WO2014114303A1 - Protéines à répétitions de dipeptides comme cible thérapeutique dans des maladies neurodégénératives avec expansion de répétitions hexanucléotidiques - Google Patents

Protéines à répétitions de dipeptides comme cible thérapeutique dans des maladies neurodégénératives avec expansion de répétitions hexanucléotidiques Download PDF

Info

Publication number
WO2014114303A1
WO2014114303A1 PCT/EP2013/000190 EP2013000190W WO2014114303A1 WO 2014114303 A1 WO2014114303 A1 WO 2014114303A1 EP 2013000190 W EP2013000190 W EP 2013000190W WO 2014114303 A1 WO2014114303 A1 WO 2014114303A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
repeats
seq
terminus
amino acids
Prior art date
Application number
PCT/EP2013/000190
Other languages
English (en)
Inventor
Dieter EDBAUER
Christian Haass
Shig-Ming WENG
Kohji Mori
Thomas ARZTBERGER
Elisabeth Kremmer
Original Assignee
Deutsches Zentrum Für Neurodegenerative Erkrankungen
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 Deutsches Zentrum Für Neurodegenerative Erkrankungen filed Critical Deutsches Zentrum Für Neurodegenerative Erkrankungen
Priority to PCT/EP2013/000190 priority Critical patent/WO2014114303A1/fr
Priority to US14/762,623 priority patent/US10066007B2/en
Priority to PT14701089T priority patent/PT2948777T/pt
Priority to DK14701089.6T priority patent/DK2948777T3/da
Priority to RSP20191145 priority patent/RS59283B1/sr
Priority to HUE14701089A priority patent/HUE046434T2/hu
Priority to LTEP14701089.6T priority patent/LT2948777T/lt
Priority to SI201431324T priority patent/SI2948777T1/sl
Priority to PL14701089T priority patent/PL2948777T3/pl
Priority to ES14701089T priority patent/ES2744804T3/es
Priority to PCT/EP2014/051204 priority patent/WO2014114660A1/fr
Priority to EP14701089.6A priority patent/EP2948777B1/fr
Priority to EP19180100.0A priority patent/EP3588091A1/fr
Publication of WO2014114303A1 publication Critical patent/WO2014114303A1/fr
Priority to HRP20191608 priority patent/HRP20191608T1/hr
Priority to CY20191100935T priority patent/CY1122079T1/el

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders

Definitions

  • Dipeptide-repeat proteins as therapeutic target in neurodegenerative diseases with hexanucleotide repeat expansion
  • the present invention relates to a method of detecting a disease characterized by an expansion of genomic hexanucleotide repeats as well as polypeptides of said hexanucleotide repeats, ligands specifically binding to the polypeptide, methods of identifying an inhibitor preventing the expression, toxicity and/or aggregation of said polypeptide.
  • Neurodegenerative disorders are generally characterized by disease signifying protein deposits. Moreover, in a number of neurodegenerative diseases mutations causing genetically inherited variants of the disease were associated with the genes encoding the protein deposits, their precursors or their modulating enzymes. Functional analysis of these genetic variants fundamentally helped to understand disease associated mechanisms of Alzheimer's disease (AD) and Parkinson's disease (Gasser et al., 201 1, Haass et al., 2007).
  • AD Alzheimer's disease
  • Parkinson's disease Garr et al., 201 1, Haass et al., 2007.
  • Frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are the extreme ends of a spectrum of overlapping neurodegenerative disorders variably associated with dementia, personality changes, language abnormalities and progressive muscle weakness (Josephs et al., 201 1 ; Mackenzie et al., 2010; Rademakers et al, 2012).
  • RNA/DNA binding protein TDP-43 (Tar DNA binding protein of 43 kDa) as an abundant deposited protein (Arai et al., 201 1; Neumann et al., 2006) and by the discovery that mutations in TARDBP cause familial variants of both diseases (Benajiba et al., 2009; Sreedharan et al., 2008). The majority of cases show intracellular inclusions that are strongly positive for phosphorylated TDP-43.
  • ALS and FTLD are multisystem disorders with overlapping clinical and pathological characteristics and similar functional and genetic causes (Rademakers et al., 2012; Sieben et al., 2012) and which are therefore classified as FTLD- TDP, FTLD/ALS-TDP or ALS- TDP.
  • the hexanucleotide repeat expansion is located upstream of the C9orf72 open reading frame, either in the first intron or the promoter region, depending on the transcript isoform (see Fig. 8A).
  • the extreme GC-content precludes sequencing in patients, the number of GGGGCC repeat units is believed to be at least several hundred compared to less than 25 in healthy controls (van der Zee et al. 2012). The pathomechanisms leading to disease however remained unclear.
  • ATXN8 encodes a natural poly-Q stretch that can cause poly-Q inclusions upon repeat expansion in spinocerebellar ataxia type 8 (SCA8) patients.
  • SCA8 spinocerebellar ataxia type 8
  • the expanded CAG-repeat is translated in all three reading frames (poly-Q, poly-A and poly-S) even after removal of the endogenous start codon.
  • Poly-Q and poly-A have been found in patient aggregates.
  • myotonic dystrophy type 1 (DM1) is caused by CTG-expansion in the 3'UTR of the gene DMPK. The translation into rare poly-Q aggregates in DM1 patients and mouse models was discovered.
  • RAN repeat-associated non-ATG-initiated translation
  • WO2010/1 15033 A9 The underlying mechanism was named repeat-associated non-ATG-initiated translation (RAN) and is patented for tri-, tetra-, and penta-nucleotide repeat disorders (WO2010/1 15033 A9).
  • RAN translation has not been shown for intronic repeats or hexanucleotide repeats.
  • Laura Ranum speculates about RAN-translation in C9orf72 patients (Ashizawa and Ranum 2012). It is also possible that translation of DPR proteins is initiated from non-canonical start codon 5' of the repeat region (Ivanov, I. P. et al. 201 1 ; Peabody D. S. 1989; Touriol C. et al., 2003)
  • siRNA anti-sense, alter C9orf72 splicing), inhibiting repeat transcription and/or translation into DPR (screen for RAN-specific translation inhibitors), and preventing DPR aggregation or promoting degradation or clearance (through chemical compounds or immunotherapy) .
  • polypeptide indicates the presence of or susceptibility to a disease characterized by an expansion of genomic hexanucleotide repeats.
  • the present invention provides a polypeptide comprising or consisting of dipeptide-repeats with a sequence selected from the group consisting of (Gly- Ala) a , (Gly-Pro) b , (Gly-Arg) e , (Ala-Pro) d , (Pro-Arg) e , (Gly-Leu) f , (Ala-Trp) g , (Pro-Gly) h , (Ala-Gln) j , (Gly-Pro) k , and (Pro-Arg)i wherein a is an integer of 16 or more, b is an integer of 28 or more, c is an integer of 21 or more, d is an integer 17 or more, e is an integer of 24 or more, f is an integer of 15 or more, g is an integer of 9 or more, h is an integer of 28 or more, j is an integer of 16 or more, k is an integer of 28 or more, and 1 is
  • the present invention provides a polypeptide comprising or consisting of
  • (ix) (Ala-Gln) w dipeptide repeats, wherein w is an integer of 10 or more, at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 11 are comprised at its C-terminus, and/or at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 22 are comprised at its N-terminus,
  • (x) (Gly-Pro) x dipeptide repeats, wherein x is an integer of 10 or more, at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 12 are comprised at its C-terminus, and/or at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 23 are comprised at its N-terminus, or
  • (xi) (Arg-Pro) y dipeptide repeats, wherein y is an integer of 10 or more, at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 13 are comprised at its C-terminus, and/or at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 24 are comprised at its N-terminus.
  • the present invention provides a ligand which binds specifically to a polypeptide of the second or third aspect of the present invention.
  • the present invention provides a method of identifying an inhibitor of expression of a polypeptide comprising or consisting of dipeptide-repeats, comprising the steps of:
  • the present invention provides a method of identifying an inhibitor of the toxic effect of a polypeptide comprising or consisting of dipeptide-repeats, comprising the steps of:
  • the present invention provides a method of identifying an inhibitor of formation of aggregates comprising a polypeptide comprising or consisting of dipeptide- repeats, comprising the steps of:
  • the present invention provides an inhibitor of expression of a polypeptide comprising or consisting of dipeptide-repeats or an inhibitor of formation of aggregates comprising or consisting of a polypeptide comprising dipeptide-repeats or an inhibitor of the toxic effects caused by the expression or the formation of aggregation of the polypeptide comprising or consisting of dipeptide-repeats for treating or preventing a disease characterized by an expansion of genomic hexanucleotide repeats.
  • the present invention provides a polypeptide according to the second or third aspect, the ligand according to the fourth aspect, the inhibitor according to the eighth aspect and/or a nucleotide sequence encoding the polypeptide according to the second or third aspect, encoding the ligand according to the fourth aspect or encoding the inhibitor according to the eighth aspect for use in diagnosis, prophylaxis, or treatment of a disease characterized by an expansion of genomic hexanucleotide repeats.
  • the present invention provides a kit of parts for diagnosing, treating or preventing a disease characterized by an expansion of genomic hexanucleotide repeats comprising the polypeptide according to the second or third aspect, the ligand according to the fourth aspect, the inhibitor according to the eighth aspect and/or a nucleotide sequence encoding the polypeptide according to the second or third aspect, encoding the ligand according to the fourth aspect or encoding the inhibitor according to the eighth aspect, and optionally further comprising a container, and/or a data carrier, preferably comprising instructions for one or more of the methods of the first, fifth to seventh aspect.
  • the present invention provides a pharmaceutical comprising the polypeptide according to the second or third aspect, the ligand according to the fourth aspect, the inhibitor according to the eighth aspect and/or a nucleotide sequence encoding the polypeptide according to the second or third aspect, encoding the ligand according to the fourth aspect or encoding the inhibitor according to the eighth aspect.
  • the present invention provides a pharmaceutical comprising the polypeptide according to the second or third aspect, the ligand according to the fourth aspect, the inhibitor according to the eighth aspect and/or a nucleotide sequence encoding the polypeptide according to the second or third aspect, encoding the ligand according to the fourth aspect or encoding the inhibitor according to the eighth aspect for use in diagnosis, prophylaxis, or treatment of a disease characterized by an expansion of genomic hexanucleotide repeats.
  • Fig l Extended GGGGCC-repeats are translated into aggregating dipeptide- repeat (DPR) proteins.
  • A Validation of DPR-specific antibodies by immunoblotting with purified GST-fusion proteins containing (GA) 15 , (GP) i5 or (GR) 15 .
