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WO2014077693A1 - Moyens et procédés pour réduire un effet du vieillissement dans une cellule de mammifère - Google Patents

Moyens et procédés pour réduire un effet du vieillissement dans une cellule de mammifère Download PDF

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Publication number
WO2014077693A1
WO2014077693A1 PCT/NL2013/050827 NL2013050827W WO2014077693A1 WO 2014077693 A1 WO2014077693 A1 WO 2014077693A1 NL 2013050827 W NL2013050827 W NL 2013050827W WO 2014077693 A1 WO2014077693 A1 WO 2014077693A1
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arih2
cell
pabpnl
protein
mrna
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Vered RAZ
Silvère Maria VAN DER MAAREL
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Leids Universitair Medisch Centrum LUMC
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Leids Universitair Medisch Centrum LUMC
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    • 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
    • C12N9/93—Ligases (6)
    • 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
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y603/00—Ligases forming carbon-nitrogen bonds (6.3)
    • C12Y603/02—Acid—amino-acid ligases (peptide synthases)(6.3.2)
    • C12Y603/02019—Ubiquitin-protein ligase (6.3.2.19), i.e. ubiquitin-conjugating enzyme

Definitions

  • the invention relates to the field of aging.
  • the invention in particular relates to means and methods for preventing or delaying the reduced vitality or increased degeneration of cells during aging.
  • Protein aggregation characterizes a large spectrum of late-onset neuromuscular degenerative disorders, such as Alzheimer's disease, Huntington's and Parkinson's disease. In these disorders misfolded proteins accumulate in insoluble inclusions or nuclear inclusions (reviewed in (Bingol & Sheng 2011)). It is unclear whether nuclear inclusions or oligomers that precede inclusion formation are pathogenic and harmful to the cell (Shao & Diamond 2007).
  • An alternative explanation may be that irreversible protein entrapment in insoluble bodies depletes the cell from soluble and functional protein (Junghans 2009; de Mezer et al. 2011; Raz et al. 2011b).
  • WT and exp PABPNl are used to discriminate between pathogenic and non-pathogenic age-associated protein-aggregation.
  • both WT- and exp PABPNl form indistinguishable insoluble inclusions, but the aggregation process of WT- and exp PABPNl significantly differs (Raz et al. 2011a).
  • a slower protein turnover of expPABPNl is caused by reduced poly-ubiquitination (Raz et al. 2011b).
  • expPABPNl is associated with down-regulation of proteasome encoding genes (Anvar et al. 2011a; Anvar et al. 2011b; Raz et al. 2011b).
  • the ubiquitin proteasome system UPS
  • PABPNl nuclear inclusions Cbeil-Girard et al. 2005; Tavanez et al. 2005; Anvar et al. 2011a.
  • E3-ligases Being the substrate-specific component of the UPS, the expression of E3-ligases is also significantly deregulated in OPMD, and E3-ligases are also entrapped in PABPNl nuclear inclusions (Anvar et al. 2011a). Protein entrapment in PABPNl aggregates is gradual (Raz et al., 2011a).
  • Erisernbi:ENSG00000177479) co-localizes with PABPNl at an early step of the aggregation process, and regulates PABPNl turnover with a preference for the WT protein.
  • Down-regulation of ARIH2 induces a decrease in PABPNl expression.
  • a decrease in PABPNl for instance by mRNA down-regulation or by entrapment of soluble PABPNl in aggregates, causes a change in ARIH2 3'- untranslated region (UTR) length through a proximal instead of a distal
  • ARIH2 is a regulator of PABPNl expression, and that PABPNl regulates ARIH2 3'-UTR length and expression levels in a feed forward loop.
  • the invention provides a method for increasing the level of PABPN1 mRNA and/or protein in a cell, said method comprising increasing the level of ARIH2 protein in said cell.
  • the invention also provides a method for modifying poly-adenylation site usage in a cell comprising modifying the level of ARIH2 protein in said cell.
  • the invention further provides a method for inhibiting a molecular effect of aging in an adult cell, said method comprising increasing the level of ARIH2 protein in said cell.
  • PASMD 14 SLC1A4; E2F1; BMI1; RBI; TP63; SLC2A4; ING1; MIB1; PTEN ;
  • RRM2B CD55; HDAC4; KSR1; CD59; HMGB1; GCLC; PSMD14; CADM1; TFRC;
  • the gene symbols as used herein are the official gene symbols for the genes at the time of filing of the application.
  • the genes exhibit significant changes in expression upon aging and upon disease progression in the OPMD model. The changes are correlated to alternate poly-adenylation site (PAS) usage. With the method it is possible reduce at least the effect of aging on the altered gene expression of the targeted gene.
  • PAS poly-adenylation site
  • the invention further provides a method for modifying PAS usage in pre-mRNAs expressed by a cell, comprising providing said cell with an anti-sense oligonucleotide that is complementary to and capable of hybridizing to a PAS of a pre-mRNA encoded by the gene ARIH2; HILFA; EGFR; SUMOl; PASMD14;
  • VEGFA VEGFA ; MORF4L1; DNM1L; SOD2; RAC1; ITGB1; HIF1A; SRF; MCL1; RAD17;
  • GSK3B RYR1; MEF2A; GRB2; ZMYND11; DNMT3A; CDK7; DIABLO; JARID2; EIF4E; UBE3A; BECNl; KHDRBS3; NOLCl; LPL; NF2; ROCK2; STAT5A; ITSNl;
  • PSMA2 PSMA2 ; PLCB4; LAMP1; RHOA ; RAP- 1; UGCG; H2AFZ; CANX; UBE2I; TPP2; REV1; EMB; COL5A1; RAB1A; IMPACT; VAMP2; TANK; HSPE 1; MYLl;
  • PRKAR1A WASL; HNRNPK; SGCB; GABPA; MLF1; SMEK2; M6PR; PERP;
  • said method characterized in that said cell is an adult cell, preferably a muscle cell, preferably a skeletal muscle cell, a senescent cell, neuronal cell, a satellite cell, an adult stem cell, preferably a mesenchymal stem cell.
  • the invention also provides an isolated oligonucleotide having 12-40 bases, wherein said oligonucleotide comprises a continuous stretch of at least 7 bases that is complementary to and capable of hybridizing to a poly-adenylation site or comprises a continuous stretch of at least 7 bases that is complementary to and capable of hybridizing to a miRNA target sequence, or comprises a continuous stretch of at least 7 bases that is complementary to and capable of hybridizing to a regulatory 3' UTR target sequence, of an ARIH2 pre-mRNA or of a (pre-)mRNA encoded by the gene HILFA; EGFR; SUMOl; PASMD14; SLC1A4; E2F1; BMI1;
  • MEF2A GRB2; ZMYND11; DNMT3A; CDK7; DIABLO; JARID2; EIF4E; UBE3A;
  • HUS l HSPA9; LPL; ABI3; CAMK2D; HSPDl; ITPRl; CSNK2A1; PAKl; BIRC2; RSL1D1; GCLM; CDS 1; HDAC3; YY1; RPA1; RRM2B; CD55; HDAC4; KSR1; CD59; HMGBl; GCLC; PSMD14; CADMl; TFRC; RBX- 1; PDKl; AGFGl; PSMA2 ; PLCB4; LAMP1; RHOA ; RAP- 1; UGCG; H2AFZ; CANX; UBE2I; TPP2; REV1; EMB; COL5A1; RAB1A; IMPACT; VAMP2; TANK; HSPE1; MYL1; PRKAR1A; WASL; HNRNPK; SGCB; GABPA; MLFl; SMEK2; M6PR; PERP; P
  • oligonucleotide having 12-40 bases, comprising a continuous stretch of at least 7 bases of sequence:
  • GRB2 5'-GACAAGAAACCAAGTGGGC-3' STAT5B 5'-GAAGTGTTAATACTAGTTGT-3'.
  • the invention further provides a compound for increasing the level of ARIH2 protein in a cell for use in the treatment of an individual suffering from aging.
  • said individual is suffering from a
  • said disease is Sarcopenia,
  • Said disease is Sarcopenia.
  • Said compound is preferably selected from an antisense oligonucleotide is that is complementary to and capable of hybridizing to ARIH2 (pre-)mRNA produced by said cell; a nucleic acid that encodes ARIH2 protein; an MDM2 inhibitor that inhibits the level or activity of MDM2; a compound that increases the level or activity of or of HoxAlO in said cell; and/or all-trans retinoic acid.
