Autism Spectrum Disorder-Associated Protein, PTCHD1
Mann The role of RNA in antagonizing aberrant phase transitions of RNA-binding proteins in ALS/FTD
El-Bazzal et al. Imbalance of Neuregulin1-ErbB2/3 signaling underlies altered myelin homeostasis in models of Charcot-Marie-Tooth disease type 4H
Aladesuyi Arogundade Nucleolar Stress and RNA Toxicity in C9ORF72 Amyotrophic Lateral Sclerosis and Frontotemporal Dementia
Logan The Role of the Cajal Body Protein Coilin in MiRNA Biogenesis, NF-κB Activation and Cajal Body Condensation
Arogundade Nucleolar Stress and RNA Toxicity in C9ORF72 Amyotrophic Lateral Sclerosis and Frontotemporal Dementia
Hamze et al. Altered NRG1/ErbB4 signaling and cholesterol metabolism dysregulation are key pathomechanisms in VRK1-related motor neuropathies and motor neuron diseases

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18812350

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18812350

Country of ref document: EP

Kind code of ref document: A1