  • B GGGGCC-repeat constructs with indicated repeat length lacking an upstream ATG were transfected into HEK293 cells. Restriction digest to estimate the repeat length of the transfected constructs (upper panel). Immunoblots show length-dependent expression of poly-GA and poly-GP proteins. Poly-GA products were detectable starting from ⁇ 38 repeats (arrow). Asterisk indicates nonspecific band. Poly-GR products were not detected (not shown).
  • Fig 2 Summary of findings in FTLD/ALS cases and controls. Summary of the immunohistochemical findings for DPR, p62 and TDP-43 inclusions for all analyzed patients. TDP-43 pathology was examined with phospho-TDP-43 or pan-TDP-43 antibodies. NE, not examined. NA, not available.
  • Fig 3 Overview of DPR-pathology in the cerebellum. Immunohistochemistry in the cerebellar granular layer comparing inclusion stained with p62, poly-GA, poly-GP and poly-GR specific antibodies in an FTLD/ALS patient with C9orf72 mutation (TJ-1).
  • the number of p62-positive aggregates (A) is similar to the number of poly-GA aggregates (B), whereas poly-GP and poly-GR aggregates are less frequent (C, D).
  • poly-GR antibodies additionally showed faint nuclear and cytoplasmic staining, probably due to cross-reactivity with natural short Gly-Arg repeats present in several DNA/RNA-binding proteins. Scale bars denote 20 ⁇ . CBL-GL cerebellar granular layer.
  • Fig 4 DPR proteins form the characteristic TDP-43 negative inclusions in C9orf72 patients.
  • Immunohistochemistry with DPR-specific antibodies reveals poly-GA, poly-GP and poly-GR inclusions resembling the p62-positive aggregates in FTLD/ALS patients with C9orpi mutation (compare Fig. 2).
  • Dot-like and thread-like inclusions in cerebellar granular layer (CBL-GL) A-E).
  • CBL-GL cerebellar granular layer
  • F-I Star-shaped cytoplasmic
  • J dot-like intranuclear
  • Fig 5 Preincubation with poly-(Gly-Ala) and poly-(Gly-Pro) antigens confirms the specificity of anti-GA and anti-GP antibodies.
  • Preincubation with GST-iGA) ⁇ and GST-(GP) 15 completely blocked staining signals (compare (A) and (B) for anti-GA and (D) and (E) for anti-GP).
  • GST-(GR)i5 strongly enhanced apparent poly-GR signals (compare (G) and (H)). This was due to direct binding of the positively charged GST-(GR)i 5 protein to the tissue (presumably to DNA, RNA and phospholipids), since an anti-GST antibody showed identical enhanced staining pattern when preincubated with GST-(GR)i 5 (compare panel (J) and (K)). Preincubation with GST did not impair the immunohistochemical detection of poly-GA, poly- GP and poly-GR (C, F, I). Scale bars denote 20 ⁇ .
  • Fig 6 DPR-aggregates colocalize with p62 but not TDP-43.
  • Immunofluorescence of C9orf72 patient TJ-1 with indicated antibodies reveals no co-aggregation of poly-GA and poly-GP with phospho-TDP-43 -positive aggregates in the hippocampus (A-C).
  • poly-GP and poly-GR colocalized with p62-positive aggregates (D, E).
  • Poly-GA deposition was occasionally observed within phospho-TDP-43 aggregates, but never vice versa, indicating that poly-GA deposition precedes TDP-43 aggregation (F).
  • Fig 7 DPR pathology is specific to patients with C9orf72 hexanucleotide repeat expansion.
  • A, B Immunohistochemistry with poly-GA specific antibodies (GA) detects no aggregates in an FTLD-TDP patient (TJ-13) without C9orf72 repeat expansion and a case with Huntington's disease (TJ-11). Phospho-TDP-43 and Huntingtin inclusions are readily detectable. Granular layer of dentate gyrus (DG-GL) and frontal cortex, respectively.
  • C Poly-GA-positive inclusions but no phospho-TDP-43 inclusions in temporal cortex of patient TJ-10 with C9orf72 mutation diagnosed with FTLD-UPS. Scale bars denote 20 ⁇ .
  • Figure 8 Differential regulation of C9orf72 transcripts in mutation carriers.
  • A Genomic structure of C9orf72 (drawn not to scale).
  • B qPCR analysis shows decreased expression of spliced mature C9orf72 mR A in cerebellum of C9orf72 mutation cases. Primers from exon 2 to 3 detect all C9orf72 isoforms. Primers from exon lb to 2 detect alternative transcripts lacking the repeat expansion.
  • C Strongly increased expression of sense and antisense transcripts containing C9orf72 intron 1. Primers used for strand-specific reverse transcription and qPCR are depicted in (A). Data are mean +/- SEM.
  • Fig 9 Identification of GGGGCC-hexanucIeotide repeat specific binding proteins.
  • A Representative silver-stained gels showing proteins pulled down by the respective repeat containing RNAs.
  • HEK293 nuclear extracts were incubated with indicated RNA probes with (+) or without (-) 50 fold excess of non-biotinylated RNA competitor.
  • RNA/protein bindings were weakened with increasing concentration of NaCl.
  • G+ RNA-protein binding was inhibited, and proteins in the flow through (FL) fraction increased. Boxed lanes in 500 mM NaCl elution fractions were excised for protein identification by LC-MS MS.
  • Fig. 10 List of 20 selected proteins specifically binding to the GGGGCC-repeat 20 proteins were selected as specific GGGGCC repeat binding proteins based on the stringent criteria described in Example 9.
  • the Quantitative Value reflects the relative protein amount estimated from the intensity of LC-MS/MS signal derived from the 500mM elution using Scaffold software. In the presence of non-biotinylated GGGGCC repeat competitor (competition) binding of all proteins is efficiently suppressed. Furthermore, these proteins show at least 2 times more binding to the GGGGCC RNA repeat (GC) compared to AAAACC RNA repeat (AC). Quantitative Values shown here were the averages of three independent experiments. Detailed Description of the Invention
  • an "individual” means any mammal, reptile or bird that may benefit from the present invention.
  • an individual is selected from the group consisting of laboratory animals (e.g. mouse, rat or rabbit), domestic animals (including e.g. guinea pig, rabbit, horse, donkey, cow, sheep, goat, pig, chicken, duck, camel, cat, dog, turtle, tortoise, snake, or lizard), or primates including chimpanzees, bonobos, gorillas and human beings. It is particularly preferred that the "individual” is a human being.
  • a “patient” is any recipient of health care services. Typically, the patient is ill or injured, or susceptible to illness or injury or at risk of developing a disease and thus, in need of treatment by a physician, physician assistant, advanced practice registered nurse, veterinarian, or other health care provider.
  • a patient means any mammal, reptile or bird that may benefit from the invention described herein.
  • a “patient” is selected from the group consisting of laboratory animals (e.g. mouse or rat), domestic animals (including e.g.
  • guinea pig rabbit, horse, donkey, cow, sheep, goat, pig, chicken, camel, cat, dog, turtle, tortoise, snake, or lizard), or primates including chimpanzees, bonobos, gorillas and human beings. It is particularly preferred that the "patient” is a human being.
  • tissue refers to an ensemble of cells of the same origin which fixlfil a specific function conceitedly.
  • a tissue include but are not limited to nervous tissue, muscle tissue, bone, cartilage, connective tissue, and epithelial tissue. Multiple tissues together form an "organ” to carry out a specific function.
  • organ include but are not limited to brain, muscle, heart, blood, skeleton, joint, liver, kidney, stomach, and skin.
  • cell may either refer to a prokaryotic (e.g. a bacterial cell) or a eukaryotic cell (e.g. a fungal, plant or animal cell).
  • Multicellular organisms comprise several types of cells differentiated to fulfil different function in said organism. These include but are not limited to stem cell, embryonic stem cells, cells of the nervous system, blood cells, cells of the immune system, mesenchymal cells, epithelial cells, interstitial cells, metabolism and storage cells, gland cells, extracellular matrix cells, contractile cells, pigment cells, germ cells and tumour cells.
  • the term "cell” as used herein also refers to those cells being removed from their natural environment, such as isolated primary cell or cell lines of any of the above named cell types. Typically, cells such as bacterial cell, yeast cell, isolated primary cell or cell line are used in biotechnological assays. In the context of the present invention isolated primary cell or cell lines are preferably of mammalian origin.
  • cell-free system refers to an in vitro tool widely used to study biological reactions normally occurring in a cell.
  • Cell free systems aim to reduce the complex interactions found in a whole cell by isolating subcellular fractions e.g. by ultracentrifugation, to provide a molecular machinery that can be used in reactions in the absence of many of the other cellular components.
  • Such subcellular fraction may comprise one or more of the known cellular compartments suitable for the designed assay, including but not limited to the plasma membrane, cytosol, nucleus, nucleoli, nucleular membrane, endoplasmatic reticulum, ribosomes, Golgi apparatus, endosomes, lysosmes, mitochondria, vacuoles, cytoskeletal elements (e.g. actin filaments, microtubuli, intermediate filaments) and the like.
  • cell-free systems also encompass cell-free expression systems wherein the transcription and translation as well as protein folding are performed in vitro in absence of any cellular or subcelluar fractions.
  • nucleic acid molecules are understood as a polymeric or oligomeric macromolecule made from nucleotide monomers.
  • Nucleotide monomers are composed of a nucleobase, a five- carbon sugar (such as but not limited to ribose or 2'-deoxyribose), and one to three phosphate groups.
  • a polynucleotide is formed through phosphodiester bonds between the individual nucleotide monomers.
  • nucleic acid molecules include but are not limited to ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and mixtures thereof such as e.g. RNA-DNA hybrids, as well as cDNA, genomic DNA, recombinant DNA, cRNA and mRNA.
  • a nucleic acid may consist of an entire gene, or a portion thereof, the nucleic acid may also be a microRNA (miRNA) or small interfering RNA (siRNA).
  • miRNAs are short ribonucleic acid (RNA) molecules, on average only 22 nucleotides long, found in all eukaryotic cells.
  • miRNAs are post-transcriptional regulators that bind to complementary sequences on target messenger RNA transcripts (mRNAs), usually resulting in translational repression and gene silencing.
  • mRNAs target messenger RNA transcripts
  • siRNAs sometimes known as short interfering RNA or silencing RNA
  • RNA molecules RNA molecules
  • RNAi RNA interference pathway
  • Artificial nucleic acids include polyamide or peptide nucleic acid (PNA), morpholino and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA). Each of these is distinguished from naturally-occurring DNA or RNA by changes to the backbone of the molecule.