  • the invention further provides a method for the treatment of an individual suffering from an age related degenerative disease comprising administering to the individual in need thereof a compound for increasing the level of ARIH2 protein in a cell.
  • said individual is suffering from a neurodegenerative disease.
  • said disease is Sarcopenia, Alzheimer or Parkinson.
  • said disease is Sarcopenia.
  • Said compound is preferably selected from an antisense oligonucleotide is that is complementary to and capable of hybridizing to ARIH2 (pre-)mRNA produced by said cell; a nucleic acid that encodes ARIH2 protein; an MDM2 inhibitor that inhibits the level or activity of MDM2; a compound that increases the level or activity of or of HoxAlO in said cell; and/or all-trans retinoic acid.
  • PABPNl is a regulator of mRNA processing: it regulates
  • ARIH2 a regulator of PABPNl protein turnover.
  • ARIH2 contains a ring between ring fingers (RBR) domain, and is part of the largest family of E3-ligases.
  • RBR E3- ligases have attracted interest because of their involvement in late onset protein aggregation disorders such as Parkinson' disease, Lewy body dementia, and Alzheimer's disease (Eisenhaber et al. 2007).
  • ARIH2 or PABPNl affects the levels of the other gene.
  • the means and methods of the invention can be applied to a variety of different cells and cell types.
  • a preferred example of such a cell or cell type is selected from the group of a muscle cell, preferably a skeletal muscle cell, a senescent cell, a neuronal cell, a satellite cell, an adult stem cell, preferably a mesenchymal stem cell.
  • the cell is a skeletal muscle cell.
  • the cell is preferably a cell of an animal, preferably of a mammal or a bird.
  • the cell is a primate cell, preferably a human cell.
  • the PABPNl level declines with age and is associated with muscle weakness.
  • An adult human cell of the present invention is preferably a cell derived from or of an individual that is at least 40 years old, preferably at least 50 years old. In a particularly preferred embodiment the cell is derived from or of a 60 old individual. An adult cell of a different organism is preferably derived from an individual of a comparable age is indicated herein above for the human.
  • the effect that the concerted decline of the levels of ARIH2 and PABPNl has in a cell can be decreased and/or reversed by increasing the level of ARIH2 mRNA and/or protein in the cell.
  • Increasing the level of ARIH2 mRNA and/or protein in a cell has the effect of elevating the level of PABPNl mRNA and/or protein in the cell.
  • elevating or increasing the level of PABPNl is meant a higher level when compared to the same circumstances in the absence of ARIH2 manipulation.
  • the term also encompasses a stabilization or slower decrease of the level of PABPNl over time, when in the absence of ARIH2 mRNA and/or protein increase in otherwise similar circumstances the level of PABPNl decreases or decreases more, respectively.
  • the level of PABPNl preferably refers to the level of soluble PABPNl. Maintaining of PABPNl in turn rejuvenates the cell. Without being bound by theory, it is believed that due to a genome-wide change in mRNA stability, the expression of many different genes, including HUB molecules shifts the cell from functional/healthy to less functional/unhealthy. Re-adjusting mRNA stability of one or more of those HUB molecules pushes a cell into a normally functional state.
  • PABPNl is a HUB in the spliceosome and ARIH2 regulates PABPNl. Increasing the level of ARIH2 mRNA and/or protein in a cell can be used in a treatment of an individual suffering from aging. In a preferred
  • said individual is suffering from a neurodegenerative disease.
  • said disease is Sarcopenia, Alzheimer or Parkinson.
  • said disease is Sarcopenia.
  • poly-adenylation site usage is regulated by PABPNl, and as levels of PABPNl are regulated by ARIH2, poly-adenylation site usage in a cell can be modified by modifying the level of ARIH2 protein in a said cell.
  • Lower PABPNl levels results in proximal poly-adenylation site usage.
  • Increasing the level of ARIH2 protein in a cell with lower levels of PABPNl results in an increase in the level of PABPNl in the cell.
  • This, in turn, reverses the utility of a more distal PAS in a gene is as indicated herein. Particularly in the gene HILFA; EGFR; SUMOl; PASMD 14; SLC1A4; E2F1; BMI1; RBI; TP63; SLC2A4; ING1; MIB1; PTEN ;
  • an antisense oligonucleotide is that is complementary to and capable of hybridizing to ARIH2 (pre-)mRNA produced by said cell;
  • the level of ARIH2 protein in a cell is preferably increased by providing the cell with an antisense oligonucleotide is that is complementary to and capable of hybridizing to ARIH2 (pre-)mRNA produced by said cell.
  • the antisense oligonucleotide is preferably complementary to and capable of hybridizing to the proximal poly-adenylation signal sequence located at position 314-337 in the ARIH2 sequence of figure 8.
  • the level of ARIH2 mRNA and/or protein is increased by providing the cell with an antisense oligonucleotide (AON) that is complementary to and capable of hybridizing to an ARIH2 pre-mRNA, preferably to ARIH2 3' UTR produced by said cell.
  • AON antisense oligonucleotide
  • the antisense oligonucleotide is complementary to and capable of hybridizing to a PAS of the ARIH2 pre-mRNA.
  • PAS is the proximal PAS.
  • said antisense oligonucleotide is complementary to and capable of hybridizing to the proximal poly-adenylation signal sequence located at position 314-337 in the ARIH2 sequence of figure 8 (highlighted in BOLD).
  • the oligonucleotide is complementary to and capable of hybridizing to a miRNA binding site, located in the ARIH2 (pre-)mRNA.
  • the binding site is preferably located in the 3' UTR of the ARIH2 (pre-)mRNA.
  • the miRNA binding site is a miR19 binding site of the ARIH2 (pre-)mRNA.
  • said miR19 binding site is located downstream of the proximal PAS of the ARIH2 pre-mRNA.
  • the AON directed towards the miR19 binding site in the ARIH2 pre- mRNA is directed towards the sequence located at position 1055- 1074 in the ARIH2 sequence of figure 8 (underlined).
  • the oligonucleotide can also be directed towards a regulatory sequence in the 3' UTR of ARIH2.
  • ARIH2 pre-mRNA preferably comprises 12-40 bases.
  • the AON preferably comprises a continuous stretch of at least 7 bases of sequence 5'-GTA TAA TTG TAC AAC CTT TGA AAG-'3.
  • the AON comprises a continuous stretch of at least 15 bases of sequence 5'-GTA TAA TTG TAC AAC CTT TGA AAG-'3.
  • the AON comprises a continuous stretch of at least 20 bases of sequence 5'-GTA TAA TTG TAC AAC CTT TGA AAG-'3.
  • the AON comprises the sequence:
  • the AON comprises the sequence 5' GTA TAA TTG TAC AAC CTT TGA AAG.
  • An antisense oligonucleotide directed towards the miR19 binding site downstream of the proximal PAS of the ARIH2 pre-mRNA preferably comprises 12-40 bases.
  • the AON preferably comprises a continuous stretch of at least 7 bases of sequence 5'-TAA CTT GTG CAA ACA CAG CC-3'.
  • the AON comprises a continuous stretch of at least 15 bases of sequence 5'- TAA CTT GTG CAA ACA CAG CC -'3. In a particularly preferred embodiment the AON comprises a continuous stretch of at least 20 bases of sequence 5'- TAA CTT GTG CAA ACA CAG CC -'3. In a particularly preferred embodiment the AON comprises the sequence:
  • the AON comprises the sequence 5'TAA CTT GTG CAA ACA CAG CC-3'.
  • the nucleic acid preferably comprises an ARIH2 coding region together with the appropriate in cis required expression signal sequences for transcription and/or translation of the coding region in the target cell.
  • sequences can encompass for instance, a suitable promoter, a shine delgano sequence, poly-adenylation sequences and the like.
  • the ARIH2 coding region is preferably a coding region encoding a mammalian ARIH2, preferably a primate ARIH2 and more preferably a human ARIH2.
  • the species of that ARIH2 protein is derived from is the same as the species the cell is derived from.
  • ARIH2 protein levels in a cell are also influenced by other factors. For instance the level of ARIH2 protein is increased when the level or activity of
  • MDM2 in the cell is decreased.
  • the level of ARIH2 mRNA and/or protein in a cell is increased by decreasing the level and/or activity of MDM2 in said cell.
  • ARIH2 protein levels in a cell are also influenced by the level or activity of HoxAlO in the cell.
  • the level of ARIH2 protein is increased when the level or activity of HoxAlO in the cell is increased.
