WO2016134246A2 - Compositions et méthodes de traitement de maladies conformationnelles des protéines - Google Patents

Compositions et méthodes de traitement de maladies conformationnelles des protéines Download PDF

Info

Publication number
WO2016134246A2
WO2016134246A2 PCT/US2016/018657 US2016018657W WO2016134246A2 WO 2016134246 A2 WO2016134246 A2 WO 2016134246A2 US 2016018657 W US2016018657 W US 2016018657W WO 2016134246 A2 WO2016134246 A2 WO 2016134246A2
Authority
WO
WIPO (PCT)
Prior art keywords
ube4b
protein
lsdl
sirna
lsd1
Prior art date
Application number
PCT/US2016/018657
Other languages
English (en)
Other versions
WO2016134246A3 (fr
Inventor
Jiou Wang
Goran PERIZ
Original Assignee
The Johns Hopkins University
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 The Johns Hopkins University filed Critical The Johns Hopkins University
Publication of WO2016134246A2 publication Critical patent/WO2016134246A2/fr
Publication of WO2016134246A3 publication Critical patent/WO2016134246A3/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
    • A01K67/0333—Genetically modified invertebrates, e.g. transgenic, polyploid
    • A01K67/0335—Genetically modified worms
    • A01K67/0336—Genetically modified Nematodes, e.g. Caenorhabditis elegans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • 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
    • C12Y—ENZYMES
    • C12Y603/00—Ligases forming carbon-nitrogen bonds (6.3)
    • C12Y603/02—Acid—amino-acid ligases (peptide synthases)(6.3.2)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/05—Animals modified by non-integrating nucleic acids, e.g. antisense, RNAi, morpholino, episomal vector, for non-therapeutic purpose
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/20—Animals treated with compounds which are neither proteins nor nucleic acids
    • 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.