  • Suitable methods of detecting nucleic acids such as DNA and/or RNA include e.g. Northern blot analysis, Southern blot analysis, nuclease protection assays (NPA), in situ hybridization (ISH), polymerase chain reaction (PCR for detection of DNA) and reverse transcription-polymerase chain reaction (RT-PCR for detection of RNA).
  • NPA nuclease protection assays
  • ISH in situ hybridization
  • PCR polymerase chain reaction
  • RT-PCR reverse transcription-polymerase chain reaction
  • Hybridization can be used in order to detect a certain nucleic acid sequences.
  • a nucleic acid sequence encoding the complementary sequence of the sequence to be detected may be used as a hybridization probe according to standard hybridization techniques.
  • “In situ hybridization” uses a labeled complementary nucleic acid molecule, e.g. a DNA or RNA strand (i.e. a probe) to localize a specific nucleic acid molecule, e.g. a DNA or RNA sequence, in a sample, e.g. in a portion or section of tissue (in situ).
  • Hybridization conditions are known to those skilled in the art and can be found, for example, in Current Protocols in Molecular Biology, John Wiley & Sons, N.
  • Mode hybridization conditions are defined as equivalent to hybridization in 2X sodium chloride/sodium citrate (SSC) at 30°C, followed by a wash in IX SSC, 0.1% SDS at 50°C.
  • Highly stringent conditions are defined as equivalent to hybridization in 6X sodium chloride/sodium citrate (SSC) at 45°C, followed by a wash in 0.2 X SSC, 0.1 % SDS at 65°C.
  • RNA samples may be first separated by size via electrophoresis in an agarose gel under denaturing conditions. The RNA is then transferred to a membrane, crosslinked and hybridized with a labeled probe.
  • Nonisotopic or high specific activity radio labeled probes can be used including random-primed, nick-translated, or PCR- generated DNA probes, in vitro transcribed RNA probes, and oligonucleotides. Additionally, sequences with only partial homology (e.g., cDNA from a different species or genomic DNA fragments that might contain an exon) may be used as probes.
  • NPA Nuclease Protection Assay
  • RNA template is copied into a complementary DNA (cDNA) using a retroviral reverse transcriptase.
  • the cDNA is then amplified exponentially by PCR.
  • Relative quantitative RT-PCR (“qPCR”) involves amplifying an internal control simultaneously with the gene of interest. The internal control is used to normalize the samples. Once normalized, direct comparisons of relative abundance of a specific mRNA can be made across the samples.
  • Competitive RT-PCR is used for absolute quantitation. This technique involves designing, synthesizing, and accurately quantitating a competitor RNA that can be distinguished from the endogenous target by a small difference in size or sequence. Known amounts of the competitor RNA are added to experimental samples and RT-PCR is performed. Signals from the endogenous target are compared with signals from the competitor to determine the amount of target present in the sample.
  • the above methods may include nucleic acid labeling.
  • a series of techniques are known to the skilled person allowing for labeling of DNA, RNA or oligonuleotides. These include for example Nick translational labeling, random primed DNA labeling, PCR labeling of DNA probes and oligonucleotide 375' end labeling, transcriptional labeling of RNA probes, oligonucleotide 375' end labeling and oligonucleotide tailing.
  • ORF open reading frame
  • ORF refers to a sequence of nucleotides, that can be translated into amino acids.
  • such an ORF contains a start codon, a subsequent region usually having a length which is a multiple of 3 nucleotides, but does not contain a stop codon (TAG, TAA, TGA, UAG, UAA, or UGA) in the given reading frame.
  • stop codon TAG, TAA, TGA, UAG, UAA, or UGA
  • ORFs occur naturally or are constructed artificially, i.e. by gene-technological means.
  • An ORF codes for a peptide, polypeptide, or protein where the amino acids into which it can be translated forms a peptide-linked chain.
  • gene expression or “expression” are used interchangeably herein and refer to the process by which the genetic information is used to synthesize a functional gene product.
  • gene product is a peptide, polypeptide, or protein, or a nucleic acid such as a ribosomal RNA (rRNA), transfer RNA (tRNA) or small nuclear RNA (snRNA).
  • rRNA ribosomal RNA
  • tRNA transfer RNA
  • snRNA small nuclear RNA
  • Gene expression includes the steps of transcription, RNA splicing, translation, and post- translational modification.
  • a DNA sequence encoding a gene is first transcribed into mRNA from which the introns are removed via RNA splicing and the exons are joined and subsequently translated to produce an amino acid chain, which then folds into a protein.
  • transcription refers to the process wherein a particular segment of DNA, typically a gene, is transcribed into RNA by the enzyme RNA polymerase. During transcription, a DNA sequence is read by an RNA polymerase, which produces a complementary, antiparallel RNA strand. As opposed to DNA replication, transcription results in an RNA complement that includes uracil (U) in all instances where thymine (T) would have occurred in a DNA complement. If the gene transcribed encodes a protein, the result of transcription is a pre-messenger RNA (mRNA) or mRNA molecule, which will then be translated into a peptide, polypeptide or protein.
  • mRNA pre-messenger RNA
  • RNA splicing occurs concurrently or after the transcription process and refers to the process wherein the "introns” comprised in the pre-mRNA are removed and the “exons” are covalently joined.
  • intron refers to any nucleotide sequence within a gene which is removed by RNA splicing.
  • intron is typically used to refer to both, the DNA sequence within a gene and the corresponding sequence in the RNA transcript which is removed by RNA splicing. Sequences that are joined together in the final mature RNA after RNA splicing are referred to as "exons". Again the term exon is typically used in the art to refer to both, the DNA sequences within a gene and the corresponding sequences in the RNA transcript which are joined during RNA splicing after removal of the intron.
  • Ribosomes facilitate the process of "translation" of mRNA into an amino acid chain by inducing the binding of tRNAs with complementary anticodon sequences to that of the mRNA.
  • the tRNAs carry specific amino acids that are chained together into a polypeptide as the mRNA passes through and is "read” by the ribosome.
  • translation is an AUG-dependent process, wherein an AUG codon of the mRNA (corresponding to an ATG codon of the DNA) is recognized as translation initiation site resulting in methionine being the first amino acid in the produced amino acid chain.
  • AUG-independent translation mechanism exist, e.g.
  • AUG-independent translations mechanism includes the use of internal ribosome entry sites (IRES) which structurally mimic the initiator tRNA and manipulate the ribosomes to initiate the translation at a non-AUG site.
  • IRS internal ribosome entry sites
  • RAN-translation refers specifically to an AUG-independent translation mechanism of nucleotide repeats.
  • RAN-translation occurs for exonic RNA comprising, trinucleotide, tetranucleotide and pentanucleotide repeats.
  • polypeptides which were RAN-translated of such nucleotide repeats may (but do not necessarily have to) differ from peptides or polypeptides that were translated in an AUG- dependent manner in that they lack the initial methionine. For instance, RAN-translation may be facilitated in that the presence of said nucleotide repeats promotes the formation of a hairpin structure which subsequently triggers the RAN-translation.
  • amino acid chain and “polypeptide chain” are used synonymously in the context of present invention.
  • peptide refers to a short polymer of amino acids linked by peptide bonds. It has the same chemical (peptide) bonds as proteins, but is commonly shorter in length.
  • the shortest peptide is a "dipeptide", consisting of two amino acids joined by a single peptide bond.
  • Peptide may also have a length of up to 8, 10, 12, 15, 18 or 19 amino acids.
  • a peptide has an amino end and a carboxyl end, unless it is a cyclic peptide.
  • DPR dipeptide repeat
  • a dipeptide repeat may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000 or more dipeptides joined together by peptide bonds.
  • polypeptide refers to a single linear chain of amino acids bonded together by peptide bonds and preferably comprises at least about 20 amino acids.
  • a polypeptide can be one chain of a protein that is composed of more than one chain, or it can be the protein itself if the protein is composed of a single chain.
  • protein refers to a molecule comprising one or more polypeptides that resume a secondary and tertiary structure and additionally refers to a protein that is made up of several polypeptides, i.e. several subunits, forming quaternary structures.
  • the protein has sometimes non-peptide groups attached, which can be called prosthetic groups or cofactors.
  • isolated peptide refers to a peptide, polypeptide or protein which has been removed from its natural environment in a cell such that other cellular material normally nearby is not present anymore.
  • isolated peptides, polypeptides or proteins produced outside their natural cellular environment e.g. via chemical means or via recombinant means in a non-natural environment, are considered as isolated peptides, polypeptides or proteins.
  • Polypeptides or proteins can be further modified by chemical modification.
  • a chemically modified polypeptide may comprise chemical groups other than the residues found in the 20 naturally occurring amino acids. Examples of such other chemical groups include without limitation glycosylated amino acids and phosphorylated amino acids.
  • Chemical modifications of a polypeptide may provide advantageous properties as compared to the parent polypeptide, e.g. one or more of enhanced stability, increased biological half-life, or increased water solubility. Chemical modifications include without limitation: PEGylation, glycosylation of non-glycosylated parent polypeptides. Such chemical modifications applicable to the variants usable in the present invention may occur co- or post-translational.
  • an "antigenic protein” as referred to in the present application is a polypeptide as defined above which contains at least one epitope.
  • An "antigenic fragment” of an antigenic protein is a partial sequence of said antigenic protein comprising at least one epitope.
  • the nucleic acid construct does not need to encode the full-length antigenic protein as it is found in e.g. a diseased cell, a cancer cell, or a pathogen.
  • a shortened fragment of such a protein is sufficient as long as its amino acid sequence comprises the epitope or epitopes responsible for the recognition of the antigenic protein by the immune system.
  • antigen refers to any molecule or part of a molecule, including but not limited nucleic acid, amino acid, peptide, polypeptide, protein, carbohydrate, and lipid, to which a ligand of the invention binds.
  • epitope refers to a antigenic determinant, which is part of an antigen that is specifically bound by a ligand of the invention, preferably an antibody or antigen binding-fragment thereof.
  • Epitopes typically consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.
  • the epitopes of an antigen may be a conformational epitope or a non-conformational, i.e. a linear, epitope. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.
  • a conformational epitope is composed of discontinuous sections of the antigen's amino acid sequence. These epitopes interact with the ligand based on the 3-D surface features and shape or tertiary structure of the antigen. Most epitopes are conformational. By contrast, linear epitopes interact with the ligand based on their primary structure. A linear epitope is formed by a continuous sequence of amino acids from the antigen. Conformational epitopes preferably comprise between 8 and 20 discontinuous amino acids, preferably between 8 and 15 amino acids. Linear epitopes have a length of between 6 to 20 amino acids, more preferably between 8 and 15 amino acids.