  • the level of ARIH2 mRNA and/or protein in a cell is be increased by increasing the level and/or activity of HoxAlO in said cell.
  • ARIH2 protein levels in a cell are also influenced by contacting said cell with resveratrol.
  • the level of ARIH2 protein is increased by contacting the cell with an effective amount of resveratrol.
  • the level of ARIH2 mRNA and/or protein in a cell is increased by contacting said cell with an effective amount of resveratrol.
  • ARIH2 protein levels in a cell are also influenced by contacting said cell with a SIRT drug.
  • the level of ARIH2 protein is increased by contacting the cell with a SIRT drug.
  • the level of ARIH2 mRNA and/or protein in a cell is increased by contacting said cell with a SIRT drug.
  • ARIH2 RNA and/or protein levels in a cell are also influenced by contacting said cell with all-trans retinoic acid (Pietschmann et al., The
  • the level of ARIH2 protein is increased by contacting the cell with an effective amount of a ⁇ -trans retinoic acid.
  • the level of ARIH2 mRNA and/or protein in a cell is increased by contacting said cell with all- trans retinoic acid.
  • PABPNl expression in a cell can be down-regulated by decreasing the level of ARIH2 mRNA and/or protein in that cell.
  • This feature is for instance useful in artificially aging cells.
  • This property can be used, for instance, to the treatment of cancerous cells.
  • the invention further provides a method for the treatment of an individual suffering from cancer said method comprising administering a PABPNl, and/or an ARIH2 inhibitor to the individual in need thereof.
  • a preferred inhibitor is an antibody that binds to PABPNl or ARIH2, or a derivative or analogue of said antibody.
  • the antibody is preferably an antibody that neutralizes the activity of PABPNl or ARIH2.
  • said antibody is an intracellular antibody; in a particularly preferred embodiment said intracellular antibody is an ScFv antibody.
  • the antibody may be used as such or be provided to the cell by means of a nucleic acid delivery vehicle comprising one or more nucleic acids encoding said antibody, or derivative or analogue thereof.
  • a nucleic acid delivery vehicle comprising one or more nucleic acids encoding said antibody, or derivative or analogue thereof.
  • PABPNl or ARIH2 inhibitor is an RNA inhibitor.
  • RNA inhibitor Presently there are many different RNA molecules that can inhibit translation of an mRNA and/or decrease the stability of the RNA.
  • the RNA inhibitor is preferably an RNAi molecule specific for PABPNl or ARIH2 mRNA; shRNA molecule specific for PABPNl or ARIH2 mRNA; an AON that induces exon skipping of a PABPNl or ARIH2 exon,
  • the exon to be skipped preferably introduces a frameshift resulting in premature termination of the protein.
  • the invention further provides a method for the treatment of an individual suffering from cancer comprising administering to the individual in need thereof an effective amount of a PABPNl inhibitor and/or an ARIH2 inhibitor.
  • VEGFA VEGFA ; MORF4L1; DNM1L; SOD2; RAC1; ITGB1; HIF1A; SRF; MCL1; RAD17; GSK3B; RYR1; MEF2A; GRB2; ZMYNDll; DNMT3A; CDK7; DIABLO; JARID2;
  • PRKAR1A WASL; HNRNPK; SGCB; GABPA; MLF1; SMEK2; M6PR; PERP;
  • the invention further provides a method for modifying PAS usage in a (pre-)mRNA expressed by a cell, comprising providing said cell with an antisense oligonucleotide that is complementary to and capable of hybridizing to a poly- adenylation site of a pre-mRNA encoded by the gene ARIH2; HILFA; EGFR;
  • the gene is preferably selected from ARIH2; PSMD14; STAT5B;
  • HILFA HILFA
  • PTEN VEGFA
  • GSK3B ITGB1
  • EGFR YY1
  • SUMOl SUMOl
  • PASMD14 PASM14
  • the gene is selected from ARIH2; PSMD14; STAT5B; TDP- 43; C90RF72; or GRB2. In a particularly preferred embodiment the gene is ARIH2.
  • ARIH2 is also referred to as ARI2 or TRIAD 1.
  • An antisense oligonucleotide that is complementary to and capable of hybridizing to a PAS decreases usage of the thus targeted PAS in the cell. As a result other PAS will be used.
  • the ratio of the amount of mRNA of the specific gene with poly-adenylation at the targeted PAS over the total amount of mRNA of the specific gene decreases.
  • An antisense oligonucleotide is preferably directed towards the proximal PAS. In that way usage of the more proximal PAS is at least in part inhibited and a more downstream PAS is utilized.
  • the proximal PAS in an mRNA of a gene is the PAS that is downstream of the translation stop codon of the coding region in the mRNA and it is the PAS that is closest to that translation stop codon.
  • An antisense oligonucleotide that is complementary to and capable of hybridizing to a miRNA target sequence (directed or targeted to a miRNA target sequence) inhibits the effect of the miRNA that it competes with in the cell.
  • the miRNA target sequence is preferably located in the 3' untranslated region of the (pre-)mRNA.
  • the miRNA target sequence is preferably located downstream or distal to the proximal PAS.
  • a molecular effect of aging in an adult cell is preferably modified by providing the cell with an AON directed towards a pre-mRNA encoded by the gene ARIH2; Psmdl4; Stat5b; HILFA; PTEN; VEGFA; GSK3B; ITGB1; EGFR; YY1; SUMOl; PASMD14; SLC1A4; RBI; HDAC3; RHOA; GRB2; E2F1; RAC1 TDP-43; or C90RF72.
  • PAS usage in a (pre-)mRNA expressed by a cell is preferably modified by providing the cell with an AON directed towards a pre- mRNA encoded by the gene ARIH2; Psmdl4; Stat5b; HILFA; PTEN; VEGFA; GSK3B; ITGB1; EGFR; YY1; SUMOl; PASMD14; SLC1A4; RBI; HDAC3; RHOA; GRB2; E2F1; RAC1.
  • the AON is directed towards a pre-mRNA encoded by the gene ARIH2; Psmdl4; Stat5b; or Grb2.
  • An AON of the invention is preferably directed towards the proximal PAS in a pre-mRNA.
  • the default (and normal) mRNA processing is via the distal poly-adenylation site, the proximal poly-adenylation site usage alters mRNA metabolism in a cell.
  • Masking the proximal PAS by specific AON revert mRNA processing to a distal poly-adenylation site usage, and can restore cellular defects (Figure 18).
  • An antisense oligonucleotide or AON of the invention preferably comprises a sequence that is complementary to the target site on the (pre-)mRNA.
  • complementary is meant capable of hybridizing to the sense strand comprising the target RNA.
  • Such a complementary AON is typically the reverse complement of the sense strand.
  • the AON preferably contains a continuous stretch of between 8-50 nucleotides that is complementary to the target site on the pre- mRNA.
  • An AON of the invention preferably comprises a continuous stretch of at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28 , 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides that is complementary to the target site.
  • said AON contains a continuous stretch of between 12-45 nucleotides that is complementary to the target site on the pre-mRNA. More preferably a stretch of between 15-41 nucleotides.
  • the complementary stretch may be at the smaller side of the range or at the larger side.
  • a preferred antisense oligonucleotide according to the invention comprises a T-0 alkyl phosphorothioate antisense oligonucleotide, such as 2'-0- methyl modified ribose (RNA), 2'-0-ethyl modified ribose, 2'-0-propyl modified ribose, and/or substituted derivatives of these modifications such as halogenated derivatives.
  • RNA 2'-0- methyl modified ribose
  • 2'-0-ethyl modified ribose 2'-0-propyl modified ribose
  • substituted derivatives of these modifications such as halogenated derivatives.
  • a most preferred AON according to the invention comprises of 2'-0- methyl phosphorothioate ribose. Such AON typically do not need to have a very large complementary stretch. Such AON typically contain a stretch of between 15- 25 complementary nucleotides. Such AON also render the target (pre-mRNA) resistant to the action of RNAse H.
  • another preferred AON of the invention comprises a morpholino backbone. AON comprising such backbones typically contain somewhat larger stretches of complementarity. Such AON typically contain a stretch of between 25-40 complementary nucleotides. When in this invention reference is made to range of nucleotides, this range includes the number(s) mentioned. Thus, by way of example, when reference is made to a stretch of between 8-50, this includes 8 and 50.
  • An AON of the invention that is complementary to a target RNA is capable of hybridizing to the target RNA under stringent conditions.