Definitions

  • the present invention relates to the field of protein conformational diseases. More specifically, the present invention provides compositions and methods for treating protein conformational diseases including amyotrophic lateral sclerosis (ALS).
  • ALS amyotrophic lateral sclerosis
  • ALS amyotrophic lateral sclerosis
  • the cell coordinates several major quality control systems to guard against proteotoxicity including molecular chaperones, ubiquitin-proteasome system (UPS) and autophagy (Bukau and Horwich, 1998; Ciechanover and Brundin, 2003; Hartl et al, 2011; Mizushima et al, 2008).
  • the regulation of protein quality control occurs at different scales from individual proteins to whole organisms (Wolff et al, 2014).
  • the protein quality control systems might be harnessed to defend against proteotoxicity associated with neurodegenerative diseases. However, how the cell might reprogram its protein quality control systems is not fully understood.
  • Mutant Cu/Zn superoxide dismutase (SOD1), linked to -20% of familial ALS, represents a simple molecular model for protein misfolding and aggregation.
  • the wild-type (WT) SOD1 protein has a stable ⁇ -barrel structure with a two-state folding process (Parge et al., 1992), whereas mutant SOD1 proteins gain heightened propensity to aggregate in vitro and in vivo (Bruijn et al, 1997; Lindberg et al, 2005; Wang et al, 2003a).
  • neurodegeneration-associated proteins including ALS-linked TDP-43 and FUS (Kwiatkowski et al, 2009; Vance et al., 2009). Identifying mechanisms that suppress the toxicity of protein misfolding and aggregation may help understand the pathogenesis of neurodegenerative diseases and also provide potential targets for corrections.
  • the present invention is based, at least in part, on the discovery that Lysine-Specific Demethylase 1 (LSD1) and Ubiquitination Factor E4B (Ube4B), can be targeted to suppress proteotoxicity and treat protein conformational diseases.
  • LSD1 Lysine-Specific Demethylase 1
  • Ube4B Ubiquitination Factor E4B
  • Protein conformational diseases refer to all the pathological conditions that are associated with protein misfolding, including major forms of neurodegenerative diseases.
  • Neurodegenerative diseases in humans present daunting medical and economic challenge. With a doubling of the average human lifespan over the last century, neurodegenerative diseases have become a major aging-related public health challenge in the US and many other countries. Unfortunately, no curative treatments exist for these debilitating conditions.
  • the present inventors identified novel molecular targets of neurodegeneration, the Lysine- Specific Demethylase 1 (LSD1) and Ubiquitination Factor E4B (Ube4B). As described herein, the present inventors showed in a mammalian cell-culture model system that reduction of levels of these molecular targets lead to reduction in protein aggregation, improved proteasome function and activation of pathways beneficial to cells under stress.
  • Mouse models of neurodegeneration are being used to test the efficacy of RNAi and drug inhibitors in reducing Ube4b/LSD1 function and reducing neurotoxic burden of aggregated proteins. The outcome of such efficacy studies in is instructive towards collaborative human clinical studies.
  • the present invention provides compositions and methods for treating a protein conformational disease.
  • a method comprises the step of administering to a patient an effective amount of a Ube4B inhibitor and a LSD1 inhibitor.
  • the method further comprises the step of administering a p53 agonist.
  • the present invention also provides methods for treating a protein
  • a method for treating a protein conformational disease comprises the step of administering to a patient an effective amount of a p53 agonist, a Ube4B inhibitor and a LSD1 inhibitor.
  • a method for treating a protein conformational disease comprises the step of administering to a patient an effective amount of a p53 agonist.
  • the method further comprises administering an effective amount of a Ube4B inhibitor and/or a LSD1 inhibitor.
  • the protein conformational disease comprises a
  • the neurodegenerative disease is Creutzfeldt- Jakob disease, Alzheimer's disease, Parkinson's disease, Huntington's disease, frontotemporal dementia, or amyotrophic lateral sclerosis (ALS).
  • the inhibitor is a small molecule, an antibody or an inhibitory nucleic acid molecule.
  • the inhibitory nucleic acid molecule can be an siRNA, shRNA, antisense RNA or a ribozyme.
  • the inhibitory nucleic acid molecule is an siRNA. Examples of LSD 1 and Ube4B siRNA molecules are shown in SEQ ID NOS: 14-19 and SEQ ID NOS:20-49, respectively.
  • the inhibitory nucleic acid molecules is an shRNA. Examples of LSD1 and Ube4B shRNA molecules are shown in SEQ ID NOS:4-8 and SEQ ID NOS:9-13, respectively.
  • a pharmaceutical composition comprises one or more siRNA encoded by SEQ ID NOS: 15-19 and a pharmaceutical carrier.
  • a pharmaceutical composition comprises one or more siRNA encoded by SEQ ID NOS: 20-49 and a pharmaceutical carrier.
  • a pharmaceutical composition can also comprise one or more shRNA encoded by SEQ ID NOS:4-8 and a pharmaceutical carrier.
  • a pharmaceutical composition comprises one or more shRNA encoded by SEQ ID NOS:9-13 and a pharmaceutical carrier.
  • the composition can comprise a vector encoding a siRNA and/or shRNA.
  • p53 agonists/activating drugs include, but are not limited to, RG7112 (Ro5045337 (Roche), RG7112 with cytarabine (Roche), RG7112 with doxorubicin (Roche), RO5503781 (Roche), RO5503781 with cytarabine (Roche), MI-773 (SAR405838) (Sanofi), DS-3032b (Daiichi Sankyo), and PRIMA-l ⁇ 1 (APR246) (Aprea).
  • p53 activating drugs can include the molecules listed above whose mechanism of action is antagonizing MDM2.
  • Other p53 agonists include XI-011 (NSC146109), CGM097 (Novartis), MK-8242 (SCH900242) (Merck), Tenovin-1, Tenovin-6, and CP31398.
  • LSD1 inhibitors include, but are not limited to, poly amine analogs (see Huang et al, 104 PROC. NATL. ACAD. SCI. U.S.A. 8023-28 (2007), CBB-1007 (see Wang et al, 71 CANCER RES. 7238-49 (2011), namoline (see Willmann et al., 131 INT. J. CANCER 2704-09 (2012)), amidoximes (see Hazeldine et al, 55 J. MED. CHEM. 7378-91 (2012)), phenyl oxazoles (see Dulla et al, 11 ORG. BlOMOL. CHEM.
  • FIG. 1 A-1H Identification and characterization of a robust suppressor that ameliorates the locomotion defects in the C. elegans model of SOD 1 -associated ALS.
  • A Work flow of the suppressor screen identifying mutant C. elegans (red) with saliently improved movement.
  • FIG. 2A-2E Neuron-specific suppression of aggregation of diverse proteins is correlated with improved locomotion in ufd-2;spr-5 mutant animals.
  • A Schematic drawing at the top depicts pan-neuronal expression of YFP in head and ventral neurons in the context of the C. elegans body plan. Micrographs show the SOD1G85R-YFP (top panels) and TDP- C25-YFP (bottom panels) proteins expressed in the WT or the double mutant spr- 5(byl34);ufd-2(tml380) background. The double-mutant worms show a marked decrease in protein aggregation in neurons.
  • B Schematic drawing of muscle-directed YFP expression.
  • Micrographs show the polyQYFP proteins expressed in the C. elegans neurons (top panels) and body wall muscles (bottom panels). Only the neuronal protein aggregates are significantly decreased by spr-5(byl34);ufd- 2(tml380).
  • C A decrease in aggregated SOD1G85R-YFP or TDP-c25-YFP protein in the presence of spr-5(byl34);ufd-2(tml380), as shown by western blot analyses of the supernatant fractions (S) and the pellet fractions (P).
  • D Quantification of locomotion in the spr-5(byl34);ufd- 2(tml380) and the WT C.
  • FIG. 3A-3E UBE4B and LSD1 double-knockdown accelerates SOD1G85R protein degradation.
  • A Western blots of cell lysates derived from mock (CTRL), single UBE4B or LSD1, or double UBE4B and LSD1 knockdowns. Supernatant (S) and pellet (P) fractions were probed with indicated antibodies. While the LSDl or UBE4B single-knockdown reduces the SOD1G85R aggregates in both supernatant and pellet fractions, the combined knockdown produces the strongest reduction in the aggregates.
  • FIG. 4A-4H UBE4B and LSDl knockdown activates transcription mediated by p53 and FOXOs.
  • A Venn diagram of upstream activators (z-score >2) that are differentially activated in single, UBE4B or LSDl, and double UBE4B and LSDl knockdowns, compared to the control. Activation state of an upstream regulator is predicted from differential mRNA levels of its downstream target genes.
  • B The volcano scatter plot indicates fold changes in the levels of gene transcripts affected differentially by the UBE4B and LSDl double- knockdown versus control shRNA. Gray spots represents 22,148 annotated transcripts. Red spots are predicted p53- activated targets, and blue spots are predicted p53-inhibited targets.
  • FIG. 5A-5D UBE4B and LSD1 double-knockdown activates both proteasomes and autophagy.
  • A Increased protein levels of proteasome subunits upon the UBE4B and LSD1 double knockdown in HEK293T cells.
  • C The autophagy activity is significantly increased in cells with the UBE4B and LSD1 double-knockdown.
  • FIG. 6A-6C p53 promotes the clearance of misfolded SODl mutant proteins.
  • A p53 small molecule activators
  • Tenovin-1 and CP-31398 reduce the levels of misfolded SODl proteins, as determined by the SOD1G85R aggregation assay in HEK293 cells.
  • Increasing concentrations of the p53 activators significantly decrease the levels of SOD1G85R but not the endogenous WT SODl proteins in western blots of both supernatant and pellet fractions.
  • the middle graph indicates the ratio of G85R to WT SODl proteins in the presence or absence of p53 with varying amounts of transfected mutant SODl .
  • the right graph panel shows the same data as shown in the middle panel, but normalized to the average SOD1G85R level for each amount of the transfected plasmid. See also FIG. 12.
  • FIG. 7 Suppression of protein aggregates by UBE4B and LSDl knockdown depends on p53.
  • p53 knockdown reverses the suppression of SOD1G85R protein aggregates in the UBE4B and LSDl double-knockdown. Quantification of SOD1G85R in pellet fractions from HEK293T cells transfected with mock, double (UBE4B/LSD1), or triple
  • TDP-43M337V eye phenotype is exacerbated by the knockdown of the Drosophila homolog of p53 (p53-RNAi), or by the overexpression of a dominant negative p53 mutant (p53.R155H). Expression of p53-RNAi, p53.R155H, and TDP-43M337V are driven by GMR-Gal4.
  • C The p53 activator drug Tenovin-1 (TEN1) protects spinal cord motor neurons from SODlG85R-induced proteotoxicity. Toxicity assay on mixed spinal cord cultures treated with vehicle (DMSO, VEH) or Tenovin-1 (TEN1).
  • FIG. 8A-8C Knockdown of UBE4B and LSDl reduces TDP-43 ⁇ 2 1K protein aggregation in mammalian cells (related to FIG. 3).
  • A The flow chart of the mammalian cell-based protein aggregation assay as described herein.
  • B The western blots of TDP- 43 ⁇ 2 331K protein aggregation assay.
  • HEK293 cells were transfected with a TDP-43 Q 1K expression plasmid, together with a control shRNA (CTRL) or the mixed UBE4B and LSDl shRNA plasmids. Following cell lysis and fractionation, supernatant (S) and pellet (P) fractions were run on 15% SDS-PAGE gels.
  • FIG. 9A-9D The transcriptional profiling and network analysis (related to FIG. 4).
  • A The heat map of microarray signals of differentially regulated genes (p ⁇ 0.05) upon the knockdown of LSDl alone, UBE4B alone, or both, in triplicates. Hierarchical clustering of the samples indicates that the single UBE4B knockdown induces similar transcriptional changes as the double-knockdown, consistent with the pattern of anti-proteotoxic activities shown in FIGS. 3A and 3B.
  • B The heat map of p53 transcriptional targets. The hierarchical clustering of the samples demonstrates the same partem as shown above for all differentially regulated genes.
  • the transcriptional targets with changes consistent with p53 activation are shown, with up- regulated genes in red and downregulated genes in green.
  • FIG. 1 lA-11C The proteasomal and autophagic activity assays (related to FIG. 5).
  • FIG. 3 The flow chart of an autophagic activity assay to measure LC3 cleavage based on a luciferase (GLuc) reporter.
  • GLuc luciferase reporter.
  • Cells were transfected with a set of plasmids to knockdown LSD1 and UBE4B (or non- targeting shRNA, CTRL) and to express the GLuc reporters and SEAP (Secreted Embryonic Alkaline Phosphatase). SEAP is constitutively secreted and serves as a transfection normalization control. S0D1 G85R is expressed concurrently to match the condition with the increased burden of misfolded proteins, as described earlier (FIG. 3).
  • C A schematic of the LC3 cleavage and Glue release assay.
  • a cleavable fusion protein, Actin(Act)-LC3-GLuc, or its uncleavable negative control, Act-GLuc is anchored to the actin cytoskeleton inside the cell.
  • Act-LC3-GLuc is cleaved by the autophagy-associated protease ATG4B, the GLuc fragment is released from its actin anchor and rapidly secreted out of the cell.
  • the activity of GLuc in the cell medium is assayed over a period of several days using the Dual Luminescence Assay kit.
  • FIG. 12 p53-activating drugs enhance protein clearance of misfolded mutant SOD1 (related to FIG. 6).
  • VH vehicle-treated controls
  • FIG. 13A-13B p53 mediates improved protein clearance induced by the knockdown of UBE4B and LSDl (related to FIG. 7).
  • Stable knockdown of p53 partially blocks the improved clearance of SOD 1 G85R proteins conferred by the knockdown of UBE4B and LSDl.
  • a stable cell line with inducible knockdown of p53 via an integrated shRNA is used to conditionally remove p53 upon the induction of Doxycycline (DOX).
  • DOX Doxycycline
  • the protein aggregation assay was used to analyze the S0D1 G85R protein levels in S and P fractions.
  • Protein quality control is essential for clearing misfolded and aggregated proteins from the cell, or its failure would lead to numerous neurodegenerative disorders. How to boost protein quality control to enhance cellular defense against proteotoxicity is not well explored.
  • the human homologues, UBE4B and LSD1 encoding a ubiquitin ligase and a lysine-specific demethylase, when inactivated, suppress aggregation of disease-associated proteins in mammalian cells.
  • Caenorhabditis elegans model that expresses neuronal ALS-linked SODl mutant proteins and develops robust movement defects, and performed an unbiased forward genetic screen for potent suppressors of the behavioral defects.
  • the present inventors identified mutations in two genes, ufd-2, encoding a ubiquitin ligase, and spr-5, encoding a lysine-specific demethylase, that synergistically attenuate the neurotoxicity of mutant human SOD1 and other misfolded proteins.
  • these two post-translational lysine modifiers were found to be part of a pathway regulating protein quality control in human cells. Further analysis showed that this pathway acts through transcription factors such as p53 that mediate cellular stress responses. Together these results describe a new mechanism involving previously unrecognized players for the cell to reprogram cellular stress responses towards protein quality control.