  • Peptides, polypeptides or proteins may be detected via various methods including but not limited to a filter trap assay, Western Blotting, enzyme-linked immunosorbent assay (ELISA), Immunohistochemistry (IHC), Immunocytochemistry (ICC), and size exclusion chromatography (SEC).
  • a filter trap assay Western Blotting
  • ELISA enzyme-linked immunosorbent assay
  • IHC Immunohistochemistry
  • ICC Immunocytochemistry
  • SEC size exclusion chromatography
  • Immunostaining including but not limited to immunohistochemistry (IHC) or immunocytochemistry (ICC), is an antibody-based method to detect a specific protein in a sample.
  • immunostaining was originally used to refer to the immunohistochemical staining of tissue sections. Now however, immunostaining encompasses a broad range of techniques used in histology, cell biology, and molecular biology that utilize antibody-based staining methods. While the first cases of IHC staining used fluorescent dyes, other non- fluorescent methods using enzymes such as peroxidase and alkaline phosphatase are now used more often. These enzymes are capable of catalyzing reactions that give a coloured product that is easily detectable by light microscopy.
  • radioactive elements can be used as labels, and the immunoreactions can be visualized by autoradiography.
  • Tissue preparation or fixation is essential for the preservation of cell morphology and tissue architecture. Inappropriate or prolonged fixation may significantly diminish the antibody binding capability.
  • Many antigens can be successfully demonstrated in formalin-fixed paraffin- embedded tissue sections. Optimization of fixation methods and times, pre-treatment with blocking agents, incubating antibodies with high salt, and optimizing post-antibody wash buffers and wash times may be important for obtaining high quality immunostaining.
  • SEC Size-exclusion chromatography
  • Gel-filtration chromatography typically refers to an SEC wherein an aqueous solution is used to transport the sample through the column, whereas “gel permeation chromatography” refers to SEC wherein an organic solvent is used as mobile phase.
  • filter retention assay and "filter trap assay” are used simultaneously herein referring to the analysis of protein aggregates. Purified proteins are filtered through a cellulose-acetate filters resulting in big aggregates being retained on the filter. Subsequently these membrane-bound aggregates may be characterized by any means or methods known in the art.
  • “Western blotting” allows the detection of specific proteins (native or denatured) from extracts made from cells or tissues, before or after any purification steps. Proteins are generally separated by size using gel electrophoresis before being transferred to a synthetic membrane (typically nitrocellulose or PVDF) via dry, semi-dry, or wet blotting methods. The membrane can then be probed using antibodies using methods similar to immunohistochemistry, but without a need for fixation. Detection is typically performed using peroxidase linked antibodies to catalyze a chemiluminescent reaction.
  • Western blotting is a routine molecular biology method that can be used to semi quantitatively or quantitatively compare protein levels between extracts.
  • Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non- denaturing conditions).
  • the "enzyme-linked immunosorbent assay (ELISA)" is a diagnostic method for quantitatively or semi-quantitatively determining protein concentrations from blood plasma, serum or cell/tissue extracts in a multi-well plate format (usually 96-wells per plate). Broadly, proteins in solution are adsorbed to ELISA plates. Antibodies specifically binding to the protein of interest are used to probe the plate.
  • EM Electromon microscopy
  • a "marker”, “tag”, or “label” is any kind of substance which is able to indicate the presence of another substance or complex of substances.
  • the marker can be a substance that is linked to or introduced in the substance to be detected. Detectable markers are used in molecular biology and biotechnology to detect e.g. a protein, a product of an enzymatic reaction, a second messenger, DNA, interactions of molecules etc.
  • marker or labels examples include a fluorophore, a chromophore, a radiolabel, a metal colloid, an enzyme, or a chemiluminescent or bioluminescent molecule.
  • fluorophores include various forms of green fluorescent protein (GFP) such as EnGFP, RFP, CYP, BFP, YFP, dsRed etc., phycobiliproteins (allophycocyanin, phycocyanin, phycoerythrin and phycoerythrocyanin), fluorescein (fluorescein isothiocyanate, FITC), rhodamine (tetramethyl rhodamine isothiocyanate, TRITC), and cyanine dyes (such as C2, Cy3 Cy5, Cy7).
  • GFP green fluorescent protein
  • FITC fluorescein isothiocyanate
  • rhodamine tetramethyl rhodamine isothiocyanate
  • TRITC
  • radiolabels examples include 3 H, 14 C, 32 P, 33 P, 35 S, 99m Tc or l25 I.
  • enzymes include luciferase, beta-galactosidase, horseradish peroxidase, alkaline phosphatase, glucose oxidase, and urease.
  • selection marker refers to a reporter gene which conveys a certain trait to a cell or organism which allows such cell or organism to survive in an environment in which it would not survive without said reporter gene. Accordingly, the expression of a selection marker allows for the artificial selection of cells or organism whose genome comprises said reporter gene. Selection markers include but are not limited to auxotrophic marker genes and resistance marker genes conferring resistance to a toxin or an antibiotic. The terms “selection marker”, “reporter gene”, and “marker gene” are used interchangeably herein. Selection markers may be auxotrophic marker genes or resistance marker genes conferring resistance to a toxin or an antibiotic.
  • auxotrophy marker gene is understood as a gene which enables growth of such auxotrophic cells or organism under selective culture conditions by encoding a molecule required for the synthesis of a product necessary for the survival of an auxotrophic prokaryote on a selective medium used for cell culturing. Any marker gene which is capable of complementing the corresponding gene which is nonfunctional in an auxotrophic cell or organism may be useful in the context of this invention.
  • the terms "resistance marker genes conferring resistance to a toxin and/or an antibiotic”, or variations thereof such as “toxin-resistance marker genes” or “antibiotica-resistance genes” refer to reporter genes which encode an enzyme that functionally inactivates a toxin or an antibiotic.
  • the functional inactivation of a toxin or antibiotic may be achieved by expressing a marker gene which carries mutation(s) rendering the respective gene product insensitive to a toxin or antibiotic.
  • the functional inactivation of a toxin or antibiotic may be achieved by expressing a marker gene which inhibits the toxin or antibiotic e.g. by interacting or binding to it.
  • the functional inactivation of a toxin or antibiotic may also be achieved by expressing a marker gene which counteracts the effects of the toxin or antibiotic.
  • Antibiotic compounds include but are not limited to tetracyclines, sulfonamides, penicillins, cephalosporins, ansamycins, carbapenems, macrolides, quinolones, aminonucleoside, aminoglycosides, peptides, glycopeptides, and lipopeptides.
  • hygromycin B, neomycin, kanamycin, gentamicin, and G418 are aminoglycoside antibiotics which are similar in structure. In general, neomycin and kanamycin are used for prokaryotes, whilst G418 is needed for eukaryotes.
  • Radioisotopes were used extensively in the past, but they are expensive, have a short shelf-life, offer no improvement in signal to noise ratio and require special handling and disposal.
  • Enzymes and fluorophores have largely replaced radioactive isotopes as detectable tags for assays. A number of advancements in reagents and instrumentation make these newer technologies more versatile and powerful.
  • Enzymatic tags such as horseradish peroxidase (HRP) are most commonly used for blotting, immunoassays and irnmunohistochemistry methods. Fluorescent tags are used predominately for cellular imaging, nucleic acid amplification and sequencing and microarrays; however, fluorescence technology is developing rapidly for application in all types of assays.
  • HRP horseradish peroxidase
  • expression level refers to the amount of gene product (e.g. DPR) present in the body or a sample at a certain point of time.
  • the expression level can e.g. be measured/quantified/detected by means of the amounts of the protein or of the mRNA encoding the protein.
  • the expression level can be quantified by normalizing the amount of gene product of interest (e.g. DPR) present in a sample with the total amount of gene product of the same category (total protein or mRNA) in the same sample or in a reference sample (e.g.
  • the expression level can be measured/quantified/detected by means of any method as known in the art, e.g. methods for the direct detection and quantification of the gene product of interest (such as mass spectrometry) or methods for the indirect detection and measurement of the gene product of interest that usually work via binding of the gene product of interest with one or more different molecules or detection means (e.g. primer(s), probes, antibodies, scaffold-proteins) specific for the gene product of interest (e.g. DPR).
  • the expression level is determined on the basis of the protein rather than on the basis of the mRNA.
  • toxicity refers to the degree to which a compound/substance can damage an organism or a substructure of the organism, such as a cell (cytotoxicity), tissue or an organ. Accordingly, the term “toxic effect” refers to the damaging effect a compound/substance has on an organism, organ, tissue or cell.
  • a compound may exhibit a toxic effect in that it damages the function and/or structure of an organism, organ, tissue or cell, which may result in an altered function or a loss of function of certain elements or parts of the organism, organ, tissue or cell, or may even result in the death of said organism, organ, tissue or cell.
  • the term “toxic compound” thus, refers to a substance, e.g. a nucleic acid, a peptide, polypeptide or protein, or a chemical substance or compound, which exhibits a toxic effect on the organism, organ, tissue or cell.
  • the toxicity or the toxic effect of a compound may be measured using one of various viability assays known in the art including but not limited to formazan-based assays (MTT/XTT), Lactate dehydrogenase (LDH) assay, ATP test, Calcein AM, Clonogenic assay, Ethidium homodimer assay, evans blue, Fluorescein diacetate hydrolysis/Propidium iodide staining (FDA/PI staining), Flow cytometry, TUNEL assay, with green fluorescent protein (GFP), methyl violet, propidium iodide, trypan blue, or resazurin. Further, DNA stainings may be used to differentiate between necrotic, apoptotic and normal cells.
  • MTT/XTT formazan-based assays
  • LDH Lactate dehydrogenase
  • ATP test Calcein AM
  • Calcein AM Calcein AM
  • Clonogenic assay Ethidium homodimer as
  • disease and “disorder” are used interchangeably herein, referring to an abnormal condition, especially an abnormal medical condition such as an illness or injury, wherein a cell, a tissue, an organ, or an individual is not able to efficiently fulfil its function anymore.
  • a disease is associated with specific symptoms or signs indicating the presence of such disease. The presence of such symptoms or signs may thus, be indicative for a cell, a tissue, an organ, or an individual suffering from a disease. An alteration of these symptoms or signs may be indicative for the progression of such a disease.
  • a progression of a disease is typically characterised by an increase or decrease of such symptoms or signs which may indicate a "worsening" or “bettering” of the disease.