  • the reverse complement of the AON is at least 90% and preferably at least 95% and more preferably at least 98% and most preferably at least 100% identical to the nucleotide sequence of the target at the targeted sited.
  • An AON of the invention thus preferably has two or less mismatches with the reverse complement of the target RNA, preferably it has one or less mismatches with the reverse complement of the target RNA. Preferably it has no mismatch with the reverse complement of the target RNA.
  • a mismatch is defined herein as a nucleotide or nucleotide analogue that does not have the same base pairing capacity in kind, not necessarily in amount, as the nucleotide it replaces. For instance the base of uracil that replaces a thymine and vice versa is not a mismatch.
  • a preferred mismatch comprises an inosine.
  • An inosine nucleotide is capable of pairing with any natural base in an RNA, i.e. capable of pairing with an A, C, G or U in the target RNA.
  • the oligonucleotide comprises RNA, as RNA/RNA hybrids are very stable. Since one of the aims of the present invention is to alter poly-adenylation in subjects it is preferred that the oligonucleotide comprises a modification providing the AON with an additional property.
  • Some types of anti- sense oligonucleotides render the target (pre-mRNA) sensitive to nucleases such as RNAseH.
  • Oligonucleotides that render the duplex formed by the antisense oligonucleotide and the target pre-mRNA sensitive to RNAseH degradation are not preferred for the present invention as these can reduce the amount of target RNA in the cell via RNAi type mechanisms.
  • the anti-sense oligonucleotide does not have this property and thus comprises a modification that renders the renders the duplex formed by the antisense oligonucleotide and the target pre-mRNA resistant to RNAseH.
  • Various oligonucleotides and modifications and variants thereof do not promote the nuclease degradation of the target (pre-)mRNA.
  • oligonucleotides examples include 2'0-methyl phosphorothioate oligonucleotides and morpholinos.
  • Other features that may be added to or enhanced in said anti-sense oligonucleotide are increased stability (for instance in a bodily fluid), increased or decreased flexibility, reduced toxicity, increased intracellular transport, tissue-specificity, etc.
  • the AON comprises a modified backbone.
  • backbones are provided by morpholino backbones, carbamate backbones, siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioform acetyl backbones, methyleneformacetyl backbones, riboacetyl backbones, alkene containing backbones, sulfamate, sulfonate and sulfonamide backbones, methyleneimino and methylenehydrazino backbones, and amide backbones.
  • Phosphorodiamidate morpholino oligomers are modified backbone oligonucleotides that have previously been investigated as antisense agents.
  • Morpholino oligonucleotides have an uncharged backbone in which the deoxyribose sugar of DNA is replaced by a six membered ring and the phosphodiester linkage is replaced by a phosphorodiamidate linkage.
  • Morpholino oligonucleotides are resistant to enzymatic degradation and appear to function as antisense agents by arresting translation or interfering with pre-mRNA splicing rather than by activating RNase H.
  • Morpholino oligonucleotides have been successfully delivered to tissue culture cells by methods that physically disrupt the cell membrane, and one study comparing several of these methods found that scrape loading was the most efficient method of delivery; however, because the morpholino backbone is uncharged, cationic lipids are not effective mediators of morpholino oligonucleotide uptake in cells.
  • a recent report demonstrated triplex formation by a morpholino oligonucleotide, because of the non-ionic backbone; these studies showed that the morpholino oligonucleotide was capable of triplex formation in the absence of magnesium.
  • a modified backbone is typically preferred to increase nuclease resistance of the AON, the target RNA or the AON/target RNA hybrid or a combination thereof.
  • a modified backbone can also be preferred because of its altered affinity for the target sequence compared to an unmodified backbone.
  • An unmodified backbone can be RNA or DNA, preferably it is an RNA backbone.
  • the linkage between the residues in a backbone does not include a phosphorus atom, such as a linkage that is formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain
  • a preferred nucleotide analogue or equivalent comprises a Peptide Nucleic Acid (PNA), having a modified polyamide backbone (Nielsen, et al. (1991) Science 254, 1497-1500). PNA-based molecules are true mimics of DNA molecules in terms of base-pair recognition.
  • the backbone of the PNA is composed of 7V-(2- aminoethyl)- glycine units linked by peptide bonds, wherein the nucleobases are linked to the backbone by methylene carbonyl bonds.
  • An alternative backbone comprises a one- carbon extended pyrrolidine PNA monomer (Govindaraju and Kumar (2005) Chem. Commun, 495-497).
  • PNA-RNA hybrids are usually more stable than RNA- RNA or RNA-DNA hybrids, respectively (Egholm et al (1993) Nature 365, 566-568).
  • a further preferred backbone comprises a morpholino nucleotide analog or equivalent, in which the ribose or deoxyribose sugar is replaced by a 6- membered morpholino ring.
  • a most preferred nucleotide analog or equivalent comprises a phosphorodiamidate morpholino oligomer (PMO), in which the ribose or deoxyribose sugar is replaced by a 6-membered morpholino ring, and the anionic phosphodiester linkage between adjacent morpholino rings is replaced by a non- ionic phosphorodiamidate linkage.
  • PMO phosphorodiamidate morpholino oligomer
  • a nucleotide analogue or equivalent of the invention comprises a substitution of one of the non-bridging oxygens in the phosphodiester linkage. This modification slightly destabilizes base-pairing but adds significant resistance to nuclease degradation.
  • a preferred nucleotide analogue or equivalent comprises phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, H-phosphonate, methyl and other alkyl phosphonate including 3'-alkylene phosphonate, 5'-alkylene phosphonate and chiral phosphonate, phosphinate, phosphoramidate including 3'- amino phosphoramidate and aminoalkylphosphoramidate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphate or boranophosphate.
  • a further preferred nucleotide analogue or equivalent of the invention comprises one or more sugar moieties that are mono- or disubstituted at the 2', 3' and/or 5' position such as a -OH; -F; substituted or unsubstituted, linear or branched lower (C1-C10) alkyl, alkenyl, alkynyl, alkaryl, allyl, aryl, or aralkyl, that may be interrupted by one or more heteroatoms; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, S-or N-alkynyl; 0-, S-, or N-allyl; O-alkyl-0- alkyl, -methoxy, - aminopropoxy; -amino xy; methoxyethoxy; -dimethylaminooxyethoxy; and - dimethylaminoethoxyethoxy.
  • sugar moieties
  • the sugar moiety can be a pyranose or derivative thereof, or a deoxypyranose or derivative thereof, preferably a ribose or a derivative thereof, or a deoxyribose or a derivative thereof.
  • Such preferred derivatized sugar moieties comprise Locked Nucleic Acid (LNA), in which the 2'-carbon atom is linked to the 3' or 4' carbon atom of the sugar ring thereby forming a bicyclic sugar moiety.
  • LNA Locked Nucleic Acid
  • a preferred LNA comprises 2'-0,4'-C-ethylene-bridged nucleic acid (Morita et al. 2001. Nucleic Acid Res Supplement No. 1: 241-242). These substitutions render the nucleotide analogue or equivalent RNase H and nuclease resistant and increase the affinity for the target RNA.
  • an antisense oligonucleotide of the invention has at least two different types of analogues or equivalents.
  • a preferred AON according to the invention comprises a T-0 alkyl phosphorothioate antisense oligonucleotide, such as 2'-0-methyl modified ribose (RNA), 2'-0-ethyl modified ribose, 2'-0-propyl modified ribose, and/or substituted derivatives of these modifications such as halogenated derivatives.
  • RNA 2'-0-methyl modified ribose
  • a most preferred AON according to the invention comprises of 2'-0-methyl phosphorothioate ribose.
  • An AON of the invention can be linked to a moiety that enhances uptake of the antisense oligonucleotide in cells.
  • moieties are cholesterols, carbohydrates, vitamins, biotin, lipids, phospholipids, cell- penetrating peptides including but not limited to antennapedia, TAT, transportan and positively charged amino acids such as oligoarginine, poly-arginine, oligolysine or polylysine, antigen-binding domains such as provided by an antibody, a Fab fragment of an antibody, or a single chain antigen binding domain such as a cameloid single domain antigen-binding domain, or a recombinant affinity binder such as a affibody.
  • flanking sequences are used to modify the binding of a protein to said AON, or to modify a thermodynamic property of the AON, more preferably to modify target RNA binding affinity.
  • AON administration in humans is typically well tolerated.