  • Ranges may be expressed herein as from “about” one particular value, and/or to "about” another particular value.
  • the term “about” is used herein to mean approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as being within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, 0.5%, 0.1 %, 0.05%, or 0.01 % of the stated value. Unless otherwise clear from context, all numerical values provided herein can be modified by the term "about.”
  • an "agonist” is a type of modulator and refers to an agent that can activate one or more functions of the target.
  • an agonist of a protein can activate the protein in the absence of its natural or cognate ligand.
  • an "antagonist” is a type of modulator and is used interchangeably with the term “inhibitor.”
  • the term refers to an agent that can inhibit a one or more functions of the target.
  • an antagonist of an enzymatic protein can inhibit the enzymatic activity of the protein.
  • the term "antibody” is used in reference to any immunoglobulin molecule that reacts with a specific antigen. It is intended that the term encompass any immunoglobulin (e.g., IgG, IgM, IgA, IgE, IgD, etc.) obtained from any source (e.g., humans, rodents, non-human primates, caprines, bovines, equines, ovines, etc.).
  • antibodies include polyclonal, monoclonal, humanized, chimeric, human, or otherwise-human-suitable antibodies.
  • Antibodies also includes any functional fragment or derivative of any of the herein described antibodies. Functional fragments include antigen- binding fragments. In specific embodiments, antibodies may be raised against Ube4B and/or LSD 1 and used as Ube4B and/or LSD1 modulators. As used herein, the term "effective,” means adequate to accomplish a desired, expected, or intended result.
  • a "therapeutically effective amount” as provided herein refers to an amount of a Ube4B and/or LSD1 modulator of the present invention, either alone or in combination with another therapeutic agent, necessary to provide the desired therapeutic effect, e.g., an amount that is effective to prevent, alleviate, or ameliorate symptoms of disease or prolong the survival of the subject being treated.
  • the term “therapeutically effective amount” as provided herein refers to an amount of a Ube4B and/or LSD1 modulator, necessary to provide the desired therapeutic effect, e.g., an amount that is effective to prevent, alleviate, or ameliorate symptoms of disease or prolong the survival of the subject being treated.
  • the disease or condition is a protein conformation disease.
  • the exact amount required will vary from subject to subject, depending on age, general condition of the subject, the severity of the condition being treated, the particular compound and/or composition administered, and the like.
  • An appropriate "therapeutically effective amount” in any individual case can be determined by one of ordinary skill in the art by reference to the pertinent texts and literature and/or by using routine experimentation.
  • high stringency conditions conditions that allow hybridization comparable with that resulting from the use of a DNA probe of, for example, at least 40 nucleotides in length, in a buffer containing 0.5 M NaHP04, pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA (Fraction V), at a temperature of 65°C, or a buffer containing 48% formamide, 4.8XSSC, 0.2 M Tris-Cl, pH 7.6, lXDenhardt's solution, 10% dextran sulfate, and 0.1% SDS, at a temperature of 42°C
  • Other conditions for high stringency hybridization such as for PCR, Northern, Southern, or in situ hybridization, DNA sequencing, etc., are well-known by those skilled in the art of molecular biology. (See, for example, F. Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1998).
  • inhibitor is a type of modulator and is used interchangeably with the term “antagonist.”
  • the term “inhibitor” includes any type of molecule or agent that directly or indirectly inhibits the expression or activity of a target gene or protein.
  • An inhibitor can be any type of compound, such as a small molecule, polypeptide, polynucleotide and the like including an antibody or an RNA interference compound.
  • the target gene or protein is Ube4B and/or LSD1.
  • the term also includes agents that have activity in addition to Ube4B and/or LSD1 inhibitory activity.
  • the term “modulate” indicates the ability to control or influence directly or indirectly, and by way of non-limiting examples, can alternatively mean inhibit or stimulate, agonize or antagonize, hinder or promote, and strengthen or weaken.
  • Ube4B modulator and “LSD1 modulator” refers to an agent that modulates the expression and/or activity of Ube4B and LSD1 , respectively.
  • Inhibitors may be organic or inorganic, small to large molecular weight individual compounds, mixtures and combinatorial libraries of inhibitors, agonists, antagonists, and biopolymers such as peptides, nucleic acids, or oligonucleotides.
  • a modulator may be a natural product or a naturally-occurring small molecule organic compound. In particular, a modulator may be a carbohydrate;
  • polypeptide protein; receptor; nucleic acid; nucleoside; nucleotide; oligonucleotide;
  • a modulator identified according to the invention is preferably useful in the treatment of a disease disclosed herein.
  • nucleic acid refers to a naturally occurring or synthetic oligonucleotide or polynucleotide, whether DNA or RNA or DNA-RNA hybrid, single- stranded or double-stranded, sense or antisense, which is capable of hybridization to a complementary nucleic acid by Watson-Crick base-pairing.
  • Nucleic acids of the invention can also include nucleotide analogs (e.g., BrdU), and non-phosphodiester internucleoside linkages (e.g., peptide nucleic acid (PNA) or thiodiester linkages).
  • nucleic acids can include, without limitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA or any combination thereof.
  • patient refers to a mammal, particularly, a human.
  • the patient may have a mild, intermediate or severe disease or condition, he patient may be an individual in need of treatment or in need of diagnosis based on particular symptoms or family history.
  • the terms may refer to treatment in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters; and primates.
  • rodents including mice, rats, and hamsters; and primates.
  • rodents including mice, rats, and hamsters
  • primates primates.
  • the term also includes mammals diagnosed with a protein conformational disease, disorder or condition.
  • normal subject is meant an individual who does not have a protein conformational disease as well as an individual who has increased susceptibility for developing a protein conformational disease.
  • Polypeptide refers to any peptide, oligopeptide, polypeptide, gene product, expression product, or protein. A polypeptide is comprised of consecutive amino acids.
  • the term “polypeptide” encompasses naturally occurring or synthetic molecules.
  • the term “polypeptide” refers to amino acids joined to each other by peptide bonds or modified peptide bonds, e.g., peptide isosteres, etc., and may contain modified amino acids other than the 20 gene-encoded amino acids.
  • the polypeptides can be modified by either natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art.
  • Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini.
  • the same type of modification can be present in the same or varying degrees at several sites in a given polypeptide.
  • a given polypeptide can have many types of modifications.
  • Modifications include, without limitation, acetylation, acylation, ADP-ribosylation, amidation, covalent cross-linking or cyclization, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphytidylinositol, disulfide bond formation, demethylation, formation of cysteine or pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristolyation, oxidation, pergylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and transfer- RNA mediated addition of amino acids to protein such as arginylation.
  • probe By “probe,” “primer,” or oligonucleotide is meant a single-stranded DNA or RNA molecule of defined sequence that can base-pair to a second DNA or RNA molecule that contains a complementary sequence (the “target”).
  • target a complementary sequence
  • the stability of the resulting hybrid depends upon the extent of the base-pairing that occurs.
  • the extent of base-pairing is affected by parameters such as the degree of complementarity between the probe and target molecules and the degree of stringency of the hybridization conditions.
  • the degree of hybridization stringency is affected by parameters such as temperature, salt concentration, and the concentration of organic molecules such as formamide, and is determined by methods known to one skilled in the art.
  • Probes or primers specific for Ube4B and/or LSDl nucleic acids have at least 80%-90% sequence complementarity, preferably at least 9 ⁇ %-95% sequence complementarity, more preferably at least 96%-99% sequence complementarity, and most preferably 100% sequence complementarity to the region of the Ube4B and/or LSDl nucleic acid to which they hybridize.
  • Probes, primers, and oligonucleotides may be detectably -labeled, either radioactively, or non-radioactively, by methods well-known to those skilled in the art.
  • Probes, primers, and oligonucleotides are used for methods involving nucleic acid hybridization, such as: nucleic acid sequencing, reverse transcription and/or nucleic acid amplification by the polymerase chain reaction, single stranded conformational polymorphism (SSCP) analysis, restriction fragment polymorphism (RFLP) analysis, Southern hybridization, Northern hybridization, in situ hybridization, electrophoretic mobility shift assay (EMSA).
  • SSCP single stranded conformational polymorphism
  • RFLP restriction fragment polymorphism
  • Southern hybridization Southern hybridization
  • Northern hybridization in situ hybridization
  • ESA electrophoretic mobility shift assay
  • protein conformational disease refers to all the pathological conditions that are associated with protein misfolding and aggregation, or proteotoxicity, and specifically include neurodegenerative diseases.
  • protein conformational diseases include, but are not limited to, Creutzfeldt-Jakob disease, Alzheimer's disease, Parkinson's disease, Huntington's disease, frontotemporal dementia, and amyotrophic lateral sclerosis (ALS).
  • binding refers to that binding which occurs between such paired species as antibody/antigen, enzyme/substrate, receptor/agonist, and lectin/carbohydrate which may be mediated by covalent or non-covalent interactions or a combination of covalent and non-covalent interactions.
  • the binding which occurs is typically electrostatic, hydrogen-bonding, or the result of lipophilic interactions. Accordingly, "specific binding” occurs between a paired species where there is interaction between the two which produces a bound complex having the characteristics of an antibody/antigen or enzyme/substrate interaction.
  • the specific binding is characterized by the binding of one member of a pair to a particular species and to no other species within the family of compounds to which the corresponding member of the binding member belongs.
  • an antibody typically binds to a single epitope and to no other epitope within the family of proteins.
  • specific binding between an antigen and an antibody will have a binding affinity of at least 10 "6 M.
  • the antigen and antibody will bind with affinities of at least lO "7 M, 10 "8 M to 10 "9 M, 10 "10 M, 10 "11 M, or 10 "12 M.
  • a probe, primer, or oligonucleotide recognizes and physically interacts (that is, base-pairs) with a substantially complementary nucleic acid (for example, a Ube4B and/or LSD1 nucleic acid) under high stringency conditions, and does not substantially base pair with other nucleic acids.
  • a substantially complementary nucleic acid for example, a Ube4B and/or LSD1 nucleic acid
  • treatment refers to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse affect attributable to the disease.
  • Treatment covers any treatment of a disease in a subject, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, e.g., causing regression of the disease, e.g., to completely or partially remove symptoms of the disease.
  • the disease or condition is a protein conformational disease.
  • the Ube4B and/or LSD1 inhibitor is selected from the group consisting of a small molecule, a polypeptide, a nucleic acid molecule, a peptidomimetic, or a combination thereof.
  • the agent can be a polypeptide.
  • the polypeptide can, for example, comprise an antibody.
  • the agent can be a nucleic acid molecule.
  • the nucleic acid molecule can, for example, be a Ube4B and/or LSD1 inhibitory nucleic acid molecule.
  • the Ube4B and/or LSD1 inhibitory nucleic acid molecule can comprise a short interfering RNA (siRNA) molecule, a microRNA (miRNA) molecule, or an antisense molecule.
  • RNA Interference Compositions for Targeting Ube4B and LSD1 mRNA may be inhibited by the use of RNA interference techniques (RNAi).
  • RNAi is a remarkably efficient process whereby double-stranded RNA (dsRNA) induces the sequence-specific degradation of homologous mRNA in animals and plant cells. See Hutvagner and Zamore, 12 CURR. OPIN. GENET. DEV. 225-32 (2002); Hammond et al, 2 NATURE REV. GEN. 110-19 (2001); Sharp, 15 GENES DEV. 485-90 (2001).
  • RNAi can be triggered, for example, by nucleotide (nt) duplexes of small interfering RNA (siRNA) (Chiu et al, 10 MOL. CELL. 549-61 (2002); Elbashir et al, 411 Nature 494-98 (2001)), micro-RNAs (miRNA), functional small-hai in RNA (shRNA), or other dsRNAs which are expressed in-vivo using DNA templates with RNA polymerase III promoters. See, e.g., Zeng et al, 9 MOL. CELL. 1327-33 (2002);
  • a Ube4B and/or LSDl inhibitory nucleic acid sequence can be a siRNA sequence or a miRNA sequence.
  • a 21-25 nucleotide siRNA or miRNA sequence can, for example, be produced from an expression vector by transcription of a short-hairpin RNA (shRNA) sequence, a 60-80 nucleotide precursor sequence, which is processed by the cellular RNAi machinery to produce either an siRNA or miRNA sequence.
  • shRNA short-hairpin RNA
  • a 21-25 nucleotide siRNA or miRNA sequence can, for example, be synthesized chemically.
  • siRNA sequences Chemical synthesis of siRNA or miRNA sequences is commercially available from such corporations as Dharmacon, Inc. (Lafayette, Colo.), Qiagen (Valencia, Calif), and Ambion, Inc. (Austin, Tex.).
  • An siRNA sequence preferably binds a unique sequence within the Ube4B and/or LSDl mRNA with exact complementarity and results in the degradation of the Ube4B and/or LSDl mRNA molecule.
  • An siRNA sequence can bind anywhere within the mRNA molecule.
  • An miRNA sequence preferably binds a unique sequence within the
  • Ube4B and/or LSDl mRNA with exact or less than exact complementarity and results in the translational repression of the Ube4B and/or LSDl mRNA molecule.