  • the "worsening" of a disease is characterised by a decreasing ability of a cell, tissue, organ or individual/patient to fulfil its function efficiently, whereas the “bettering" of a disease is typically characterised by an increase in the ability of a cell, tissue, an organ or an individual/patient to fulfil its function efficiently.
  • a cell, a tissue, an organ or an individual being "susceptible" to a disease is in a healthy state but especially vulnerable to the emergence of a disease, e.g. due to genetic predisposition, lacking vaccination, poorly developed or immature immunity, poor nutritional status, or the like. The outbreak of the disease may still be prevented by prophylaxis or pre-cautionary treatment.
  • a cell, a tissue, an organ or an individual may be "suspected of having" a disease wherein said cell, tissue, organ or individual typically shows early or weak signs or symptoms of such disease. In such case, the onset of the disease may still be prevented or its progression may be reduced or prevented by treatment.
  • detect refers to establishing the presence or absence of a disease in a patient.
  • a moiety used in the detection of a disease is able to identify the presence or absence of an indicator of a disease in a sample or in an individual or patient.
  • a disease may be detected by means of a ligand or a tagged ligand interacting with, i.e. binding to or forming a complex with, a disease specific nucleic acid or peptide, polypeptide or protein.
  • a disease may also be detected by means of an inhibitor blocking the mechanism of action underlying the disease and thus, altering the symptoms such that the underlying disease may be identified.
  • treat means accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting or preventing development of symptoms characteristic of the disorder(s) being treated; (c) inhibiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting or preventing recurrence of the disorder(s) in an individual that has previously had the disorder(s); and (e) limiting or preventing recurrence of symptoms in individuals that were previously symptomatic for the disorder(s).
  • a therapy treats a disease or disorder, or the symptoms of a disease or disorder by accomplishing one or more of above named effects (a)-(e).
  • a disease may be treated by means of inhibiting blocking the mechanism of action underlying the disease, e.g. via compounds that inhibit the expression of disease specific polypeptide/proteins or by inhibiting further processes said polypeptide/protein is involved in.
  • a disease may also be treated by activating the patients immune system, e.g. via active immunization, or by supporting the patients immune system, e.g. via passive immunization.
  • a disease or disorder means preventing that such disease or disorder occurs in a patient. Accordingly, a moiety having a prophylactic effect prevents the onset of a disease or disorder in a patient.
  • a disease or disorder may be prevented by immunizing, e.g. actively or passively immunizing, a healthy individual such that the onset of disease is avoided.
  • a disease or disorder may be prevented through ligands or inhibitors undermining the function of disease related molecules or processes, e.g. via ligands or inhibitors that block the expression of disease specific polypeptide/proteins or by inhibiting further processes said polypeptide/protein is involved in.
  • Immunization refers to the process of activating, strengthening or boosting the immune system of an individual against an agent which typically causes or induces a disease or disorder. Hence, by immunizing a healthy individual against said agent, the onset of a disease or disorder may be prevented. Immunizing a patient suffering from a disease or disorder may treat said disease or disorder or may prevent the further progression of said disease. Immunization may be achieved through various techniques, most commonly immunization is achieved through vaccination of the healthy individual or the patient suffering from a disease or disorder.
  • Passive immunization refers to the process wherein pre-synthesized elements of the immune system are transferred to an individual/patient such that the body does not need to produce these elements itself. Passive immunization aims at the treatment of a disease or the prevention of the progression of a disease, in particular in cases where the patient is not able to combat such disease or disorder due to an inefficient immune system (e.g. deficient immune system, or unrecognizable immunogen, e.g. of a tumour).
  • an inefficient immune system e.g. deficient immune system, or unrecognizable immunogen, e.g. of a tumour.
  • antibodies e.g. animal or humanized antibodies produced in vitro by cell culture directed against a disease-specific immunogen, or nucleic acids encoding said antibody and allowing for its expression, are means of passive immunization.
  • active immunization refers to the immunization via the introduction of a foreign molecule (immunogen) into the body, which causes the body itself to generate an immune response against the immunogen.
  • Active immunization aims at the prophylaxis of a disease or the prevention of the progression of a disease, as the immune system of an individual is primed/activated/strengthened to react against said immunogen resulting in a more efficient or faster immune response to the immunogen.
  • the principle underlying active immunization is the generation of an immunological "memory”. Challenging an individual's immune system with e.g.
  • a vaccine comprising a disease specific immunogen, induces the formation and/or propagation of immune cells which specifically recognize the immunogen comprised by the vaccine. At least a part of said immune cells remains viable for a period of time which can extend to 10, 20 or 30 years after vaccination. If the individual's immune system encounters the immunogen again within the aforementioned period of time, the immune cells generated by vaccination are reactivated and enhance the immune response against the immunogen as compared to the immune response of an individual which has not been challenged with the vaccine and encounters said immunogen for the first time. In many cases, a single administration of a vaccine is not sufficient to generate the number of long-lasting immune cells which is required for effective protection against said diseases or disorder.
  • Prime-boost vaccination regimen An administration regimen comprising the repeated administration of a vaccine directed against the same disease is referred to in the present application as "prime-boost vaccination regimen".
  • a prime-boost vaccination regimen may involve at least two administrations of a vaccine or vaccine composition directed against a specific pathogen, group of pathogens or diseases.
  • the first administration of the vaccine is referred to as "priming” and any subsequent administration of the same vaccine or a vaccine directed against the same pathogen as the first vaccine is referred to as "boosting".
  • the period of time between prime and boost is, preferably, 1 week, 2 weeks, 4 weeks, 6 weeks or 8 weeks.
  • Prime-boost vaccination regimens may be homologous or heterologous. In homologous prime-boost regimens both the priming and the at least one boosting is performed using the same means of administration of the antigenic protein or antigenic fragment thereof, i.e. priming and boosting are performed using a polypeptide or priming and boosting are performed using a nucleic acid construct comprised by the same vector.
  • a heterologous prime-boosting regimen involves the use of different means for priming and for boosting the immune response.
  • Two or more antigenic proteins or antigenic fragments thereof are "immunologically identical" if they are recognized by the same antibody, T-cell or B-cell.
  • the recognition of two or more immunogenic polypeptides by the same antibody, T-cell or B-cell is also known as "cross reactivity" of said antibody, T-cell or B-cell.
  • the recognition of two or more immunologically identical polypeptides by the same antibody, T-cell or B-cell is due to the presence of identical or similar epitopes in all polypeptides. Similar epitopes share enough structural and/or charge characteristics to be bound by the Fab region of the same antibody or B-cell receptor or by the V region of the same T-cell receptor.
  • the binding characteristics of an antibody, T-cell receptor or B-cell receptor are, preferably, defined by the binding affinity of the receptor to the epitope in question.
  • Two immunogenic polypeptides are "immunologically identical" as understood by the present application if the affinity constant of polypeptide with the lower affinity constant is at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 %, at least 95 % or at least 98 % of the affinity constant of the polypeptide with the higher affinity constant.
  • Methods for determining the binding affinity of a polypeptide to a receptor such as equilibrium dialysis or enzyme linked immunosorbent assay (ELISA) are well known in the art.
  • two or more "immunologicaly identical" polypeptides comprise at least one identical epitope.
  • the strongest vaccination effects can usually be obtained, if the immunogenic polypeptides comprise identical epitopes or if they have an identical amino acid sequence.
  • the term "vaccine” refers to a biological preparation, typically a pharmaceutical, which improves immunity to a specific disease. Said preparation may comprise one or more disease specific immunogens suitable for eliciting an immune response.
  • said compound may be a polypeptide which is substantially identical or immunologically identical to a polypeptide as specified below comprising the specified dipeptide repeat (DPR).
  • DPR dipeptide repeat
  • the vaccine may comprise a nucleic acid construct which encodes an immunogenic polypeptide which is substantially identical or immunologically identical to a polypeptide comprising the specified dipeptide repeat.
  • the polypeptide is expressed in the individual treated with the vaccine.
  • said nucleic acid construct may be a vector.
  • the term “vector” refers to a protein or a polynucleotide or a mixture thereof which is capable of being introduced or of introducing the proteins and/or nucleic acid comprised therein into a cell. Moreover, the term “vector” refers to at least one polynucleotide formulated with a preparation of liposomes or lipid nanoparticles which is capable of transfecting a cell with the at least one polynucleotide as described, e.g. by Geall et al., 2012. In addition to the polynucleotide encoding the gene of interest, additional polynucleotides and/or polypeptides may be introduced into the cell.
  • polynucleotides and/or polypeptides are especially preferred if said additional polynucleotides and/or polypeptides are required to introduce the nucleic acid construct into the cell or if the introduction of additional polynucleotides and/or polypeptides increases the expression of the immunogenic polypeptide encoded by the nucleic acid construct of the present invention.
  • genes of interest encoded by the introduced polynucleotide are expressed within the cell upon introduction of the vector or vectors.
  • suitable vectors include but are not limited to plasmids, cosmids, phages, viruses or artificial chromosomes.
  • Examples of a disease include but are not limited to neurological disorders, inflammatory diseases, infectious diseases, cutaneous conditions, endocrine diseases, intestinal diseases, genetic disorders, autoimmune diseases, traumatic diseases, joint diseases, and various types of cancer.
  • Neurological disorders refers to any disorder of the nervous system wherein structural, biochemical or electrical abnormalities occur in the brain, the spinal cord or other nerves which affect a range of symptoms including but not limited to paralysis, muscle weakness, poor coordination, loss of sensation, seizures, confusion, pain and altered levels of consciousness.
  • neurological disorders include but are not limited to damage of the brain or individual parts of the brain (e.g. damage of the prefrontal cortex, frontal lobe, parietal lobe, temporal lobe, occipital lobe, cerebellum, hippocampus, brain stem, limbic system), dysfunction of the brain or individual parts of the brain (e.g.
  • aphasia aphasia, dysarthria, apraxia, agnosia, amnesia, ataxia
  • inflammation of the brain e.g. encephalitis, viral encephalitis, cavernous sinus thrombosis, brain abscess, amoebic.
  • "Neurodegenerative diseases” such as Alzheimer's, Parkinson's disease, Huntington's disease, Amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), which is also called frontotemporal lobar degeneration (FTLD), amyotrophic lateral sclerosis-frontotemporal dementia (ALS-FTD, also called ALS-FTLD), and spinocerebellar ataxia are further examples of neurological disorders.