  • Clinical manifestations of the administration of AON in human clinical trials have been limited to the local side effects following subcutaneous (SC) injection (on the whole intravenous (i.v.) administration seems to be better tolerated) and generalized side effects such as fever and chills that similar to the response to interferon
  • AON phosphorothioate backbone
  • new generation AON uniform phosphorothioated backbone with flanking 2' methoxyethoxy wing
  • AON Delivery of AON to cells of the brain can be achieved by various means. For instance, they can be delivered systemically through (coated) liposomes that are targeted to the brain or, directly to the brain via intracerebral inoculation (Schneider et al, journal of Neuroimmunology 195 (2008) 21-27). Alternatively, the AON can be coupled to a single domain antibody or the variable domain thereof (VHH) that has the capacity to pass the Blood Brain barrier.
  • VHH variable domain thereof
  • the complementary part is at least 50% of the length of the oligonucleotide of the invention, more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90% or even more preferably at least 95%, or even more preferably 98% and most preferably up to 100% of the length of the oligonucleotide of the invention.
  • An AON of the invention preferably comprises a sequence that is complementary to part of said pre-mRNA as defined herein.
  • the length of said complementary part of said oligonucleotide is of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28 , 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides.
  • additional flanking sequences are used to modify the binding of a protein to said molecule or oligonucleotide, or to modify a
  • An AON of the invention may further comprise additional nucleotides that are not complementary to the target site on the target pre-mRNA.
  • an AON contains between 8-50 nucleotides.
  • An AON of the invention preferably comprises a stretch of at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28 , 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • said AON contains a continuous stretch of between 12-45 nucleotides, more preferably a stretch of between 15-41 nucleotides.
  • an AON of the invention contains between 15-25 nucleotides.
  • An AON of the invention with a morpholino backbone typically contains a stretch of between 25-40 nucleotides.
  • the indicated amounts for the number of nucleotides in the AON refers to the length of the complementarity to the target pre-mRNA. In another preferred embodiment the indicated amounts refer to the total number of nucleotides in the AON.
  • the total number of nucleotides typically does not exceed 50, and the additional nucleotides preferably range in number from between 5-25, preferably from 10-25, more preferably from 15-25.
  • the additional nucleotides typically are not complementary to the target site on the pre-mRNA but may be complementary to another site on said pre-mRNA or may serve a different purpose and not be complementary to the target pre-mRNA, i.e., less then 95% sequence identity of the additional nucleotides to the reverse complement of the target pre-mRNA.
  • Hela cells were transfected with YFP-Alal6-PABPN1, and immunofluorescence was performed with anti-Arih2 (red) and anti-ubiquitin (Ubi; blue) antibodies. Co- localization is shown in the merged image and in the fluorescence intensity plots. Small PABPNl foci are indicated with arrows in both images and intensity plots.
  • Hela cells were transduced with shRNA specific to human ARIH2 (272, 273). Cells transduced with shRNA to mouse Arih2 (261) or with HI empty vector were used as controls. Histogram shows RT-qPCR oiARIH2 and PABPNl mRNA expression. Fold change was normalized to GUSB housekeeping gene and to non- transfected cells. Averages are of 3 biological replicates. Asterisks indicate significant down-regulation (pvalue ⁇ 0.05). Lower panel shows protein levels of ARIH2 in the transduced cell cultures. Actin was used as a loading control.
  • B. and C. Stably HI- or sh273- transduced cells were transfected with YFP- AlalO- or YFP-Alal6-PABPN1 constructs.
  • B. Cells were immunolabeled with anti-
  • Controls (NT), and Arih2-DR (sh073, sh023) C2C12 cells were treated with 15 ⁇ cyclohexamide (CHX) for 6 hours.
  • Immunoblots are of soluble protein extracts. Tubulin shows equal loading. MyoD is used as a control for CHX treatment.
  • Control (NT) and Arih2-DR sh073 cell cultures were transduced with AlalO- or Alal6-PABPN1 fused to YFP.
  • YFP-PABPN1 fused proteins were
  • E. Robust quantification of cell fusion Images show segmentation of myosin heavy chain (Myol) (red) and nuclei (blue) in 6 days fused mock or sh073 Arih2-DR C2C12 cultures. Myonuclei in fused cells are depicted in green. Histogram shows the percentage of nuclei in Myol expressing cells in mock, sh023 or sh027 C2C1 cultures. N indicates that number of quantified nuclei. Averages and SD are of 4 replications.
  • FIG. 5 Arih2 expression decreases in OPMD and during muscle aging and correlates with PABPNl expression.
  • B-box plots show fold changes, which are normalized to the average of GUSB housekeeping gene. Student's T-Test statistical significance (p ⁇ 0.05) is indicated with an asterisk.
  • a switch in poly(A) site usage oiArih2 is regulated by PABPN1.
  • PAS polyadenylation sites
  • PABPN1 protein accumulation is regulated by ARIH2 and depends on the proteasome.
  • ARIH2 expression level declines and induces PABPN1 accumulation and a subsequent aggregation.
  • Reduced levels of soluble PABPN1 cause alternative PAS usage in ARIH2 transcript, and a decline in ARIH2 expression.
  • a reduction in soluble PABPNl results in genome-wide changes in mRNA stability, leading to muscle dysfunctionality.
  • pre- aggregated structures of expPABPNl cause faster aggregation of PABPNl and reduced levels of soluble PABPNl, causing a decrease in ARIH2 expression.
  • the control AON 47/2 is fluorescence-conjugated and was use to evaluate transfection efficiency. &2 hours post-transfection RNA was extracted from cells and cDNA was made with oligo d(T). Expression levels were determined with RT-qPCR using primers specific to ARIH2 last exon. Fold change was calculated by normalization to GUSB house keeping gene and control transfection. levels of GapDH are used as control for specific effect of AON.
  • ARIH2 AON3 has the sequence 5'-GTATAATTGTACAACCTTTGAAAG-'3.
  • Mir 19 AON has the sequence 5'-TAACTTGTGCAAACACAGCC-3';
  • AON binding site for the AON directed towards the proximal PAS is indicated in bold.
  • the AON directed towards the miR19 binding site is underlined.
  • Figure 9. PABPNl protein accumulation significantly decreases in aging in senescent cells.
  • A) Chart bar shows PABPNl accumulation in human mesenchymal stem cells. PABPNl accumulation was determined from western blot analysis of total protein extracts from 5 young (19-29 years) and 6 old (79-87 years) donors. PABPNl expression was normalized to actin. Averages from all samples represent two independent experiments. Cultures were propagated in vitro for a single passage.
  • B) Chart bar shows PABPNl accumulation senescent human fibroblasts.
  • PABPNl accumulation was determined from western blot analysis of soluble protein extracts from cultures at passage 13 and passage 27. PABPNl expression was normalized to actin. Averages are from four independent experiments.
  • C) Chart bar shows nuclear PABPNl accumulation in primary muscle cell cultures that were fused for 6 days. PABPNl was visualized with the immuno-fluorescence procedure and nuclear PABPNl was measured from segmented nuclei. PABPNl integrated fluorescence was normalized to DAPI staining. Averages are from 100 nuclei and three experiments.
  • Figure 10 Pabpnl and Arih2 expression in aging control mice. Expression levels were normalized to Hrpt house keeping gene and mice at 4 month. The age- associated increase in expression is highly significant. P ⁇ 0.0005.
  • Figure 11 ARIH2 expression decreases in OPMD and during muscle aging and correlates with PABPNl expression.
  • B-box plots show fold changes, which are normalized to GUSB housekeeping gene. Student's T-Test statistical significance (p ⁇ 0.05) is indicated with an asterisk.
  • GUSB housekeeping gene.
  • HeLa cells were transfected with YFP-Alal6-PABPN1, and immunofluorescence was performed with anti-ARIH2 (red) and anti-ubiquitin (Ubi; blue) antibodies. Co- localization is shown in the merged image and in the fluorescence intensity plots. Small PABPNl foci are indicated with arrows in both images and intensity plots.
  • Figure 13 PABPN1 aggregation is increased in ARIH2 down-regulated cells.
  • HeLa cells were transduced with shRNA specific to human ARIH2 (272, 273). Cells transduced with shRNA to mouse ARIH2 (261) or with HI empty vector were used as controls. Histogram shows RT-qPCR of ARIH2 and PABPN1 mRNA expression. Fold change was normalized to GUSB housekeeping gene and to non- transfected cells. Averages are of 3 biological replicates. Asterisks indicate significant down-regulation (p-value ⁇ 0.05). Lower panel shows protein levels of ARIH2 in the transduced cell cultures. Actin was used as a loading control.