  • An miRNA sequence can bind anywhere within the mRNA molecule, but preferably binds within the 3'UTR of the mRNA molecule.
  • Methods of delivering siRNA or miRNA molecules are known in the art. See, e.g., Oh and Park, Adv. Drug Deliv. Rev. 61(10):850-62 (2009); Gondi and Rao, J. Cell. Physiol. 220(2):285-91 (2009); and Whitehead et al, Nat. Rev. Drug Discov. 8(2)129-38 (2009).
  • a Ube4B and/or LSDl inhibitory nucleic acid sequence can be an antisense nucleic acid sequence.
  • Antisense nucleic acid sequences can, for example, be transcribed from an expression vector to produce an RNA which is complementary to at least a unique portion of the Ube4B and/or LSDl mRNA and/or the endogenous gene which encodes Ube4B and/or LSDl. Hybridization of an antisense nucleic acid molecule under specific cellular conditions results in inhibition of Ube4B and/or LSDl protein expression by inhibiting transcription and/or translation. i. Small Interfering RNA
  • the present invention features "small interfering RNA molecules" ("siRNA molecules” or “siRNA”), methods of making siRNA molecules and methods for using siRNA molecules (e.g., research and/or therapeutic methods).
  • siRNA molecules small interfering RNA molecules
  • methods of making siRNA molecules e.g., research and/or therapeutic methods.
  • the siRNAs of this invention encompass any siRNAs that can modulate the selective degradation of Ube4B and/or LSD1 mRNA. Examples of LSD 1 and Ube4B siRNA are shown in SEQ ID NOS: 14-19 and SEQ ID NOS:20-49, respectively.
  • the siRNA of the present invention may comprise double- stranded small interfering RNA molecules (ds-siRNA).
  • ds-siRNA double- stranded small interfering RNA molecules
  • a ds-siRNA molecule of the present invention may be a duplex made up of a sense strand and a complementary antisense strand, the antisense strand being sufficiently complementary to a target Ube4B or LSD1 mRNA to mediate RNAi.
  • the siRNA molecule may comprise about 10 to about 50 or more nucleotides. More specifically, the siRNA molecule may comprise about 16 to about 30, e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in each strand.
  • the strands may be aligned such that there are at least 1, 2, or 3 bases at the end of the strands which do not align (e.g., for which no complementary bases occur in the opposing strand) such that an overhang of 1, 2 or 3 residues occurs at one or both ends of the duplex when strands are annealed.
  • the siRNA of the present invention may comprise single-stranded small interfering RNA molecules (ss-siRNA). Similar to the ds-siRNA molecules, the ss-siRNA molecule may comprise about 10 to about 50 or more nucleotides. More specifically, the ss-siRNA molecule may comprise about 15 to about 45 or more nucleotides. Alternatively, the ss-siRNA molecule may comprise about 19 to about 40 nucleotides.
  • ss-siRNA single-stranded small interfering RNA molecules
  • the ss-siRNA molecules of the present invention comprise a sequence that is "sufficiently complementary" to a target mRNA sequence to direct target-specific RNA interference (RNAi), as defined herein, e.g., the ss-siRNA has a sequence sufficient to trigger the destruction of the target mRNA by the RNAi machinery or process.
  • RNAi target-specific RNA interference
  • the ss-siRNA molecule can be designed such that every residue is complementary to a residue in the target molecule.
  • substitutions can be made within the molecule to increase stability and/or enhance processing activity of the molecule. Substitutions can be made within the strand or can be made to residues at the ends of the strand.
  • the 5 '-terminus may be phosphorylated (e.g., comprises a phosphate, diphosphate, or triphosphate group).
  • the 3' end of an siRNA may be a hydroxyl group in order to facilitate RNAi, as there is no requirement for a 3' hydroxyl group when the active agent is a ss-siRNA molecule.
  • the 3' end (e.g., C3 of the 3' sugar) of ss-siRNA molecule may lack a hydroxyl group (e.g., ss-siRNA molecules lacking a 3 ' hydroxyl or C3 hydroxyl on the 3 ' sugar (e.g., ribose or deoxyribose).
  • the siRNA molecules of the present invention may be modified to improve stability under in vitro and/or in vivo conditions, including, for example, in serum and in growth medium for cell cultures.
  • the 3 '-residues may be stabilized against degradation, e.g., they may be selected such that they consist of purine nucleotides, particularly adenosine or guanosine nucleotides.
  • substitution of pyrimidine nucleotides by modified analogues e.g., substitution of uridine by 2'- deoxythymidine is tolerated and does not affect the efficiency of RNA interference.
  • the absence of a 2' hydroxyl may significantly enhance the nuclease resistance of the siRNAs in tissue culture medium.
  • siRNAs of the present invention may include modifications to the sugar-phosphate backbone or nucleosides. These modifications can be tailored to promote selective genetic inhibition, while avoiding a general panic response reported to be generated by siRNA in some cells. In addition, modifications can be introduced in the bases to protect siRNAs from the action of one or more endogenous enzymes.
  • the siRNA molecule may contain at least one modified nucleotide analogue.
  • the nucleotide analogues may be located at positions where the target-specific activity, e.g., the RNAi mediating activity is not substantially effected, e.g., in a region at the 5 '-end and/or the 3 '-end of the RNA molecule. Particularly, the ends may be stabilized by incorporating modified nucleotide analogues.
  • examples of nucleotide analogues include sugar- and/or backbone-modified ribonucleotides (e.g., include modifications to the phosphate-sugar backbone).
  • the phosphodiester linkages of natural RNA may be modified to include at least one of a nitrogen or sulfur heteroatom.
  • the phosphoester group connecting to adjacent ribonucleotides may be replaced by a modified group, e.g., a phosphothioate group.
  • the 2' OH-group may be replaced by a group selected from H, OR, R, halo, SH, SR, NH 2 , NHR, NR 2 or ON, wherein R is Ci-Ce alkyl, alkenyl or alkynyl and halo is F, CI, Br or I.
  • Nucleobase-modified ribonucleotides may also be utilized, e.g., ribonucleotides containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase. Bases may be modified to block the activity of adenosine deaminase.
  • modified nucleobases include, but are not limited to, uridine and/or cytidine modified at the 5-position, e.g., 5-(2-amino)propyl uridine, 5-bromo uridine; adenosine and/or guanosines modified at the 8 position, e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; O- and N-alkylated nucleotides, e.g., N6-methyl adenosine are suitable. It should be noted that the above modifications may be combined.
  • siRNA derivatives may also be utilized herein.
  • cross-linking can be employed to alter the pharmacokinetics of the composition, e.g., to increase half-life in the body.
  • the present invention includes siRNA derivatives that include siRNA having two complementary strands of nucleic acid, such that the two strands are crosslinked.
  • the present invention also includes siRNA derivatives having a non-nucleic acid moiety conjugated to its 3' terminus (e.g., a peptide), organic compositions (e.g., a dye), or the like.
  • Modifying siRNA derivatives in this way may improve cellular uptake or enhance cellular targeting activities of the resulting siRNA derivative as compared to the corresponding siRNA, are useful for tracing the siRNA derivative in the cell, or improve the stability of the siRNA derivative compared to the corresponding siRNA.
  • siRNAs of the present invention can be enzymatically produced or totally or partially synthesized. Moreover, the siRNAs can be synthesized in vivo or in vitro. For siRNAs that are biologically synthesized, an endogenous or a cloned exogenous RNA polymerase may be used for transcription in vivo, and a cloned RNA polymerase can be used in vitro. siRNAs that are chemically or enzymatically synthesized are preferably purified prior to the introduction into the cell.
  • siRNA molecules that contain some degree of modification in the sequence can also be adequately used for the purpose of this invention. Such modifications may include, but are not limited to, mutations, deletions or insertions, whether spontaneously occurring or intentionally introduced.
  • siRNAs not all positions of a siRNA contribute equally to target recognition.
  • mismatches in the center of the siRNA may be critical and could essentially abolish target RNA cleavage.
  • the 3' nucleotides of the siRNA do not contribute significantly to specificity of the target recognition.
  • residues 3 ' of the siRNA sequence which is complementary to the target RNA may not critical for target RNA cleavage.
  • Sequence identity may be determined by sequence comparison and alignment algorithms known to those of ordinary skill in the art. To determine the percent identity of two nucleic acid sequences (or of two amino acid sequences), the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first sequence or second sequence for optimal alignment). The nucleotides (or amino acid residues) at corresponding nucleotide (or amino acid) positions are then compared. When a position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the alignment generated over a certain portion of the sequence aligned having sufficient identity but not over portions having low degree of identity e.g., a local alignment.
  • a non-limiting example of a local alignment algorithm utilized for the comparison of sequences is the algorithm of Karlin and Altschul, 87 PROC. NATL. ACAD. SCI. USA 2264-68 (1990), and as modified as in Karlin and Altschul 90 PROC. NATL. ACAD. SCI. USA 5873-77 (1993). Such an algorithm is incorporated into the BLAST programs (version 2.0) of Altschul, et al, 215 J. MOL. BIOL. 403-10 (1990).
  • the alignment may optimized by introducing appropriate gaps and determining percent identity over the length of the aligned sequences (e.g., a gapped alignment).
  • Gapped BLAST can be utilized as described in Altschul et al, 25(17) NUCLEIC ACIDS RES. 3389-3402 (1997).
  • the alignment may be optimized by introducing appropriate gaps and determining percent identity over the entire length of the sequences aligned (e.g., a global alignment).
  • a non-limiting example of a mathematical algorithm utilized for the global comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package.
  • a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
  • siRNA may be defined functionally as a nucleotide sequence (or oligonucleotide sequence) that is capable of hybridizing with a portion of the target gene transcript (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C hybridization for 12-16 hours; followed by washing).
  • a portion of the target gene transcript e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C hybridization for 12-16 hours; followed by washing.
  • Additional hybridization conditions include, but are not limited to, hybridization at 70°C in IxSSC or 50°C in IxSSC, 50% formamide followed by washing at 70°C in 0.3xSSC or hybridization at 70°C in 4xSSC or 50°C in 4xSSC, 50% formamide followed by washing at 67°C in IxSSC.
  • stringency conditions for polynucleotide hybridization are provided in Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11, and Current Protocols in Molecular
  • the length of the identical nucleotide sequences may be at least about 10, 12, 15, 17, 20, 22, 25, 27, 30, 32, 35, 37, 40, 42, 45, 47 50 or more bases.
  • Antisense molecules can act in various stages of transcription, splicing and translation to block the expression of a target gene. Without being limited by theory, antisense molecules can inhibit the expression of a target gene by inhibiting transcription initiation by forming a triple strand, inhibiting transcription initiation by forming a hybrid at an RNA polymerase binding site, impeding transcription by hybridizing with an RNA molecule being synthesized, repressing splicing by hybridizing at the junction of an exon and an intron or at the spliceosome formation site, blocking the translocation of an mRNA from nucleus to cytoplasm by hybridization, repressing translation by hybridizing at the translation initiation factor binding site or ribosome biding site, inhibiting peptide chain elongation by hybridizing with the coding region or polysome binding site of an mRNA, or repressing gene expression by hybridizing at the sites of interaction between nucleic acids and
  • an antisense oligonucleotide of the present invention is a cDNA that, when introduced into a cell, transcribes into an RNA molecule having a sequence complementary to at least part of the Ube4B or LSD1 mRNA.
  • antisense oligonucleotides of the present invention include
  • oligonucleotides having modified sugar-phosphodiester backbones or other sugar linkages which can provide stability against endonuclease attacks.
  • the present invention also encompasses antisense oligonucleotides that are covalently attached to an organic or other moiety that increase their affinity for a target nucleic acid sequence.
  • intercalating agents, alkylating agents, and metal complexes can be also attached to the antisense oligonucleotides of the present invention to modify their binding specificities.
  • the present invention also provides ribozymes as a tool to inhibit Ube4B and/or LSD1 expression.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
  • the characteristics of ribozymes are well-known in the art. See, e.g., Rossi, 4 CURRENT BIOLOGY 469-71 (1994).
  • the mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by an endonucleolytic cleavage.
  • the ribozyme molecules include one or more sequences
  • the expression of the Ube4B and/or LSD1 genes can also be inhibited by using triple helix formation.
  • Nucleic acid molecules to be used in triple helix formation for the inhibition of transcription can be single stranded and composed of deoxynucleotides.
  • the base composition of these oligonucleotides must be designed to promote triple helix formation via Hoogsteen base paring rules, which generally require sizeable stretches of either purines or pyrimidines to be present on one strand of a duplex.
  • Nucleotide sequences may be pyrimidine-based, which will result in TAT and CGC + triplets across the three associated strands of the resulting triple helix.
  • the pyrimidine-rich molecules provide base
  • nucleic acid molecules that are purine-rich e.g., containing a stretch of G residues, may be chosen. These molecules will form a triple helix with a DNA duplex that is rich in GC pairs, in which the majority of the purine residues are located on a single strand of the targeted duplex, resulting in GGC triplets across the three strands in the triplex.
  • the potential sequences that can be targeted for triple helix formation may be increased by creating a so-called "switchback" nucleic acid molecule.
  • Switchback molecules are synthesized in an alternating 5 '-3 ',3 '-5' manner, such that they base pair first with one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.