  • Neurological disorders also include spinal cord disorders (e.g. syringomyelia, syringobulbia, Morvan's syndrome, Vascular myelopathy, Foix- Alajouanine syndrome, spinal cord compression) and spinal cord inflammation (e.g. myelitis, poliomyelitis, demyelinating disease, transverse myelitis, tropical spastic paraparesis, epidural abscess), central and/or peripheral neuropathy, cranial nerve disorders (e.g. trigeminal neuralgia), movement disorders of the central and/or peripheral nervous system (e.g.
  • spinal cord disorders e.g. syringomyelia, syringobulbia, Morvan's syndrome, Vascular myelopathy, Foix- Alajouanine syndrome, spinal cord compression
  • spinal cord inflammation e.g. myelitis, poliomyelitis, demyelinating disease, transverse myelitis, tropical spastic paraparesis, epidural abscess
  • Parkinson's disease ALS, Tourette's Syndrome, multiple sclerosis
  • sleep disorders e.g. insomnia, hypersomnia, sleep apnea, narcolepsy, cataplexy, Kleine-Levin, circadian rhythm sleep disorder, advanced sleep phase disorder, delayed sleep phase disorder
  • headache e.g. migraine, cluster, tension
  • neuropsychiatric illnesses delirium
  • dementia e.g. Alzheimer's disease, vascular dementia, FTD, semantic dementia and dementia with Lewy bodies
  • stroke e.g. MCA, ACA, PCA, Foville's, Millard-Gubler, Lateral medullary, Weber's, Lacunar stroke
  • tumors e.g.
  • MND motor neuron diseases
  • PLS primary lateral sclerosis
  • PMA progressive muscular atrophy
  • PBP progressive bulbar palsy
  • pseudobulbar palsy e.g. ALS, primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), progressive bulbar palsy (PBP), pseudobulbar palsy
  • Symptoms of a disease are implication of the disease noticeable by a cell, tissue, organ or individual having such disease and include but are not limited to pain, weakness, tenderness, strain, stiffness, and spasm of the cell, tissue, an organ or an individual.
  • “Signs” or “signals” of a disease include but are not limited to the change or alteration such as the presence, absence, increase or elevation, decrease or decline, of specific indicators such as biomarkers or molecular markers, or the development, presence, or worsening of symptoms.
  • an “indicator” can be defined as a substance within a biological system that is used as an indicator of a biological state of said system.
  • biomarker is sometimes also applied to means for the detection of said endogenous substances (e.g. antibodies, nucleic acid probes, imaging systems). In the context of present invention, however, the term “biomarker” shall be only applied for the substance, not for the detection means.
  • biomarkers can be any kind of molecule present in a living organism, such as a nucleic acid (DNA, mR A, miRNA, rRNA etc.), a protein (cell surface receptor, cytosolic protein etc.), a metabolite or hormone (blood sugar, insulin, estrogen, etc.), a molecule characteristic of a certain modification of another molecule (e.g. sugar moieties or phosphoryl residues on proteins, methyl-residues on genomic DNA, expansion of nucleotide repeats) or a substance that has been internalized by the organism or a metabolite of such a substance.
  • a disease or disorder may be characterized by the presence or absence, increase or decrease of such an indicator. Said indicator of a disease may or may not cause the disease.
  • Indicators of the presence and/or progression of a disease include "genetic markers" such as nucleotide repeat, VNTRs (variable number tandem repeat; e.g. STR (Short tandem repeat), AFLP (amplified fragment length polymorphism), SSR (Simple sequence repeat), MLVA), SSLP (Simple sequence length polymorphism), RFLP (restriction fragment length polymorphism), RAPD (random amplification of polymorphic DNA), SNP (single nucleotide polymorphism), SFP (single feature polymorphism), DArT (diversity arrays technology), RAD markers (restriction site associated DNA markers).
  • gene markers such as nucleotide repeat, VNTRs (variable number tandem repeat; e.g. STR (Short tandem repeat), AFLP (amplified fragment length polymorphism), SSR (Simple sequence repeat), MLVA), SSLP (Simple sequence length polymorphism),
  • nucleotide repeat refers to a location in the genome wherein a short nucleotide sequence forms repeating sequences of 2, 3, 4, 5, or 6 nucleotides, i.e. dinucleotides, trinucleotides, tetranucleotides, pentanucleotides, and hexanucleotides.
  • the number of repetitions of the repeat may vary between 1 and 50, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50.
  • these repeats may be expanded to be repeated far more often than in a healthy state, e.g.
  • the expansion level of dinucleotides, trinucleotides, tetranucleotides, pentanucleotides, or hexanucleotides may be an indicator (and/or the cause) of a disease such as e.g. a neurological disease, in particular a neurogenerative disease.
  • nucleotide repeats may be present in the exon or intron of a gene and may or may not be transcribed and may or may not be translated into a peptid
  • the neurogenerative diseases ALS, FTD and ALS-FTD are characterized in that the hexanucleotide (GGGGCC) n repeats located within intron I of the C9orf72 gene are expanded.
  • GGGGCC-repeats may vary between 1 and 19, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, and 19.
  • the number "n" of these repeats may be expanded to be repeated far more often than in a healthy state, e.g. typically they may be expanded much more than 30 to 40 times. However, some patients may already show symptoms starting from 20 repeats.
  • the expansion of the hexanucleotide repeats may lead to several hundred repetition of the hexanucleotide repeat, i.e. 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, and 10000 repeats, or more.
  • the exact number of repeats cannot be determined with current sequencing techniques.
  • the five different possible DPRs are the following: poly-(Gly- Ala), poly-(Gly-Pro), poly-(Gly-Arg), poly-(Ala-Pro) and poly-(Pro-Arg).
  • poly-GA, poly-GP and poly-AP polypeptides are highly hydrophobic and form aggregates in an intracellular environment.
  • genomic sequence of the region surrounding the hexanucleotide repeat of the C9orf72 gene reads as follows in the reference sequence from NCBI (SEQ ID No: 1)
  • the neurogenerative disease spinocerebellar ataxia SCA36 is characterized in that the hexanucleotide (GGCCTG) n repeats located within the intron 1 region of the NOP56 gene are expanded.
  • GGCCTG hexanucleotide
  • the number "n" of the GGGGCC-repeats may vary between 1 and 15, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 15.
  • the number "n" of these repeats may be expanded to be repeated far more often than in a healthy state, e.g. typically they may be expanded 30 to 40, leading to several hundred repetition of the repeat, i.e.
  • RNA transcription products which comprise either GGCCUG- or CAGGCC-repeats.
  • DPR dipeptide-repeat
  • the genomic sequence of the region surrounding the hexanucleotide repeat of the NOP56 gene reads as follows (SEQ ID No: 2)
  • polypeptides comprising or consisting of dipeptide-repeats encoded by genomic hexanucleotide repeats refers to polypeptides that comprise dipeptide repeats, preferably of 9 or more of consecutive identical dipeptides. It is more preferred that the polypeptides comprise more than 10, more than 15, more than 20, more than 25, more than 30, more than 35, more than 40, more than 45, more than 50 dipeptide repeats.
  • the polypeptide comprises or consists of dipeptide-repeats with a sequence selected from the group consisting of (Gly-Ala) a , (Gly-Pro) b , (Gly-Arg) c , (Ala-Pro) d , (Pro- Arg) e , (Gly-Leu)f, (Ala-Trp) g , (Pro-Gly) h, (Ala-Gln) j , (Gly-Pro)k, and (Pro-Arg)i wherein a is an integer of 16 or more, preferably 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more; b is
  • non-repetitive regions 5' and 3' of the genomic repeat region further polypeptide encoding regions termed "non-repetitive regions" are located.
  • the sequence of the non-repetitive regions depends on the genomic location of the hexanucleotide repeat region. Accordingly, the DPRs may further comprise additional amino acid sequences at its N- and/or C-Terminus.
  • a polypeptide comprising a dipeptide repeat region further comprises additional C-terminal amino acid sequence encoded by the genomic region 3' of the repeat region.
  • the exact start site of translation is unknown. In the case of RAN-translation the start site may be within the repeat region or within the flanking region.
  • Initiation from a non-canonical start-codon may also lead to an N-terminal flanking region with the largest N-terminal region extending to the first in- frame stop-codon at the 5' end of the RNA.
  • the DPR may also comprise N-terminal amino acid sequence encoded by the genomic region 5' of the repeat region. The length of the respective non-repetitive C-terminal amino acid sequence depends on the position of the first STOP codon in the respective reading frame that gives rise to the DPR.
  • the C- and/or N-terminal non-repetitive regions comprised in the polypeptide will vary depending on the genomic context and will allow distinguishing between two diseases characterized by expansion of genomic hexanucleotide repeats.
  • the skilled person is able to determine the respective 5' and/or 3' genomic region and, thus, the amino acid sequences that may be comprised N- and/or C-terminally of the DPR.
  • Polymorphisms in the 3' region of the gGGGCC repeat may cause frame shifts in patients exhibiting an expansion of the hexanucleotide repeat in the C9orf72 gene.
  • Amino acid sequences that may be comprised at the C-terminus of the DPRs characteristic for ALS, FTD, or ALS-FTD include for
  • Amino acid sequences that may be comprised at the C-terminus of the DPRs characteristic for SCA36 include for
  • Amino acid sequences that may be comprised at the N-terminus of the DPRs characteristic for ALS, FTD, or ALS-FTD include for
  • Amino acid sequences that may be comprised at the N-terminus of the DPRs characteristic for SCA36 include for (vi) the (Gly-Leu) repeat: GVVAGRGRRDGGGFGLRSGRRQ (SEQ ID NO: 19);
  • polypeptide resulting from the hexanucleotide repeat in the C9orf72 gene in sense and/or antisense direction may have one of the following sequences:
  • SEQ ID NO: 27 greskeearspslvpappppppppgspgpgcrqfhqsleakarhpasvremrgkvkm rralI ⁇ apastrassrq npkqppamlppphsptrhrlrlmgrrrlrnrspapgp pgpp GP [GP] 0 GP
  • SEQ ID NO: 28 rltrrkqggkqpqpvassgtqesrarGR[GR] p GRgwgagpgagpgrgcgcgacarg gggagggewvseeaaswrvavwgsaagkrrg
  • SEQ ID NO: 29 geppllpaplpgsrtpnshp gcrllthplatacasaaagagtataappra ⁇ ra dh
  • SEQ ID NO: 30 s rrqg srv seprlg k raah af qa lstrgslfss qrqrsqrvpgketarvl ragkqgrgqipipcpcaaaaaaagkpgarmqaippvargesptpsfgqrneresknassseesprfyprlfpaaepqt atrqdaasslthspppappppraqapqpqp gpapgpapttP [PR] r PRplards.