  • IP immunoprecipitation
  • ARIH2 is visualized with anti-ARIH2 and Alexa-594 conjugated secondary antibody,
  • Co-localization between PABPN1 and ARIH2 is shown in the merged image and in the intensity distribution plots (lower panel) of two representative nuclei. The cross-section lines of intensity distribution are shown in the merged image. Scale bar is 10 pm.
  • Bar chat shows the fraction of transfected cells with nuclear PABPN1 nuclear inclusions. Number of nuclei is indicated in the bars. Fold enrichment shows PABPN1 nuclear inclusions in ARIH2-DR cells compared to HI control. Averages are from three independent experiments.
  • Figure 14 ARIH2 co-IP with soluble wild type PABPN1 in myotube cultures.
  • Soluble or insoluble proteins were extracted from 4 days myotube cultures that stably express wild type (AlalO) or expPABPNl (Alal7) fused to FLAG. Parental culture (IM2) was used as control. PABPN1 IP was carried out with VHH-3F5.
  • Immunoblot was carried out with goat-anti-ARIH2, mouse -anti- FLAG antibodies or antibodies to muscle actin (MSA). Loading controls are shown in the input fractions. Molecular weights are indicated in kDa. Figure 15: ARIH2 regulates PABPN1 protein turnover and affects muscle cell fusion.
  • Results are from a representative experiments down-regulated stable cultures in C2C12 myoblasts were generated with shRNA specific to mouse ARIH2 (sh023, sh073) lentiviruses.
  • Non-transduced (NT) and cells transduced with shRNA specific to human ARIH2 (sh273) were used as controls.
  • Histogram shows RT- qPCR of ARIH2 and PABPN1 mRNA expression. Fold change was normalized to HPRT housekeeping gene and to NT culture. Averages are of 4 biological replicates.
  • Lower panel shows western blot analysis of ARIH2 protein levels in the transduced cultures. Tubulin is used as a loading control. Significant down- regulation (p-value ⁇ 0.05) is indicated with asterisks.
  • PABPN1 protein turnover in C2C12 cultures Controls (NT), and ARIH2-DR (sh073, sh023) C2C12 cells were treated with 15 ⁇ cycloheximide (CHX) for 6 hours. Soluble proteins were used to western blot analysis. PABPN1 was detected with VHH-3F5. Tubulin shows equal loading and MyoD was used as a control for CHX treatment.
  • CHX cycloheximide
  • PABPN1 and ARIH2 mRNA abundance in input and PABPN1-RIP fractions from PABPN1-DR or HI control C2C12 cultures were determined with RT-qPCR. Fold change in the input fraction was determined after normalization to HRPT house keeping gene, and fold enrichment in the RIP fraction was calculated after normalization to the input fraction. RIP was carried out with VHH-3F5. Averages and SD are from four biological replicates. Statistical significance (p ⁇ 0.05) between PABPN1-DR and control cultures is indicated with an asterisk.
  • FIG. 17 A switch in poly(A) site usage of ARIH2 3'-UTR is regulated by PABPN1.
  • PAS polyadenylation sites
  • MHCl myosin heavy chain
  • Histogram shows the percentage of nuclei in MHCl expressing cells in mock, 7304 cultures after transfection with control AON, and AONs to ARIH2 proximal PAS, AON1 and AON2. Per condition, >10,000 nuclei were analyzed. Averages and SD are of three experiments. Statistical significance (p ⁇ 0.05) is indicated with one asterisk.
  • Figure 19 A feed-forward graphical model for age-regulated expression of ARIH2 and PABPN1 in muscles.
  • PABPN1 regulates ARIH2 mRNA stability (depicted in black) via PAS usage and PABPN1 protein (depicted in blue and expPABPNl is depicted in red)
  • ARIH2 protein depicted in orange.
  • ARIH2 protein With age expression levels of both PABPN1 and ARIH2 mRNAs declines due to two regulatory loops: reduced ARIH2 induces increase in PABPN1 aggregation and thus reduced educed levels of soluble and functional PABPN1.
  • Reduced PABPN1 causes proximal PAS usage in ARIH2 transcript (depicted in gray), and thus a decline in ARIH2 expression.
  • PABPNl self-regulates its mRNA levels. A decline in both ARIH2 and PABPNl levels is aging-regulated and is not affected in carriers of mutant PABPNl at a pre-symptomatic stage.
  • Protein turnover of nuclear proteins is predominantly regulated by the ubiquitin proteasome system.
  • Poly-ubiquitination levels of PABPNl differ between wild type (WT) and alanine -expanded PABPNl resulting in differences in protein
  • IP co-immunoprecipitation
  • Arih2 co-IP was enriched in WT-PABPN1 expressing myotubes compared with expPABPNl ( Figure 1A). Since functional PABPNl is soluble (Kuhn et al., 2009), we next compared Arih2 co-IP with soluble or insoluble PABPNl. PABPNl was IP from both soluble and insoluble protein fractions, while Arih2 preferentially co-IP with soluble PABPNl ( Figure 1A). Together this suggests a preference binding of Arih2 to WT and soluble PABPNl.
  • PABPNl aggregation is considered as the cause for muscle weakness in OPMD.
  • PABPNl aggregation in mitotic cells like HeLa cells is fast and is caused by expression of both WT- and expPABPNl (Abu-Baker et al. 2003; Raz et al. 2011a).
  • High-resolution fluorescence microscopy reveals that ARIH2 co-localizes with PABPNl fluorescence foci in HeLa cells expressing expPABPNl fused to yellow fluorescent protein (YFP) (Figure 2).
  • YFP yellow fluorescent protein
  • co-localization of both PABPNl and ARIH2 was also with ubiquitin, suggesting a complex of the three ( Figure 2).
  • ARIH2 expression was stably down-regulated (ARIH2-DR) in HeLa cells using different shRNA vectors.
  • a significant reduction in ARIH2 RNA and protein levels was obtained with shRNAs 272 and 273 ( Figure 3A).
  • Empty vector (HI) and an unspecific shRNA (261), were used as controls ( Figure 2A).
  • Down-regulation of ARIH2 resulted in reduced co-localization between foci of PABPNl fused to yellow fluorescent protein (YFP) and ARIH2 (Figure 3B).
  • YFP yellow fluorescent protein
  • ARIH2 co- localization with expanded (Alal6)
  • YFP-PABPN1 was less affected in ARIH2-DR cells ( Figure 3B).
  • PABPNl was significantly higher compared with control cells (Figure 3C). This suggests that ARIH2 regulates accumulation of PABPNl.
  • the increase in nuclei with PABPNl nuclear inclusions was about 2-fold higher in WT-PABPN1 transfected cells compared with expPABPNl-transfected cells ( Figure 3C), further suggesting a preference of ARIH2 for WT-PABPN1, which is consistent with preferential binding of Arih2 to WT-PABPN1 ( Figure 1).
  • Arih2 preferentially binds to WT PABPNl.
  • Arih2 co-IP with endogenous PABPNl ( Figure 4D).
  • western blot analysis in input reveals that in the Arih2-DR cells PABPNl expression levels decreases ( Figure 4D).
  • RT-qPCR of PABPNl in those cells shows that in the Arih2-DR cells expression levels of PABPNl mRNA also decreases.
  • PABPNl knockdown in muscle cells leads to reduced cell fusion (Apponi et al. 2010). Also in our cell model, down-regulation of PABPNl leads to reduced cell fusion, which is associated with reduced expression of myogenic genes
  • Age-regulated expression of both ARIH2 and PABPNl was not found in the full dataset from blood, kidney medulla, brain cortex and kidney cortex (Table 1, and Anvar 2013, respectively). Moreover, an age-regulated expression of both ARIH2 and PABPN 1 was also not found in Rectus abdominis skeletal muscles, a muscle that shows minor aging-associated changes (Marzani et al. 2005). In brain cortex we identified a small reduction in PABPN 1 expression in elderly (>70 years) (Anvar 2013). Compared with PABPN1 expression in VL muscles, the decrease in the brain cortex was smaller and delayed (Anvar 2013).
  • PABPN1 regulates mRNA stability by regulating PAS usage (E. de Klerk 2012; Jenal et al. 2012), we therefore also studied whether changes in Arih2 mRNA levels are associated with alternative PAS usage. Distal and proximal PAS generate long or short 3' UTR, respectively. A change in the ratio between long and short 3' UTR can be a measure for a change in PAS usage.