  • Co-repression refers to the phenomenon in which, when a gene having an identical or similar to the target sequence is introduced to a cell, expression of both introduced and endogenous genes becomes repressed. This phenomenon, although first observed in plant system, has been observed in certain animal systems as well.
  • the sequence of the gene to be introduced does not have to be identical to the target sequence, but sufficient homology allows the co-repression to occur. The determination of the extent of homology depends on individual cases, and is within the ordinary skill in the art.
  • siRNA and other nucleic acids designed to bind to a target mRNA e.g., shRNA, stRNA, antisense oligonucleotides, ribozymes, and the like, that are advantageously used in accordance with the present invention.
  • each AA dinucleotide sequence and the 3' adjacent 16 or more nucleotides are potential siRNA targets.
  • the siRNA is specific for a target region that differs by at least one base pair between the wild type and mutant allele or between splice variants.
  • the first strand is complementary to this sequence, and the other strand identical or substantially identical to the first strand.
  • siRNAs with lower G/C content 35-55%) may be more active than those with G/C content higher than 55%.
  • the invention includes nucleic acid molecules having 35-55% G/C content.
  • the strands of the siRNA can be paired in such a way as to have a 3' overhang of 1 to 4, e.g., 2, nucleotides.
  • the nucleic acid molecules may have a 3' overhang of 2 nucleotides, such as TT.
  • the overhanging nucleotides may be either RNA or DNA.
  • BLAST National Center for Biotechnology Information website
  • the GC content of the selected sequence should be from about 30% to about 70%, preferably about 50%.
  • sequences absent from other genes are preferred.
  • the secondary structure of the target mRNA may be determined or predicted, and it may be preferable to select a region of the mRNA that has little or no secondary structure, but it should be noted that secondary structure seems to have little impact on RNAi.
  • siRNA sbRNA or stRNA (as well as other antisense oligonucleotides)
  • sequences that bind transcription and/or translation factors should be avoided, as they might competitively inhibit the binding of a siRNA, sbRNA or stRNA (as well as other antisense oligonucleotides) to the mRNA.
  • siRNA siRNA User Guide
  • Negative control siRNAs should have the same nucleotide composition as the selected siRNA, but without significant sequence complementarity to the appropriate genome.
  • Such negative controls may be designed by randomly scrambling the nucleotide sequence of the selected siRNA; a homology search can be performed to ensure that the negative control lacks homology to any other gene in the appropriate genome.
  • compositions of the present invention e.g., siRNAs, antisense oligonucleotides, or other compositions described herein
  • Delivery of the compositions of the present invention into a patient can either be direct, e.g., the patient is directly exposed to the compositions of the present invention or compound- carrying vector, or indirect, e.g., cells are first transformed with the compositions of this invention in vitro, then transplanted into the patient for cell replacement therapy.
  • in vivo and ex vivo therapy are known as in vivo and ex vivo therapy, respectively.
  • the compositions of the present invention are directly administered in vivo, where they are expressed to produce the encoded product.
  • compositions of the present invention can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor. See, e.g., W093/14188, WO
  • Ex vivo therapy involves transferring the compositions of the present invention to cells in tissue culture by methods well-known in the art such as electroporation, transfection, lipofection, microinjection, calcium phosphate mediated transfection, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, and infection with a viral vector containing the nucleic acid sequences.
  • These techniques should provide for the stable transfer of the compositions of this invention to the cell, so that they are expressible by the cell and preferably heritable and expressible by its cell progeny.
  • the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred compositions.
  • the resulting recombinant cells can be delivered to a patient by various methods known in the art. Examples of the delivery methods include, but are not limited to, subcutaneous injection, skin graft, and intravenous injection.
  • a Ube4B and/or LSD1 inhibitor is a small molecule.
  • small molecule organic compounds refers to organic compounds generally having a molecular weight less than about 5000, 4000, 3000, 2000, 1000, 800, 600, 500, 250 or 100 Daltons, preferably less than about 500 Daltons.
  • a small molecule organic compound may be prepared by synthetic organic techniques, such as by combinatorial chemistry techniques, or it may be a naturally-occurring small molecule organic compound.
  • Specific examples of LSD1 small molecule inhibitors can be found in PCT Publication Nos. WO 2014/100818, WO 2014/100463, WO 2014/085613, WO 2014084298, WO 2012/135113, WO
  • compound libraries may be screened for Ube4B and/or LSD1 inhibitors.
  • a compound library is a mixture or collection of one or more putative inhibitors generated or obtained in any manner. Any type of molecule that is capable of interacting, binding or has affinity for Ube4B and/or LSD1 may be present in the compound library.
  • compound libraries screened using this invention may contain naturally-occurring molecules, such as carbohydrates, monosaccharides, oligosaccharides, polysaccharides, amino acids, peptides, oligopeptides, polypeptides, proteins, receptors, nucleic acids, nucleosides, nucleotides, oligonucleotides, polynucleotides, including DNA and DNA fragments, RNA and RNA fragments and the like, lipids, retinoids, steroids, glycopeptides, glycoproteins, proteoglycans and the like; or analogs or derivatives of naturally-occurring molecules, such as peptidomimetics and the like; and non-naturally occurring molecules, such as "small molecule" organic compounds generated, for
  • a library typically contains more than one putative inhibitor or member, i.e., a plurality of members or putative inhibitors.
  • a compound library may comprise less than about 50,000, 25,000, 20,000, 15,000, 10000, 5000, 1000, 500 or 100 putative inhibitors, in particular from about 5 to about 100, 5 to about 200, 5 to about 300, 5 to about 400, 5 to about 500, 10 to about 100, 10 to about 200, 10 to about 300, 10 to about 400, 10 to about 500, 10 to about 1000, 20 to about 100, 20 to about 200, 20 to about 300, 20 to about 400, 20 to about 500, 20 to about 1000, 50 to about 100, 50 to about 200, 50 to about 300, 50 to about 400, 50 to about 500, 50 to about 1000, 100 to about 200, 100 to about 300, 100 to about 400, 100 to about 500, 100 to about 1000, 200 to about 300, 200 to about 400, 200 to about 500, 200 to about 1000, 300 to about 500, 300 to about 1000, 300 to 2000, 300 to 3000
  • a compound library may be prepared or obtained by any means including, but not limited to, combinatorial chemistry techniques, fermentation methods, plant and cellular extraction procedures and the like.
  • a library may be obtained from synthetic or from natural sources such as for example, microbial, plant, marine, viral and animal materials. Methods for making libraries are well-known in the art. See, for example, E. R. Felder, Chimia 1994, 48, 512-541 ; Gallop et al, J. Med. Chem. 1994, 37, 1233-1251; R. A. Houghten, Trends Genet.
  • Compound libraries may also be obtained from commercial sources including, for example, from May bridge, ChemNavigator.com, Timtec Corporation, ChemBridge Corporation, A- Syntese-Biotech ApS, Akos-SC, G & J Research Chemicals Ltd., Life Chemicals, Interchim S.A., and Spectrum Info. Ltd.
  • antibody is used herein in a broad sense and includes both polyclonal and monoclonal antibodies.
  • the term can also refer to a human antibody and/or a humanized antibody. Examples of techniques for human monoclonal antibody production include those described by Cole et al. (Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985)) and by Boerner et al. (J. Immunol. 147(l):86-95 (1991)). Human antibodies (and fragments thereof) can also be produced using phage display libraries (Hoogenboom et al., J. Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol.
  • the disclosed human antibodies can also be obtained from transgenic animals.
  • transgenic mutant mice that are capable of producing a full repertoire of human antibodies, in response to immunization, have been described (see, e.g., Jakobovits et al, Proc. Natl. Acad. Sci. USA 90:2551-5 (1993); Jakobovits et al, Nature 362:255-8 (1993); Bruggermann et al., Year in Immunol. 7:33 (1993)).
  • Antibodies of the present invention include, but are not limited to, synthetic antibodies, polyclonal antibodies, monoclonal antibodies, recombinantly produced antibodies, intrabodies, multispecific antibodies (including bi-specific antibodies), human antibodies, humanized antibodies, chimeric antibodies, synthetic antibodies, single-chain Fvs (scFv) (including bi-specific scFvs), single chain antibodies Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.
  • synthetic antibodies polyclonal antibodies, monoclonal antibodies, recombinantly produced antibodies, intrabodies, multispecific antibodies (including bi-specific antibodies), human antibodies, humanized antibodies, chimeric antibodies, synthetic antibodies, single-chain Fvs (scFv) (including bi-specific scFvs), single chain antibodies Fab fragments, F(ab') fragments, disulfide-linked
  • antibodies of the present invention include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, e.g., molecules that contain an antigen binding site that immunospecifically binds to an antigen (e.g., one or more complementarity determining regions (CDRs) of an antibody).
  • immunoglobulin molecules e.g., molecules that contain an antigen binding site that immunospecifically binds to an antigen (e.g., one or more complementarity determining regions (CDRs) of an antibody).
  • CDRs complementarity determining regions
  • Mimetics are peptide-containing molecules that mimic elements of protein secondary structure. See, for example, Johnson et al, "Peptide Turn Mimetics” in BIOTECHNOLOGY AND PHARMACY, Pezzuto et al, Eds., Chapman and Hall, New York (1993).
  • the underlying rationale behind the use of peptide mimetics in rational design is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and antigen.
  • a peptide mimetic is expected to permit molecular interactions similar to the natural molecule.
  • peptide mapping may be used to determine "active" antigen recognition residues, and along with molecular modeling and molecular dynamics trajectory analysis, peptide mimic of the antibodies containing antigen contact residues from multiple CDRs may be prepared.
  • an antibody specifically binds an epitope of the Ube4B or LSD1 protein.
  • the peptide regions may not necessarily precisely map one epitope, but may also contain a Ube4B or LSD1 sequence that is not immunogenic.
  • Methods of predicting other potential epitopes to which an immunoglobulin of the invention can bind are well-known to those of skill in the art and include, without limitation, Kyte- Doolittle Analysis (Kyte, J. and Dolittle, R. F., 157 J. MOL. BlOL. 105-32 (1982)); Hopp and Woods Analysis (Hopp, T. P. and Woods, K. R, 78 PROC. NATL. ACAD. SCI.
  • Amino acid sequence variants of the Ube4B and LSD1 antibodies of the present invention may be prepared by introducing appropriate nucleotide changes into the polynucleotide that encodes the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the antibody. Any combination of deletions, insertions, and substitutions may be made to arrive at the final construct.
  • Amino acid sequence insertions include amino-terminal and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include an antibody with an N-terminal methionyl residue or the antibody fused to a cytotoxic polypeptide.
  • Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody of a polypeptide that increases the serum half-life of the antibody.
  • antibody variants are an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule replaced by a different residue.
  • sites of greatest interest for substitutional mutagenesis of antibodies include the hypervariable regions, but framework region (FR) alterations are also contemplated.
  • a useful method for the identification of certain residues or regions of the Ube4B and LSD 1 antibodies that are preferred locations for substitution, i.e., mutagenesis is alanine scanning mutagenesis. See Cunningham & Wells, 244 SCIENCE 1081-85 (1989). Briefly, a residue or group of target residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with antigen.
  • the amino acid locations demonstrating functional sensitivity to the substitutions are refined by introducing further or other variants at, or for, the sites of substitution.
  • the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined.
  • alanine scanning or random mutagenesis may be conducted at the target codon or region and the expressed antibody variants screened for the desired activity.
  • Substantial modifications in the biological properties of the antibody can be accomplished by selecting substitutions that differ significantly in their effect on, maintaining (i) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (ii) the charge or hydrophobicity of the molecule at the target site, or (iii) the bulk of the side chain.
  • Naturally occurring residues are divided into groups based on common side-chain properties:
  • hydrophobic norleucine, met, ala, val, leu, ile
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
  • Conservative substitutions involve exchanging of amino acids within the same class.
  • cysteine residues not involved in maintaining the proper conformation of the antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking.
  • cysteine bond(s) may be added to the antibody to improve its stability, particularly where the antibody is an immunoglobulin fragment such as an Fv fragment.
  • substitutional variant involves substituting one or more hypervariable region residues of a parent antibody.
  • the resulting variant(s), i.e., functional equivalents as defined above, selected for further development will have improved biological properties relative to the parent antibody from which they are generated.
  • a convenient way for generating such substitutional variants is by affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino substitutions at each site.
  • the antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of Ml 3 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as herein disclosed.
  • alanine- scanning mutagenesis may be performed to identify hypervariable region residues contributing significantly to antigen binding.
  • ADCC antigen- dependent cell-mediated cyotoxicity
  • CDC complement dependent cytotoxicity
  • This may be achieved by introducing one or more amino acid substitutions in an Fc region of an antibody.
  • cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region.
  • the homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC).
  • ADCC complement-mediated cell killing and antibody-dependent cellular cytotoxicity
  • an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. Stevenson et al, 3 ANTI-CANCER DRUG DESIGN 219-30 (1989).
  • a salvage receptor binding epitope refers to an epitope of the Fc region of an IgG molecule (e.g., IgGl, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.
  • Polynucleotide molecules encoding amino acid sequence variants of the antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the anti-Ube4B and LSD1 antibodies of the present invention.
  • a pharmaceutical composition of the present invention may comprise an effective amount of a Ube4B and/or LSD1 inhibitor.
  • the term "effective,” means adequate to accomplish a desired, expected, or intended result. More particularly, an "effective amount” or a “therapeutically effective amount” is used interchangeably and refers to an amount of a Ube4B and/or LSD1 inhibitor, perhaps in further combination with yet another therapeutic agent, necessary to provide the desired "treatment” (defined herein) or therapeutic effect, e.g., an amount that is effective to prevent, alleviate, treat or ameliorate symptoms of a disease or prolong the survival of the subject being treated.
  • the pharmaceutical compositions of the present invention are administered in a therapeutically effective amount to treat patients suffering from a protein conformational disease.
  • a therapeutically effective amount to treat patients suffering from a protein conformational disease.
  • the exact low dose amount required will vary from subject to subject, depending on age, general condition of the subject, the severity of the condition being treated, the particular compound and/or composition administered, and the like.
  • An appropriate "therapeutically effective amount" in any individual case can be determined by one of ordinary skill in the art by reference to the pertinent texts and literature and/or by using routine experimentation.
  • compositions of the present invention are in biologically compatible form suitable for administration in vivo for subjects.
  • the pharmaceutical compositions can further comprise a pharmaceutically acceptable carrier.
  • “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U. S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly, in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which a Ube4B and/or LSD1 inhibitor is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, including but not limited to peanut oil, soybean oil, mineral oil, sesame oil and the like. Water may be a carrier when the pharmaceutical composition is administered orally. Saline and aqueous dextrose may be carriers when the pharmaceutical composition is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions may be employed as liquid carriers for injectable solutions.
  • suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried slim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the pharmaceutical composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • compositions of the present invention can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations and the like.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
  • a pharmaceutical composition comprises an effective amount of a Ube4B and/or LSD1 inhibitor together with a suitable amount of a pharmaceutically acceptable carrier so as to provide the form for proper administration to the patient.
  • the formulation should suit the mode of administration.
  • compositions of the present invention may be administered by any particular route of administration including, but not limited to oral, parenteral, subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracelebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intraosseous, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, iontophoretic means, or transdermal means.
  • Most suitable routes are oral administration or injection. In certain embodiments, subcutaneous injection is preferred.
  • the pharmaceutical compositions comprising a Ube4B and/or LSD1 inhibitor may be used alone or in concert with other therapeutic agents at appropriate dosages defined by routine testing in order to obtain optimal efficacy while minimizing any potential toxicity.
  • the dosage regimen utilizing a pharmaceutical composition of the present invention may be selected in accordance with a variety of factors including type, species, age, weight, sex, medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular pharmaceutical composition employed.
  • a physician of ordinary skill can readily determine and prescribe the effective amount of the pharmaceutical composition (and potentially other agents including therapeutic agents) required to prevent, counter, or arrest the progress of the condition.
  • Optimal precision in achieving concentrations of the therapeutic regimen within the range that yields maximum efficacy with minimal toxicity may require a regimen based on the kinetics of the pharmaceutical composition's availability to one or more target sites. Distribution, equilibrium, and elimination of a pharmaceutical composition may be considered when determining the optimal concentration for a treatment regimen.
  • the dosages of a pharmaceutical composition disclosed herein may be adjusted when combined to achieve desired effects.
  • dosages of the pharmaceutical compositions and various therapeutic agents may be independently optimized and combined to achieve a synergistic result wherein the pathology is reduced more than it would be if either was used alone.
  • toxicity and therapeutic efficacy of a pharmaceutical composition disclosed herein may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effect is the therapeutic index and it may be expressed as the ratio LD50/ED50.
  • Pharmaceutical compositions exhibiting large therapeutic indices are preferred except when cytotoxicity of the composition is the activity or therapeutic outcome that is desired.
  • a delivery system can target such compositions to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the pharmaceutical compositions of the present invention may be administered in a manner that maximizes efficacy and minimizes toxicity.
  • Data obtained from cell culture assays and animal studies may be used in formulating a range of dosages for use in humans.
  • the dosages of such compositions lie preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose may be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (the concentration of the test composition that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information may be used to accurately determine useful doses in humans.
  • Levels in plasma may be measured, for example, by high performance liquid chromatography.
  • the dosage administration of the compositions of the present invention may be optimized using a pharmacokinetic/pharmacodynamic modeling system. For example, one or more dosage regimens may be chosen and a pharmacokinetic/pharmacodynamic model may be used to determine the pharmacokinetic/pharmacodynamic profile of one or more dosage regimens. Next, one of the dosage regimens for administration may be selected which achieves the desired pharmacokinetic/pharmacodynamic response based on the particular pharmacokinetic/pharmacodynamic profile. See WO 00/67776, which is entirely expressly incorporated herein by reference.
  • SOD1 and TDP-43 were expressed in pEF-BOS and pRK5-Myc, respectively, as previously described (Ketteler and Seed, 2008; Ketteler et al, 2008; Wang et al., 2003a; Zhang et al, 2011).
  • the UBE4B (TF308519) and LSD1 shRNA (TF316984) plasmids and the scrambled control (cat #: TR30015) were from Origene.
  • the p53 shRNA plasmid pLVTH-sip53, and control pLVTH were from D. Trono (Addgene #12239) (Wiznerowicz and Trono, 2003).
  • the p53 transcriptional reporter PG13- Luc was a generous gift from B. Vogelstein (el-Deiry et al, 1993). The autophagy luciferase release plasmids Act-LC3-Gluc and Act-Glue were kindly provided by B. Seed (Ketteler and Seed, 2008), and the control pCMV-SEAP was from A. Cochrane, (Addgene #24595).
  • cDNAs complementary DNAs
  • C. elegans Strains Suppressor Screen, and Mutation Identification.
  • the Bristol N2 C. elegans strain was used in all experiments unless otherwise specified.
  • a list of C. elegans strains is given in the Supplemental Materials and Methods.
  • Transgenic lines were generated according to standard procedures by injecting 20 ⁇ g/ml of expression plasmid DNA into hermaphrodite gonads.
  • For the suppressor screen worms were mutagenized with 47 mM ethyl methanesulfonate, and a semi-clonal strategy was used with five P0 worms in one plate. Suppressors were visually selected based on strong recovery in the movement phenotype in the F2 generation.
  • the suppressor mutations were mapped by using single nucleotide polymorphism markers in the Hawaiian strain and then identified by whole-genome deep sequencing, followed by Sanger sequencing validations (see Supplemental Materials and Methods).
  • C. elegans Locomotor Assay and Microscopy The C. elegans strains were observed stereoscopically and their motility was quantified by the thrashing assay (Zhang et al, 2011): Animals were transferred from the feeding plate into M9 buffer (3 mg/ml KH2PO4, 6 mg/ml Na2HP04, 5 mg/ml NaCl and 1 mM MgS04).
  • Protein Aggregation Assay The protein aggregation assay for C. elegans and mammalian cells was modified from a previously described protocol (Wang et al, 2003a) (see Supplemental Materials and Methods).
  • Proteasome Activity Assay Proteasome assays were performed as described previously (Kisselev and Goldberg, 2005), using the Suc-LLVY-Luciferin substrate for chymotrypsin-like activity of the proteasome (the Proteasome Glo kit, Promega). In brief, cells were detached and washed in DMEM/10, followed by several washes in cold PBS. Proteasome lysis buffer (50 mM Tris-HCl, pH 7.5, 0.025% digitonin, 250 mM sucrose, 5 mM MgCh, 0.5 mM EDTA, 2 mM ATP, and 1 mM DTT) was added to the cells and incubated on ice for 5-10 min.
  • Proteasome lysis buffer 50 mM Tris-HCl, pH 7.5, 0.025% digitonin, 250 mM sucrose, 5 mM MgCh, 0.5 mM EDTA, 2 mM ATP, and 1
  • the ly sates were then centrifuged for 15 min at 20,000 g to isolate ("squeeze-out") the cytoplasm containing the proteasomes. The supernatant was transferred to a fresh tube, and equal amounts of protein were used in each assay.
  • Autophagy Assays Autophagy was quantified with a Gaussia luciferase release assay
  • the DMEM/10 medium was replaced, and 100 ⁇ of cell growth medium was withdrawn at 24 h, 48 h, and 72 h.
  • the medium was centrifuged at 6000 g for 5 min to remove detached cells, followed by the luciferase analysis according to the manufacturer's recommendations (GeneCopoeia) using a microplate reader (Synergy HI, Bio-Tek).
  • LC3 western blot analysis cells were lysed in LC3 buffer (50 mM Tris-Cl, pH 8.0, with 1%SDS, 0.5% NP40, 150 mM NaCl, and 5 mM EDTA), and sonicated with a Diagenode Bioruptor device (set on High, 30-sec pulse, 30-sec pause, 7.5 min total).
  • the microarray data were managed using the Partek Genomic Suite (Partek Inc., St. Louis) and Spotfire DecisionSite software (TIBCO Software Inc., Palo Alto, CA) and analyzed using Ingenuity Pathways Analysis software (IP A, Ingenuity Systems).
  • IP A Ingenuity Pathways Analysis software
  • cDNAs were synthesized with the QuantiTect reverse transcription kit using RNA samples from both (Qiagen). Primers for quantitative RT-qPCR were from PrimerBank (Spandidos et al., 2010). RT-qPCRs were performed on a BioRad thermal cycler with iQ SYBER Green PCR mix (BioRad).
  • Misfolded S0D1 G85R protein is highly toxic, leading to age-dependent synaptic dysfunction, neurodegeneration, and severely impaired movement in the worms (Wang et al, 2009a). This severe locomotor defect allows us to perform a large-scale screen for genes that suppress neurodegeneration and improve worm locomotion.
  • F2 offspring which contain both heterozygous and homozygous suppressor mutations, we selected individual C.
  • SNP mapping Single nucleotide polymorphism (SNP) mapping (Davis et al, 2005), which approximately located the chromosomal regions responsible for the Ml phenotype, and subsequently performed whole-genome deep sequencing (Sarin et al, 2008). SNP mapping first localized the Ml suppressor mutations to two linkage regions: a 2.2Mb-interval on Chromosome I and an 8Mb-interval on Chromosome II (FIG. ID). Two rounds of deep sequencing were performed on the Ml strain genomic DNA, attaining 27-fold coverage. When the Ml genomic DNA sequencing data was aligned with the C.
  • ufd-2 encodes a U-box type ubiquitin ligase
  • W824X mutation results in a truncated protein lacking the C-terminal U-box
  • spr-5 encodes a lysine-specific histone demethylase
  • R646Q substitution occurs at a highly conserved residue in the C-terminal portion of an amine oxidase-like (AOL) domain (FIG. IE).
  • ufd-2 and spr-5 as the suppressor genes, we obtained independent null alleles of the two genes: a deletion mutation, ufd-2(tml380), that lacks the C-terminal 4/5ths of the protein (Janiesch et al., 2007) and a non-sense mutation, spr-5(by 134), that results in deletion of the C-terminal half of the protein (Eimer et al, 2002) (FIG. IE).
  • elegans could be related to a change in the amount of toxic misfolded proteins.
  • YFP fusions of several aggregating proteins such as S0D1 G85R -YFP (Wang et al, 2009a), TDP-43 c25 -YFP (Zhang et al., 2011), and PolyQ-YFP (Brignull et al., 2006; Morley et al, 2002), are toxic to neurons and muscles, impair the movement of C. elegans, and form large protein aggregates that are easily visualized in live animals.
  • FOG. IF C. elegans thrashing assay
  • Ubiquitination factor E4 B Ubiquitination factor E4 B
  • LSD1 lysine-specific demethylase 1
  • the human and C. elegans orthologs share 32% and 29% protein sequence identity for ufd-2/UBE4B and spr-5/LSDl, respectively, and all the major protein domains are conserved (FIG. IE).
  • the mutant S0D1 G85R had a much larger fraction of misfolded and aggregated proteins that were sensitive to UBE4B/LSD1 -dependent clearance, similar to S0D1 G85R -YFP in C. elegans (FIGS. 2A and 2C), than does the WT SOD1 protein.
  • the decrease in aggregation was not specific to S0D1 G85R but also occurred with other aggregation-prone proteins, including TDP-43 (FIGS. 8B and 8C), indicative of a general effect on misfolded proteins.
  • MDM2 a negative regulator of p53, or ⁇ - galactosidase as a control, together with the p53 activity reporter.
  • the introduction of MDM2 significantly reduced p53-dependent transcriptional activation of the luciferase reporter under the UBE4B and LSDl double-knockdown condition (FIG. 10), confirming the specificity of the up-regulation of p53 by UBE4B and LSDl .
  • FOXOs are a family of transcription factors invoked in protein quality control (Zhang et al, 2011 ; Zhao et al, 2007), and PSMDl 1 is a critical regulator of proteasome activity (Vilchez et al, 2012a; 2012b).