  • polypeptide resulting from the hexanucleotide repeat in the NOP56 gene in sense and/or antisense direction may have one of the following sequences:
  • SEQ ID NO: 32 qehqepdyyperafpsrfaacsa ⁇ gaa ⁇ rqp ⁇ paqlq ⁇ ekarspalgttqpalraippwa appkagdgwpglrlvggagawplepgsarrsalaalraepgswrhgeewlrgagdatvgvsacvraadrAW[A W] t AWacacacacacpgngfrqtlrlr
  • SEQ ID NO: 34 gsagppkeptdlthlrlqqtdllhflqrqqrvadrvlkqhvqqhlerepgrerqrnlsvcrn pfpgqAQ[AQ] w AQalsaartqaetptvaspaprnhsspwrqlpgsarnaasalrraepgsrghapapptnnpgtt pspafggaaqggiarsagwvvpsagerafsglcscaggrgcrgraapgralqaakrlgkalsg (xi) SEQ ID NO: 35: spplpsacampsrpraqtaratapgtgagarastqpqrlpepvpragagagagGP[GP] x G Pvcgpnagmphrrvarapqplltmapapgfgsqcg
  • SEQ ID NO: 36 ptacsnstcnstwngsrgasvnatsawarrsqgnrrrrrPR[PR] y PRpclrpenpkppps I ⁇ p attphhgassrvrlamrlarsgamqapeat ppp ⁇ iggrg ⁇ hhl legpprws ga ⁇ agwcra saaragwagdarppvghcrrrndlgrpfrgnspvpdvlvi,
  • m is an integer of 10 or more, preferably 15, 20 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more; "o" is an integer of 10 or more, preferably 15, 20 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,
  • r is an integer of 10 or more, preferably 15, 20 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more; "r" is an integer of 10 or more, preferably 15, 20 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500,
  • 4000, 4500, 5000 or more is an integer of 10 or more, preferably 15, 20 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more; "t” is an integer of 10 or more, preferably 15, 20 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400,
  • u is an integer of 10 or more, preferably 15, 20 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more;
  • "w” is an integer of 10 or more, preferably 15, 20 25,
  • the C-terminus of the amino acid sequences according to SEQ ID NO: 14 to 24 is connected via a peptide bond with the N-terminus of the respectively indicated DPR.
  • the DPR comprises at least three contiguous, more preferably at least five contiguous amino acids, more preferably at least ten contiguous amino acids of the amino acids according to SEQ ID NO: 14 to 24, most preferably the entire amino acid sequence.
  • aggregates refers to the intra- or extracellular accumulation of peptides, polypeptides or proteins.
  • sample or “sample of interest” are used interchangeably herein, referring to a part or piece of a tissue, organ or individual, typically being smaller than such tissue, organ or individual, intended to represent the whole of the tissue, organ or individual.
  • samples include but are not limited to fluid samples such as blood, serum, plasma, synovial fluid, lymphatic fluid, cerebrospinal fluid, meningeal fluid, glandular fluid, fine needle aspirate, spinal fluid and other body fluids (urine, saliva), as well as biopsy sample or solid samples such as tissue extracts of the brain or spinal cord.
  • samples include cell cultures (e.g. patient derived lymphoblasts) or tissue cultures.
  • Analysis of a sample may be accomplished on a visual or chemical basis.
  • Visual analysis includes but is not limited to microscopic imaging or radiographic scanning of a tissue, organ or individual allowing for the evaluation of a sample.
  • Chemical analysis includes but is not limited to the detection of the presence or absence of specific indicators or the detection of the alterations in their amount or level.
  • ligand refers to any substance or compound that is able to specifically interact with, e.g. to specifically bind to or to form a complex with, the specified molecule, e.g. a polypeptide of the invention comprising or consisting of a dipeptide repeat.
  • inhibitor refers to a substance, e.g. a ligand, which blocks the action of another compound, i.e. a receptor molecule.
  • inhibitors act by binding to the active site of the receptor molecule, or by interacting with unique binding sites not normally involved in the regulation of the activity of the receptor molecule.
  • the activity of the inhibitor may be reversible or irreversible depending on the longevity of the interaction of the inhibitor-receptor molecule complex.
  • Examples for inhibitors include but are not limited to nucleic acid molecules, such as siRNAs or miRNAs, or proteins such as transcription factors, immunoglobulin molecules, antibodies, antibody-like proteins, peptidomimetics, hormones, cytokines, growth factors, or neurotransmitters.
  • transcription factor refers to a protein which binds to a specific nucleic acid sequences and thereby controls the transcription of the genetic information. Transcription factors perform their function alone or in complex with other proteins, by promoting (as an activator), or blocking (as a repressor) e.g. by recruiting the RNA polymerase to a specific gene. Transcription factors may bind to either enhancer or promoter regions in the DNA adjacent to the ORF of the gene they regulate. Transcription factors use a variety of mechanisms to regulate gene expression, e.g.
  • transcription factors include but are not limited to Leucine zipper factors (bZIP) such as AP-l (-like) components, CREB, C/EBP-like factors, bZIP / PAR; Helix-loop-helix factors (bHLH) such as ubiquitous (class A) factors, myogenic transcription factors (MyoD), achaete-scute, tal, twist, atonal, hen; Helix-loop-helix / leucine zipper factors (bHLH-ZIP) such as Ubiquitous bHLH-ZIP factors, Cell-cycle controlling factors; NF-1 (A, B, C, X); RF-X (1 , 2, 3, 4, 5, ANK); bHSH; Zinc-coordinating DNA-binding domains such as Cys
  • RNA interacting factor is used to refer to further regulatory elements which are involved in the regulation of gene expression, i.e. in the regulation of transcription, RNA splicing, and translation.
  • RNA interacting factors include e.g. a splicing factor, a factor promoting or repressing the activity of a splicing factor, or a factor promoting or repressing cytoplasmic mRNA trafficking, factors influencing the nuclear import and export, or factors effecting the mRNA stability and turnover, and translation etc.
  • RNA interacting factors include but are not limited to heterogeneous nuclear ribonucleoproteins A2 B1 (hnRNP A2/B), heterogeneous nuclear ribonucleoproteins A3 (hnRNP A3), Nucleoporin p62 (p62), the splicing factor proline- and glutamine-rich SFPQ, the splicing factor 3B subunit 3 (SF3B3), the ELAV-like protein 1 (ELAV1), the interleukin enhancer-binding factor 3 (ILF3), the non-POU domain-containing octamer-binding protein (NONO), the heterogeneous nuclear ribonucleoprotein R (FTNRPR), the heterogeneous nuclear ribonucleoprotein A3 (ROA3), the heterogeneous nuclear ribonucleoprotein L (HNRPL), the scaffold attachment factor Bl (SAFB1), the insulin-like growth factor 2 mRNA-binding protein 1 (IF2B1), the scaffold
  • RNA interacting factors include but are not limited to EIF2S1, EIF2S2, EIF2S3, eIF4E, eIF4Gl , eIF4G2, eIF4Al, eIF4A2, eIF4A3, eIF4B, eIF2Bl, eIF2B2, eIF2B3, eIF2B4, eIF2B5, Dbrl, RPL38, HNRNPK, HNRNPE, HNRNPL, HNRNPA1, HNRNP A3 and HNRNPC.
  • ligand is used in the present invention to refer to a chemical moiety that specifically binds to an antigen, preferably a polypeptide of the invention.
  • Preferred ligands are antibodies or antigen-binding fragments thereof, antibody-like protein or peptidomimetics.
  • immunoglobulin refers to immunity conferring glycoproteins of the immunoglobulin superfamily.
  • Surface immunoglobulins are attached to the membrane of effector cells by their transmembrane region and encompass molecules such as but not limited to B-cell receptors, T-cell receptors, class I and II major histocompatibility complex (MHC) proteins, beta-2 microglobulin ( ⁇ 2 ⁇ ), CD3, CD4 and CD8.
  • MHC major histocompatibility complex
  • ⁇ 2 ⁇ beta-2 microglobulin
  • CD3, CD4 and CD8 CD3
  • antibody refers to secreted immunoglobulins which lack the transmembrane region and can thus, be released into the bloodstream and body cavities. Antibodies are grouped into different isotypes based on the heavy chain they possess.
  • human Ig heavy chains There are five types of human Ig heavy chains denoted by the Greek letters: , ⁇ , ⁇ , ⁇ , and ⁇ .
  • the type of heavy chain present defines the class of antibody, i.e. these chains are found in IgA, IgD, IgE, IgG, and IgM antibodies, respectively, each performing different roles, and directing the appropriate immune response against different types of antigens. Distinct heavy chains differ in size and composition; a and ⁇ comprise approximately 450 amino acids, while ⁇ and ⁇ have approximately 550 amino acids (Janeway et al. (2001) Immunobiology, Garland Science).
  • the term "human antibody”, as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
  • the human mAbs may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs.
  • human antibody as used herein, is not intended to include “humanized antibodies” in which the CDR sequences derived from the germline of another mammalian species (e.g., mouse), have been grafted onto human FR sequences.
  • Human antibodies also include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins.
  • the term "monoclonal antibody” as used herein refers to a preparation of antibody molecules of single molecular composition.
  • a monoclonal antibody displays a single binding specificity and affinity for a particular epitope.
  • the monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a non-human animal, e.g. mouse, fused to an immortalized cell.
  • recombinant antibody includes all antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal with respect to the immunoglobulin genes or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the antibody, e.g. from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of immunoglobulin gene sequences to other DNA sequences.
  • a “heterologous antibody” is defined in relation to a transgenic organism producing such an antibody. This term refers to an antibody having an amino acid sequence or an encoding nucleic acid sequence corresponding to that found in an organism not consisting of the transgenic organism, and being generally derived from a species other than the transgenic organism.
  • a “heterohybrid antibody” refers to an antibody having light and heavy chains originating of different organism. For example, an antibody having a human heavy chain associated with a murine light chain is a heterohybrid antibody.
  • antigen-binding fragments refers to fragments of an antibody which retain the function of specifically binding an antigen or antigenic protein but which lack some or all other structural features of an antibody or artificial constructs that comprise parts of antibodies.
  • Preferred examples of antigen-binding fragments include but are not limited to the following Fab fragments, Fc fragment, Fab' fragment, F(ab') 2 , single domain antibodies (sdAb), Nanobodies, single chain Fv, Divalent single-chain variable fragments (di-scFvs), tandem scFvs, diabodies, single-chain diabodies (scDB), triabodies, Bi-specific T-cell engagers (BiTEs), or dual affinity retargeting molecules (DART molecules).