  • RT-qPCR analysis was performed with primers that specifically amplify long or short Arih2 3' mRNA ends (figure 6A). In the OPMD mouse model, A17.1 the ratio between long and short Arih2 transcripts significantly decreased compared with the wild-type control mice ( Figure 6A).
  • the ratio between long and short Arih2 3' UTR in sh536-transduced cells indicates a higher expression of the shorter 3' UTR PCR product in cells with reduced levels of endogenous Pabpnl (Supplementary Figure 2).
  • these experiments reveal a post- transcriptional regulation oiArih2 expression by PABPNl levels, where either a decline in Pabpnl expression or aggregation of expPABPNl result in a decrease ratio between Arih2 transcripts containing long or short 3' UTR.
  • GlutamaxTM-I GlutamaxTM-I (GIBCO ® Invitrogen) with 4,5 g/L glucose, supplemented with 10% fetal calf serum (FCS) (GIBCO ® Invitrogen) and 100 U/ml antibiotics
  • IM2 cells and the PABPN1 clones were previously described (Raz 2011).
  • cells were grown in Dulbecco's modified Eagle's medium (DMEM) + GlutamaxTM-I (GIBCO ® Invitrogen) with 4,5 g/L glucose, supplemented with 20% fetal calf serum (FCS) (GIBCO ® Invitrogen), 0,5% chicken embryo extract (CEE) (PAA
  • HS horse serum
  • antibiotics streptomycin/penicillin
  • C2C12 cells were grown in DMEM supplemented with 20% FCS and antibiotics. Treatments with 5 ⁇ cyclohexamide were conducted for 10 hours in culture at 90% confluence.
  • TRCN0000034269 (269); TRCN0000034272 (272); TRCN0000034273 (273).
  • shRNA for mouse Arih2 are: TRCN0000041023 (023); TRCN0000041027 (027).
  • ShRNA for mouse PABPN1 is: TRCN0000102536 (536), and for human PABPN1: TRCN0000000121 (121), TRCN0000000122 (122) and TRCN0000000123 (123). Between brackets are the symbol for each sh-vector that are used in the document.
  • Lentiviruses for YFP-Alal6-PABPN1 or YFP-Alal0-PABPN1 were generated from the expression vectors described in (Raz et al. 2011a). Lentivirus particles were produced as described in (Raz et al. 2006). Four independent transduction experiments were performed for every virus.
  • ARIH2 and PABPN1 expression levels were determined from the previously described microarray studies in mouse (GEO GSE26604, (Trollet et al. 2010), in human quadriceps (GEO GSE26605) (Anvar et al. 2011a), frontal cortex: (GEO- GD707, GEO-GSE1572) (Lu et al. 2004), Rectus abdominis (GEO-GSE5086) (Zahn et al. 2006), blood (GEO-GSE16717) (Passtoors et al. 2012), kidney cortex and medulla (Rodwell et al. 2004).
  • Total proteins were extracted in a RIPA-buffer (20mM Tris pH 7.5, 150mM NaCl, 5mM EDTA, 1% NP40, 0.05% SDS), and soluble proteins were extracted with a buffer composed of 20mM Tris pH 7.5, 10% glycerol, 150mM NaCl and 5mM EDTA.
  • Protease inhibitor cocktail (SigmaFASTTM protease inhibitor tablets, Sigma- Aldrich ® ) was freshly added. Soluble fraction was separated from the insoluble fraction by centrifugation. Insoluble proteins were recovered with RIPA buffer supplemented with 1% triton and 0.05% SDS, and ensuing sonification.
  • First antibodies used are: Goat anti-Arih2 (Everest Biotech), mouse -anti -muscle actin (Santa Cruz Biotechnology ® ), mouse-anti-FLAG (Sigma-Aldrich ® ); mouse anti-Ubiquitin (FK2, Tebu-bio); mouse anti-tub ulin (Sigma-Aldrich ® ); MyoD (M-318 Santa Cruz).
  • the VHH-3F5 (Verheesen et al. 2006) intrabody was detected with rabbit anti-VHH.
  • Antibodies were detected with the Odyssey® Infrared Imaging System (LI-COR® Biosciences) and applicable IRDye Secondary antibodies.
  • RIP was performed using C2C12 myoblasts of shl21- or Hl-transduced and untransduced cultures. Proteins were extracted with lysis buffer (100 mM KC1, 5 mM MgC , 10 mM HEPES (pH 7.0), 0.5% NP40, 1 mM DTT, 80 U RNAse Inhibitor (Roche), Protease Inhibitor Cocktail (Roche)), and lysates were passed 5 times through a 29G needle and incubated for 10 minutes on ice. The lysates were then clarified by centrifugation at 16.000 rcf for 5 minutes at 4 °C and the supernatants were recovered. DNA was removed from the protein-RNA extracts by a DNAse I (Fermentas) treatment. Aliquots of protein extracts were used for
  • GlutamaxTM-I GlutamaxTM-I (GIBCO® Invitrogen) with 4,5 g/L glucose, supplemented with 10% fetal calf serum (FCS) (GIBCO® Invitrogen) and 100 U/ml antibiotics
  • C2C12 immortalized mouse myoblasts were cultured in DMEM supplemented with 20% FCS and antibiotics. Treatments with 5 ⁇ cycloheximide were conducted for 10 hours in culture at 90% confluence. IM2 cultures are detailed in 11 . Cell growth and fusion conditions were performed as described in 17
  • the human 7304 immortalized myoblasts generated by expressing telomerase (hTERT) and cyclin-dependent kinase 4 21 , were propagated in a medium
  • shRNA for human ARIH2 (NM_006321) are: TRCN0000034269 (269); TRCN0000034272 (272); TRCN0000034273 (273).
  • the shRNA for mouse ARIH2 (NM_011790) are: TRCN0000041023 (023); TRCN0000041073 (073).
  • the shRNA for mouse PABPNl is: TRCN0000102536, and for human PABPNl: TRCN0000000121,
  • TRCN0000000122 and TRCN0000000123 Between brackets are the names for each sh-vector that are described here.
  • the shRNA for PABPNl are described in 15 and in n .
  • Lentiviruses for YFP-Alal6-PABPN1 or YFP-Alal0-PABPN1 were generated from the expression vectors described in 22 .
  • Lentivirus particles were produced as described in 23 .
  • Four independent transduction experiments were performed for every virus. AON transfection
  • AON design was carried out as described in 24 , AON sequences are shown in Table 3.
  • AON transfection was conducted as described in 3 ⁇ 4 26_ p or RNA expression analysis 143 ng AON were transfected, and for fusion experiments 200 ng AON.
  • AON in 0.15M NaCl were transfected into 7304 cells using 2.5 ⁇ polyethylenimine (ExGen 500, MBI Fermentas) or TurboFect Transfection Reagent (Thermo
  • ARIH2 and PABPNl expression levels were determined from the previously described 18 .
  • the microarray studies in used in this paper are publically available: in human quadriceps (GEO GSE26605), frontal cortex: GEO-GD707, GEO- GSE1572 Rectus abdominis GEO-GSE5086 28 , blood GEO-GSE16717 29 , and kidney cortex and medulla 30 .
  • Statistical analyses of linear and quadratic models, in microarray studies and RT-qPCR were carried out with PASWStatistics 18.0 for Mac (IBM). Total RNA extraction from cell cultures and muscle biopsies for RT- qPCR was performed as described in 18 .
  • Total proteins for direct western blots were extracted with a RIPA-buffer (20mM Tris pH 7.5, 150mM NaCl, 5mM EDTA, 1% NP40, 0.05% SDS), and for
  • soluble proteins were extracted with a buffer composed of 20mM Tris pH 7.5, 10% glycerol, 150mM NaCl and 5mM EDTA.
  • Protease inhibitor cocktail (SigmaFASTTM protease inhibitor tablets, Sigma- Aldrich®) was freshly added. Where indicated, 10 mM iodoacetamide was added to the extraction buffer. Soluble proteins were collected in the supernatant centrifugation. Insoluble proteins were recovered with RIPA buffer supplemented with 1% triton and 0.05% SDS, and ensuing sonification. Immunoprecipitation of PABPN1 was performed with VHH-3F5 was performed as described in 11 .
  • Proteins were fractionated on SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membrane.
  • First antibodies used are: Goat anti-ARIH2 (Everest Biotech), mouse- anti-muscle actin (Santa Cruz Biotechnology®), mouse- anti-FLAG (Sigma- Aldrich®); mouse anti-Ubiquitin (FK2, Tebu-bio); mouse anti-tubulin (Sigma- Aldrich®); MyoD (M-318 Santa Cruz).