  • FOX03a is transcriptionally up-regulated by p53 (Renault et al, 2011), and PSMDl 1 is transcriptionally induced by FOXOs (Vilchez et al, 2012a; 2012b).
  • FOXOs Vanchez et al, 2012a; 2012b.
  • FOX03a, FOX04, and PSMDl 1 were all transcriptionally up-regulated when UBE4B and LSDl are knocked down (FIG. 9D), linking these positive regulators of protein quality control downstream of p53 to the UBE4B- and LSDl -dependent anti-proteotoxicity activity.
  • FOX03a-TM constitutively active form
  • FOX03a-TM FHRE-lucif erase reporter
  • HCT116 cells which are amenable to this assay, were transfected with shRNA constructs to knock down UBE4B and LSDl, with the LC3-GLuc plasmid used to measure the cleavage of LC3 and constitutively secreted control (secreted embryonic alkaline phosphatase [CMV-SEAP]) for transfecti on/secretion normalization (FIGS. 11B and 11C).
  • the LC3-dependent GLuc activity measured over a period of 72 h, showed a 2- to 3-fold increase in ATG4B proteolytic activity at the end of the time course, demonstrating the activation of autophagy by UBE4B and the double-knockdown (FIG. 5B).
  • the cells transfected with the non-cleavable, LC3-less fusion, the Act-GLuc construct showed only background levels of Glue activity, similar to the levels observed in non-transfected cells.
  • p53 Regulates Protein Quality Control. Until now, p53 has not been associated with anti-proteotoxicity activity. p53 has been shown to regulate autophagy, but in conflicting directions (Balch et al., 2008; Levine and Abrams, 2008; Prusiner, 2012). Our microarray analysis and subsequent studies establish a correlation between the activation of p53- mediated transcription and enhanced protein quality control conferred by the knockdown of UBE4B and LSD1 (FIG. 5). It has been demonstrated that p53 is a target of
  • Tenovin-1 is a SIRTl/2 deacetylase inhibitor that promotes p53 K382 acetylation, increasing its stability and activity (Lain et al, 2008; Wolff et al, 2014).
  • CP-31398 is another drug that activates p53 by stabilizing the p53 DNA-binding domain in an active conformation and inhibiting its ubiquitination (Foster, 1999; Parge et al, 1992; Wang et al., 2003b).
  • the S0D1 G85R reporter did not produce an appreciable pellet fraction, so we focused on analyzing the supernatant fraction.
  • the S0D1 G85R mutant protein was significantly increased in the p53 knockout cells when compared to the controls, indicating that the endogenous p53 promotes the clearance of misfolded proteins (FIG. 6C).
  • UBE4B forms a complex with an AAA-ATPase p97/VCP to ubiquitinate and degrade specific client proteins (Eimer et al, 2002; Kaneko et al, 2003; Morreale et al, 2009).
  • p97/V CP plays an essential role in handling unfolded proteins such as at endoplasmic reticulum-associated protein degradation (Ye et al, 2001; Zetterstrom et al, 2007), and it was recently linked to familial ALS (Johnson et al, 2010; Wang et al., 2009a).
  • Our findings thus provide a new link between p97/V CP and protein quality control, which is regulated by UBE4B.
  • p53 as a Key Switch in Protein Quality Control. Unbiased transcriptome analysis points to p53 as a central regulator of the transcriptional reprograming that mediates the effects of UBE4B and LSDl on protein quality control. Consistent with this observation, p53 has been found to have a number of direct transcriptional targets functioning in protein quality control and neuroprotection, and it also activates additional stress-response transcription factors such as FOXOs (Brignull et al, 2006; Morley et al, 2002; Renault et al, 2011).
  • p53 is elevated in the central nervous system of patients with neurodegenerative conditions such as Alzheimer's disease and ALS (Kitamura et al, 1997; Martin, 2000; Wang et al, 2009a; 2009b).
  • the transcription factors mediate the effects of this strong suppressor is reminiscent of other signaling pathways governing protein homeostasis.
  • the heat shock response activates the expression of molecular chaperones and other protein quality control machinery via the master transcription factors the heat shock factors (Morimoto, 1998; Wang et al, 2009a; 2009b).
  • the unfolded protein response promotes the endoplasmic reticulum quality control programs through the activation of a set of the transcription factors, including XBP1, ATF4, and ATF6 (Brignull et al, 2006; Morley et al, 2002; Walter and Ron, 2011).
  • the post-translational regulation by UBE4B and LSDl activates the p53 transcription factor, which is then capable of eliciting a systematic protective program against proteotoxic stress.
  • p53 has a well-established role in regulating responses to DNA damage (Lanson et al., 2011; Liu, 2001; Ritson et al., 2010; Smith et al, 1994), and recently, a neuroprotective role of activated DNA damage checkpoint has been demonstrated in a tau-dependent neurodegeneration model (Khurana et al, 2012; Wang et al, 2003a; 2006).
  • p53 is a versatile transcriptional switch that guards against both genotoxicity and proteotoxicity.
  • the specific activity of p53 may be fine-tuned at the post-translational level by upstream regulators such as UBE4B and LSDl .
  • p53 promotes apoptosis in cells with irreversible genotoxic damage (Vousden and Prives, 2009; Wu et al, 2011). p53 may also function as a dual regulator in proteotoxicity: It promotes the repair and survival of moderately damaged cells, but turns on cell death pathways in cells whose damage is irreparable. Such duality has been observed for other protein quality control systems, such as the ER stress responses (Huang et al, 2007; Walter and Ron, 2011). Thus p53 could serve as a critical regulator of cellular responses to proteotoxicity by repairing or removing damaged cells.
  • ALS- linked SOD1 mutant G85R mediates damage to astrocytes and promotes rapidly progressive disease with SOD1 -containing inclusions. Neuron 18, 327-338.
  • WAF1 a potential mediator of p53 tumor suppression. Cell 75, 817-825.
  • TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis Science 314, 130-133.
  • Drosophila p53 is a structural and functional homolog of the tumor suppressor p53.
  • TDP-43 mediates degeneration in a novel Drosophila model of disease caused by mutations in VCP/p97. J. Neurosci. 30, 7729-7739.
  • RPN-6 determines C. elegans longevity under proteotoxic stress conditions. Nature 489, 263-268.
  • CGC Caenorhabditis Genetics Center
  • P40 OD010440 NIH Office of Research Infrastructure Programs
  • Drosophila Genetics Flies were reared on standard yeast-agar-commeal medium and crosses were performed at 25°C. Drosophila transgenic strains carrying GAL4-inducible human ALS disease-causing alleles of FUS/TLS and TDP-43 were previously described (Lanson et al, 2011; Ritson et al, 2010). Standard genetic procedures were used to generate the GMR-GAL4/CyO, tub-GAL80; UAS-FUS-hR521C/TM6B, Tb and GMR-GAL4, UAS- hTDP-43-M337V/CyO, tub-GAL80 transgenic strains.
  • DN GAL4-inducible and dominant negative
  • P ⁇ w +mC UAS-p53.R155H.Ex ⁇ 2/T(2;3)TSTL, CyO: TM6B, Tb + ) (Ollmann et al., 2000).
  • the dominant effects of the reduction of CG9934, Su(Var)3-3, or Dmp53, as well as the induction of DN p53.R155H, on the degenerative eye phenotypes of GMR-GAL4;UAS-FUS-hR521C and GMR-GAL4;UAS-hTDP-43-M337V strains were assessed two weeks after the crosses were performed. Qualitative changes in pigmentation, ommatidial structure and glossiness phenotypes were monitored for enhancement or suppression.
  • the suppressor mutations were assigned to chromosomal locations through linkage mapping using single nucleotide polymorphisms between the wild-type strains N2 Bristol and CB4856 Hawaii.
  • the unique mutations within the mapping intervals were identified by deep-sequencing and comparing the genomes of the Ml suppressor mutant and the parental strain carrying the SOD1 transgene.
  • the genome sequencing data was analyzed with a bioinformatic pipeline containing Bowtie 2 (Langmead and Salzberg, 2012), SAMtools (Li et al, 2009), SnpEff (Cingolani et al, 2012), and the Integrative Genomics Viewer (IGV) (Robinson et al, 2011; Thorvaldsdottir et al, 2013).
  • the identified mutations were confirmed by Sanger sequencing of the PCR-amplified loci.
  • the phenotype-causing mutations were validated by independent alleles of the candidate genes.
  • shRNAs Gene knockdown in mammalian cells was achieved by transiently expressing shRNA plasmids, or stably expressing doxycycline-inducible shRNA in integrated cell lines when indicated.
  • a shRNA with an RFP marker Origene, pRFP-C-RS
  • pLVTH EGFP marker
  • Seydoux to contain different shRNA sequences (inserted by Agel-Hindlll digestion) under the Hl-tet (HI/TO) promoter and thus generate L4Rl-Hl/TO-shRNAi, LlL2-Hl/TO-shRNA 2 , and R2L3-Hl/TO-shRNA 3 DONR vectors.
  • the HI/TO promoter itself was derived from pTET-LKO-puro (Wieders chain et al, 2009).
  • DEST vector for constitutive shRNA expression in mammalian cells we first amplified the R4-R3 Gateway cassette from the plasmid pCG150 (a gift from G. Seydoux) and inserted it into pcDNA3.1 vector (Invitrogen) by Mfel-BstBI. To generate doxycycline-inducible DEST vector (pR4R3-TET-PURO), we replaced neo gene of pR4R3-NEO with the pkg promoter-TET-Repressor-IRES-Puromycin cassette from the pTET-LKO-puro plasmid.
  • Stable mammalian cell lines were generated by linearizing the pR4R3-TET-PURO shRNA plasmid, transfecting it into HEK293T cells, and selecting for puromycin-resistant colonies. Clones were further selected for effective knockdown of UBE4B, LSDl and p53 genes upon induction with doxycycline.
  • HEK293T and HCT1 16 cell lines were grown at 37 ° C / 5% CO2 in standard DMEM medium, supplemented with 10% FBS, 2 mM L-glutamine and lx non-essential amino acids (DMEM/10).
  • shRNAs transfections of HEK293T cells were performed by plating 3.2xl 0 5 cells in 60 mm poly(ethyleneimine) (PEI, 10 ⁇ g/ml in PBS, Sigma)-pretreated dishes one day before the transfection. 4 ⁇ g of shRNA-encoding plasmids, 350 ng of SOD1 reporter (BOS-SOD1 -G85R), and 10 ⁇ Lipofectamine 2000 (Invitrogen) were mixed in 500 ⁇ Opti-MEM I (Invitrogen), and applied to cells in 2.5 ml Opti-MEM I. One day post transfection, medium was replaced with DMEM/10. Cells were lysed 72-96 h after the start of transfections for analysis, or transfected with additional reporter plasmids for transcriptional, proteasomal, and autophagic activity assays.
  • 3-Methyladenine (Sigma) was resuspended at (10 ⁇ ) in complete DMEM, by heating to 37 ° C and vigorous vortexing. All drugs were diluted in DMEM/10 prior to cell treatments.
  • IgGs normal rabbit IgG (NeoMarkers, NC-100P). IgGs were captured using magnetic A/G beads (Pierce, #88803), washed 4x10 min with IP buffer, and eluted with boiling in 2x SDS loading buffer. Equal amount were loaded on 4-20% Tris- Glycine gel, transferred and visualized with Li-Cor's 680RD Detection Reagent.
  • C. elegans strains were collected from NGM feeding plates into M9 buffer and washed five times. Mammalian culture cells grown on 60mm plate were washed two times with cold PBS. C. elegans and mammalian cells were lysed in 200- 300 ⁇ of lysis buffer (50 mM Tris-HCl, pH 8.0, 1 mM ethylenediaminetetraacetic acid (EDTA),100 mM NaCl and 0.5% NP-40, 1/lOOth protease inhibitor cocktail (Sigma, P8340) and 25 mM iodoacetamide (Sigma, 16125), sonicated on ice in Diagenode Bioruptor (High, 30 sec pulse, 30 sec pause, 5 min total).
  • lysis buffer 50 mM Tris-HCl, pH 8.0, 1 mM ethylenediaminetetraacetic acid (EDTA),100 mM NaCl and 0.5% NP-40, 1/lOOth protease inhibitor
  • Lysates were centrifuged 5-10 min at ⁇ 130,000g (25 psi) in Airfuge (Coulter-Beckman), to separate larger pelleted aggregates (PI), from soluble proteins and smaller aggregates (SI).
  • PI pellet was resuspended in lysis buffer and sonicated as described above, except 10 min. After centrifugation (Airfuge, -130,000 g, 5-10 min), pellet (P2) was resuspended in 100 ⁇ Urea/SDS buffer (8 M Urea, 5% SDS, 40 mM Tris-Cl pH 6.8, 0.1 mM EDTA), followed by 5 min sonication.
  • Fluorescent signals from the hybridized probes were detected using the Affymetrix G3000 GeneArray Scanner, and analysis was performed through the Affymetrix GeneChip Command Console version 3.4 software.
  • the microarray data were managed and analyzed using Partek Genomic Suite (Partek) and Spotfire DecisionSite software (TIBCO Software).
  • Partek Genomic Suite Partek
  • Spotfire DecisionSite software TIBCO Software
  • IP A Ingenuity Systems
  • the microarray data set containing gene identifiers and expression values was uploaded into the application. Each identifier was then mapped to its corresponding gene product in the Ingenuity Knowledge Base.
  • the molecules with expression fold changes above the threshold ( ⁇ 1.2), and the p-values ⁇ 0.05, were overlaid onto a global molecular network developed from information contained in the Ingenuity Knowledge Base. The relevant networks of selected molecules were then algorithmically generated based on their connectivity.
  • experimental, microarray - derived expression patterns were analyzed and compared to the literature-derived expression patterns resulting from activation/inhibition of known, upstream regulatory molecules, such as, transcriptional factors, signal transducers, receptors, or chemical effectors.
  • the probability of significant overlap between microarray-derived and literature-derived sets was set to ⁇ 0.05.
  • the raw data of the present microarray analysis is deposited at the Gene Expression Omnibus (GEO) repository (accession # GSE58026).
  • Drosophila p53 is a structural and functional homolog of the tumor suppressor p53.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Zoology (AREA)
  • Neurology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Wood Science & Technology (AREA)
  • Neurosurgery (AREA)
  • General Engineering & Computer Science (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Molecular Biology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Environmental Sciences (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Virology (AREA)
  • Psychiatry (AREA)
  • Epidemiology (AREA)
  • Biophysics (AREA)
  • Hospice & Palliative Care (AREA)
  • Immunology (AREA)
  • Animal Husbandry (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Mycology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne le domaine des maladies conformationnelles des protéines. Plus spécifiquement, la présente invention concerne des compositions et des méthodes de traitement de maladies conformationnelles des protéines, y compris la sclérose latérale amyotrophique (SLA). Dans un mode de réalisation, un procédé comprend l'étape d'administration, à un patient, d'une quantité efficace d'un inhibiteur Ube4B et d'un inhibiteur LSD1. Dans un autre mode de réalisation, le procédé comprend en outre l'étape d'administration d'un agoniste de p53.
PCT/US2016/018657 2015-02-20 2016-02-19 Compositions et méthodes de traitement de maladies conformationnelles des protéines WO2016134246A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201562118503P 2015-02-20 2015-02-20
US62/118,503 2015-02-20