  • the VHH-3F5 31 intrabody was detected with rabbit anti-VHH.
  • Antibodies were detected with the Odyssey® Infrared Imaging System (LI- COR® Biosciences) and applicable IRDye Secondary antibodies. Immunohistochemistry
  • RIP was performed using C2C12 myoblasts of PABPN1-DR or Hl-transduced and un-transduced cultures as described in 15 .
  • Immunoprecipitation of PABPN1 was carried out with VHH-3F5. Immunecomplexes were isolated with Protein A
  • RNA isolated from RIP was subjected for RT-qPCR. Equal protein loading and the expression levels of PABPN1 were determined with western blot analysis of protein extracts.
  • ARIH2 mRNA levels decline in OPMD and in muscle aging and are highly associated with PABPN1 mRNA levels in muscles
  • ARIH2 E3-ligase binds to PABPNl and regulates PABPNl aggregation
  • ARIH2 binds to PABPNl and regulates PABPNl aggregation.
  • ARIH2 expression was stably down-regulated (ARIH2-DR) in HeLa cells by RNA interference using two independent shRNA lentiviruses.
  • HeLa cells as a cell model since PABPNl aggregation is highly reproducible and quantifiable 22 > 33 .
  • a significant reduction in ARIH2 RNA and protein levels was obtained with two shRNA to ARIH2 (Figure 13A).
  • An empty vector (HI) and an unspecific shRNA were used as negative controls (Figure 13A).
  • HI and ARIH2-DR cell cultures were transiently transduced with WT (AlalO) or alanine-expanded (Alal6) PABPNl fused to yellow-fluorescent protein (YFP). PABPNl IP was carried out two days after transduction and Co-IP with ARIH2 was determined in immunoblots.
  • ARIH2-DR has smaller effect on expPABPNl aggregation compared with the effect on WT- PABPNl aggregation.
  • ARIH2 binds to PABPNl and regulates its ubiquitination and aggregation, with a preference for WT-PABPN1 over expPABPNl.
  • ARIH2 regulates PABPNl protein turnover and affects muscle cell fusion
  • PABPNl protein accumulation is regulated by ARIH2.
  • CHX cyclohexamide
  • ARIH2 mRNA specifically co-IP with PABPNl protein using PABPN1-DR cultures.
  • ARIH2 mRNA levels were also significantly reduced (Figure 16, input).
  • RNA-immunoprecipitation (RIP) with antibodies to PABPNl was performed in C2C12 cell cultures, and mRNA bound to PABPNl was determined with RT-qPCR.
  • RIP reveals that ARIH2 mRNA was IP with PABPNl protein, and binding was enriched in PABPN1-DR cells ( Figure 16).
  • PABPNl mRNA was IP with PABPNl protein but was decreased in IP from PABPNl -DR cells.
  • HRPT mRNA which was used as a housekeeping gene in the input fraction, was not found in RIP fraction. This suggests that not all transcripts bind to PABPNl. This experiment suggests that PABPNl directly regulates ARiH2 mRNA levels. Moreover, PABPNl could regulate its expression levels, but in a mechanism that differ from ARIH2 regulation.
  • ARIH2 proximal PAS elevates expression levels of ARIH2 and PABPNl and restores cell fusion in PABPNl-DR cultures
  • Aging is a multi-factorial complex process, where the control of multiple cellular processes progressively loosens as age raises. Among those, genome-wide changes in RNA expression are found in aging and degenerated muscles 35 > 36 .
  • This age- regulated mRNA expression profiles are regulated, in part, by PABPNl levels n .
  • PABPNl is a multi-functional orchestrator of mRNA processing regulating different steps in mRNA processing 12. Reduced PABPNl levels causes proximal over distal PAS usage in the 3'-UTR, affecting mRNA abandnece 14 > 15 . Levels of PABPNl decline during muscle aging, and in vitro it induces myogenic defects n .
  • ARIH2-E3 ligase as a regulator of PABPNl protein
  • PABPNl levels were not found to be affected by PAS usage 15 .
  • a decrease in expression of both ARIH2 and PABPNl is exaggerated compared with age-matched control group.
  • reduced ARIH2 induces an increase in PABPNl aggregation.
  • levels of soluble PABPNl decrease 17 and proximal PAS usage increases 14 > 15 .
  • Aggregated PABPNl is associated with reduced levels of soluble PABPNl 11 , and likewise levels of proteins that are entrapped in PABPNl aggregates would deplete 22 > 39 .
  • ARIH2 E3-ligase contains a ring between ring fingers (RBR) domain, and is part of the largest family of E3-ligases.
  • RBR E3-ligases have attracted interest because of their involvement in late-onset protein aggregation disorders such as Parkinson' disease, Lewy body dementia, and Alzheimer's disease 46 .
  • RBR E3-ligases as regulators of aggregation-prone protein accumulation, could be a collective target for therapy of protein aggregation disorders.
  • proximal PAS usage in ARIH2 was demonstrated in this study by masking the proximal PAS with specific AONs, which elevated ARIH2 and PABPN1 levels and restored myogenesis in PABPN1- DR cells.
  • Antisense oligonucleotides have been widely demonstrated to be an efficient molecular tool to modulate RNA processing due to their small size, stability and high efficiency delivering into the nucleus 41 .
  • AONs application for exon-skipping is progressed as therapeutic treatment 48 .
  • AON treatment can also redirect PAS selection 49 .
  • Our results here demonstrate that manipulation of expression level by AONs to proximal PAS also cause a functional impact on cell fusion. This opens a therapeutic opportunity to restore expression levels of regulatory genes whose levels significantly change by PAS usage, and possibly could be a clinical strategy for the treatment of diseases of RNA metabolism.
  • Sandri M Signaling in muscle atrophy and hypertrophy, Physiology
  • Brais B Oculopharyngeal muscular dystrophy: a polyalanine myopathy, Current neurology and neuroscience reports 2009, 9:76-82
  • Verheesen P de Kluijver A, van Koningsbruggen S, de Brij M, de Haard HJ, van Ommen G-JB, van der Maarel SrM, Verrips CT: Prevention of oculopharyngeal muscular dystrophy- associated aggregation of nuclear poly (A) -binding protein with a single-domain intracellular antibody, Human molecular genetics 2006, 15: 105- 111
  • Fonseca M Nuclear inclusions in oculopharyngeal muscular dystrophy consist of poly(A) binding protein 2 aggregates which sequester poly(A) RNA, Human molecular genetics 2000, 9:2321-2328
  • Ciechanover A, Brundin P The ubiquitin proteasome system in
  • Eisenhaber B Chumak N
  • Eisenhaber F Hauser M-T: The ring between ring fingers (RBR) protein family, Genome biology 2007, 8:209
  • Betas ⁇ standard errors of the mean are per probe and show an age- association linear model. Values for three independent probes shown for datasets from Kidney cortex, Kidney medulla, Rectus Abdominis. P-values are adjusted for gender, except in kidney datasets and are corrected for false discovery rate. Significant changes are highlighted in bold. N indicates number of samples. Ag range is indicates in years. In Vastus lateralis muscles a linear regression model was applied on two age groups; age-range and number of samples are indicated between brackets.
  • Poly(A) tail length is controlled by the nuclear poly (A) -binding protein regulating the interaction between poly(A) polymerase and the cleavage and polyadenylation specificity factor. J Biol Chem. 284, 22803-22814.
  • Verheesen P de Kluijver A, van Koningsbruggen S, de Brij M, de Haard HJ, van Ommen G-JB, van der Maarel SrM , Verrips CT (2006).

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Abstract

Le vieillissement est associé à des changements à l'échelle du génome dans les profils d'expression. La fonction de la protéine nucléaire de liaison à la queue poly(A) 1 (PABPN1) est associée au vieillissement et à la fonctionnalité des cellules. Une mutation dans PABPN1 est la cause génétique de la dystrophie musculaire oculopharyngée (DMOP) à déclenchement tardif. Nous avons identifié la ligase de E3, ARIH2, comme régulateur de l'accumulation de PABPN1. Les effets à large spectre de PABPN1 peuvent être manipulés en modifiant la teneur en ARNm et/ou protéine ARIH2 dans une cellule.
PCT/NL2013/050827 2012-11-16 2013-11-18 Moyens et procédés pour réduire un effet du vieillissement dans une cellule de mammifère Ceased WO2014077693A1 (fr)

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