Publications (2)

Publication Number Publication Date
WO2016134246A2 true WO2016134246A2 (fr) 2016-08-25
WO2016134246A3 WO2016134246A3 (fr) 2016-11-03

Family

ID=56689486

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2016/018657 WO2016134246A2 (fr) 2015-02-20 2016-02-19 Compositions et méthodes de traitement de maladies conformationnelles des protéines

Country Status (1)

Country Link
WO (1) WO2016134246A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023231402A1 (fr) * 2022-06-01 2023-12-07 浙江大学 Utilisation médicale de la protéine ubv.e4b et composition pharmaceutique

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130303545A1 (en) * 2010-09-30 2013-11-14 Tamara Maes Cyclopropylamine derivatives useful as lsd1 inhibitors

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023231402A1 (fr) * 2022-06-01 2023-12-07 浙江大学 Utilisation médicale de la protéine ubv.e4b et composition pharmaceutique

Also Published As

Publication number Publication date
WO2016134246A3 (fr) 2016-11-03

Similar Documents

Publication Publication Date Title
D’Amico et al. The RNA-binding protein PUM2 impairs mitochondrial dynamics and mitophagy during aging
Deng et al. FUS interacts with HSP60 to promote mitochondrial damage
Zhao et al. Pharmacologically reversible zonation-dependent endothelial cell transcriptomic changes with neurodegenerative disease associations in the aged brain
Gao et al. Inactivation of PNKP by mutant ATXN3 triggers apoptosis by activating the DNA damage-response pathway in SCA3
Merrill et al. Mechanism of neuroprotective mitochondrial remodeling by PKA/AKAP1
Periz et al. Regulation of protein quality control by UBE4B and LSD1 through p53-mediated transcription
EP3071689A1 (fr) Compositions et procédés d'utilisation de transposons
Tourette et al. The Wnt receptor Ryk reduces neuronal and cell survival capacity by repressing FOXO activity during the early phases of mutant huntingtin pathogenicity
EP3575401A1 (fr) Méthodes de traitement ou de prévention d'une maladie liée au vieillissement
EP3066199B1 (fr) Sno arn, compositions et utilisations
US20210332136A1 (en) Methods for diagnosing and treating cancers
WO2016134246A2 (fr) Compositions et méthodes de traitement de maladies conformationnelles des protéines
US20230277618A1 (en) Therapeutic targeting of phosphate dysregulation in cancer via the xpr1:kidins220 protein complex
EP3121274B1 (fr) MÉTHODE PERMETTANT DE PRÉVOIR LA SENSIBILITÉ À UN TRAITEMENT ANTICANCÉREUX AU MOYEN D'UN COMPOSÉ INHIBITEUR DE p300
Pircs et al. Distinct sub-cellular autophagy impairments occur independently of protein aggregation in induced neurons from patients with Huntington’s disease
Zhou et al. Ulk4 promotes Shh signaling by regulating Stk36 ciliary localization and Gli2 phosphorylation
US10449200B2 (en) Combined inhibition of the vitamin D receptor and poly(ADP) ribose polymerase (PARP) in the treatment of cancer
US20200009149A1 (en) Methods of Treating Autism Spectrum Disorders
Fielden et al. Comprehensive Interrogation of Synthetic Relationships in the Human DNA Damage Response
Luo et al. Loss of nuclear envelope phosphatase CTDNEP1 drives aggressive medulloblastoma by triggering MYC amplification and genomic instability
US20120029055A1 (en) Modulators of apoptosis and the uses thereof
Kurup Regulation of Gene Expression and A-to-I Editing Activity by Deaminase Deficient ADAR3 in Glioblastoma
Allen Post-transcriptional regulation of BDNF and the Cdk5 pathway by the neuronal RNA binding protein HuD
Ottesen et al. A survey of transcripts generated by spinal muscular atrophy genes
Cali Genetic and Epigenetic Modifiers of C9ORF72 Expression in Neurodegenerative Disease

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: 16753137

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16753137

Country of ref document: EP

Kind code of ref document: A2