  • Fab fragments (also referred to as “Fab portion” or “Fab region”) each with a single antigen binding site, and a residual “Fc fragment” (also referred to as “Fc portion” or “Fc region”) whose name reflects its ability to crystallize readily.
  • Fab' fragment which refers to a Fab fragment additionally comprising the hinge region of an Ig molecule whilst “F(ab')2 fragments” are understood to comprise two Fab' fragments being either chemically linked or connected via a disulfide bond. Whilst sdAb (Desmyter et al.
  • scFv single chain Fv
  • scFvA-scFvB scFvA-scFvB
  • dimers usually linkers with a length of 5 residues are used to generate these dimers. This type is known as “diabodies”. Still shorter linkers (one or two amino acids) between a VH and VL domain lead to the formation of monospecific trimers, so-called “triabodies” or “tribodies”. Bispecific diabodies are formed by expressing to chains with the arrangement V H A-V L B and V H B-VLA or V L - HB and V L B-VHA, respectively.
  • Single-chain diabodies comprise a VHA- V L B and a V H B-V L A fragment which are linked by a linker peptide (P) of 12-20 amino acids, preferably 14 amino acids, (VHA-V L B-P-V H B-V L A).
  • "Bi-specific T-cell engagers (BiTEs)” are fusion proteins consisting of two scFvs of different antibodies wherein one of the scFvs binds to T cells via the CD3 receptor, and the other to a tumor cell via a tumor specific molecule ( ufer et al. 2004)).
  • Dual affinity retargeting molecules (“DART” molecules) are diabodies additionally stabilized through a C-terminal disulfide bridge.
  • antibody-like protein refers to a protein having similar properties as an antibody in that it binds to an antigen or antigenic protein without necessarily having the structural features of an antibody.
  • Antibody-like proteins may occur naturally or may be designed artificially, e.g. biotechnologically.
  • naturally occurring antibody-like proteins include but are not limited to antigen-binding proteins such as e.g. the family of lipocalins, which represent a family of diverse proteins which normally serve for the storage or transport of physiologically important compounds. They share a conserved barrel of eight antiparallel ⁇ -strands as their central folding motif and comprise at one end of this barrel structure six hypervariable loops which are connect to each pair of ⁇ -strands. These loops form the entrance to the binding pocket.
  • the structural diversity among the members of the lipocalin family reflects the differing shapes and chemical properties of their binding partner. Thus, although being composed of a single polypeptide chain and being much smaller than immunoglobulins, they exhibit a vast potential to bind antigens of differing specificities.
  • artificially designed antibody-like protein include scaffold-based proteins which are generated by fusing peptides with known affinity towards a certain target or by inserting said peptides into, a carrier protein to combine the binding properties of the peptide with the desired favorable characteristics of the scaffold carrier.
  • peptidomimetics is used to refer to any molecule whose essential elements (pharmacophore) mimic a natural peptide or protein in 3D space and which retain the ability to interact with the biological target and produce the same biological effect.
  • Peptidomimetics include small protein-like chain designed to mimic a peptide which may typically be obtained either by modifying an existing peptide, or by designing similar systems that mimic peptides, such as e.g. peptoids and ⁇ -peptides.
  • the altered chemical structure is designed to adjust the molecular properties advantageously in that e.g. the stability or biological activity is increased or decreased. According modifications involve changes to the peptide that will not occur naturally including but not limited to altered backbones and the incorporation of non-natural amino acids.
  • specific binding or “specifically binding” to an antigen refers to the ability of a ligand to bind to an antigenic determinant of an antigen with high affinity.
  • high affinity means that the K d for the interaction is below 1 x 10 '5 M, preferably below 1 x 10 "6 M, more preferably below 1 x 10 ⁇ 7 , even more preferably below 1 x 10 "8 M and most preferably below 1 x 10 "9 M.
  • “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.
  • active ingredient refers to the substance in a pharmaceutical composition or formulation that is biologically active, i.e. that provides pharmaceutical value.
  • a pharmaceutical composition may comprise one or more active ingredients which may act in conjunction with or independently of each other.
  • the active ingredient can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as but not limited to those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2- ethylamino ethanol, histidine, procaine, and the like.
  • carrier refers to a pharmacologically inactive substance such as but not limited to a diluent, excipient, or vehicle with which the therapeutically active ingredient is administered.
  • Such pharmaceutical carriers can be liquid or solid.
  • Liquid carrier include but are not limited to sterile liquids, such as saline solutions in water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • a saline solution is a preferred carrier when the pharmaceutical composition is administered intravenously. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.
  • Suitable pharmaceutical "excipients” include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • adjuvant refers to agents that augment, stimulate, activate, potentiate, or modulate the immune response to the active ingredient of the composition at either the cellular or humoral level, e.g. immunologic adjuvants stimulate the response of the immune system to the actual antigen, but have no immunological effect themselves.
  • adjuvants include but are not limited to inorganic adjuvants (e.g. inorganic metal salts such as aluminium phosphate or aluminium hydroxide), organic adjuvants (e.g. saponins or squalene), oil-based adjuvants (e.g. Freund's complete adjuvant and Freund's incomplete adjuvant), cytokines (e.g.
  • particulate adjuvants e.g. immuno-stimulatory complexes (ISCOMS), liposomes, or biodegradable microspheres), virosomes, bacterial adjuvants (e.g. monophosphoryl lipid A, or muramyl peptides), synthetic adjuvants (e.g. non-ionic block copolymers, muramyl peptide analogues, or synthetic lipid A), or synthetic polynucleotides adjuvants (e.g polyarginine or polylysine).
  • compositions are administered via a route of administration suitable to the case.
  • Administration routes include but are not limited to intranasal administration, intramuscular administration, subcutaneous administration, oral administration, and topical administration.
  • an “intranasal administration” is the administration of a pharmaceutical to the mucosa of the complete respiratory tract including the lung.
  • the pharmaceutical is administered to the mucosa of the nose.
  • An intranasal administration is achieved by means of instillation, spray or aerosol.
  • said administration does not involve perforation of the mucosa by mechanical means such as a needle.
  • intramuscular administration refers to the injection of a pharmaceutical into any muscle of an individual or a patient. Preferred intramuscular injections are administered into the deltoid, vastus lateralis or the ventrogluteal and dorsogluteal areas.
  • subcutaneous administration refers to the injection of a pharmaceutical into the hypodermis.
  • oral administration refers to the administration of a pharmaceutical via the mouth to the gastric system.
  • a “topical administration” is the administration of a pharmaceutical to any part of the skin without penetrating the skin with a needle or a comparable device.
  • the pharmaceutical may also be administered topically to the mucosa of the mouth, nose, genital region and rectum.
  • data carrier refers to any storage medium usable to provide information to the user. These may include but is not limited to manuals, books, booklets, flyers, brochures, paper, as well as computer readable storage medium.
  • computer readable storage medium refers to any medium capable of storing data in a format readable by a mechanical device such as e.g. a computer.
  • Examples of medium include but are not limited to a magnetic disk, a optical disks, a hard disk, a RAM, a ROM, an EEPROM (for example flash memory), an EPROM, a DVD, a Blu-Ray Disc, a floppy disk, a DVD-RW disc, a barcode, and magnetic ink characters.
  • polypeptide indicates the presence of or susceptibility to a disease characterized by an expansion of genomic hexanucleotide repeats.
  • the disease characterized by an expansion of genomic hexanucleotide repeats is a neurological disease, more preferably a neurodegenerative disease.
  • the genomic hexanucleotide repeats are selected from the group consisting of GGGGCC and GGCCTG on one DNA stand and the hexanucleotide repeats GGCCCC and CAGGCC on the respective complementary DNA strand, i.e. the disease is characterized by an expansion of the hexanucleotide repeats GGGGCC and/or GGCCTG on one DNA strand and the hexanucleotide repeats GGCCCC and CAGGCC on the respective complementary DNA strand.
  • the expansion of genomic hexanucleotide repeats GGGGCC characterizes a disease selected from the group consisting of ALS, FTD, ALS-FTD, and the expansion of genomic hexanucleotide repeats GGCCTG (and/or the complementary hexanucleotide repeats CAGGCC) characterizes the disease SCA36.
  • the expansion of the genomic hexanucleotide repeats GGGGCC and/or GGCCTG on one DNA strand and the hexanucleotide repeats GGCCCC and CAGGCC on the respective complementary DNA strand is an indicator of the respective diseases.
  • the sample provided in (i) may be a fluid sample or a solid sample.
  • said sample is preferably selected from the group consisting of a sample from blood, serum, plasma, synovial fluid, lymphatic fluid, cerebrospinal fluid, meningeal fluid, glandular fluid, fine needle aspirate, spinal fluid and other body fluids (urine, saliva).
  • said sample may be biopsy sample, i.e. a sample from tissue extracts of the brain or spinal cord.
  • the sample is a blood sample, plasma sample, serum sample, body fluid sample, sample of the cerebrospinal fluid, lymph fluid sample, meningeal fluid sample, glandular fluid sample, fine needle aspirate sample, spinal fluid sample, and biopsy sample.
  • the sample may be a cell culture or tissue culture. It is particularly preferred that the sample is selected from the group consisting of cerebrospinal fluid, blood, and patient-derived cells, preferably patient-derived fibroblasts or lymphoblasts. Accordingly, the sample may be provided by the patient directly before the method of the present invention is performed or may have been provided earlier and was maintained, e.g. cultured, before said method is performed.
  • the patient suspected of having a disease characterized by the expansion of genomic hexanucleotide repeats shows early signs or symptoms of said disease.
  • the patient susceptible to a disease characterized by the expansion of genomic hexanucleotide repeats does not show any symptoms of said disease yet but is vulnerable to the emergence of a disease due to genetic predisposition, lacking vaccination, poorly developed or immature immunity, and/or poor nutritional status.
  • the expression of one or more polypeptides in step (ii) is detected by detecting the polypeptide comprising dipeptide-repeats encoded by the genomic hexanucleotide repeats. If the polypeptide is detected, it is preferred that a ligand specifically binding to the polypeptide is used. Preferably an antibody or polypeptide binding fragment thereof, is used to detect the polypeptide.
  • the ligand may specifically bind to an epitope formed by dipeptide repeat itself, to an epitope formed by the junction between the dipeptide repeat and the N- or C-terminal non-repetitive sequence or to an epitope in the N- or C- terminal sequence.
  • the epitope is formed by the junction between the dipeptide repeats and the N- or C-terminal non-repetitive sequence it is preferred that the epitope comprises between 3 and 20 amino acids, preferably between 5 and 16 amino acids, more preferably between 7 and 10 amino acids, or the dipeptide repeat sequence and between 3 and 17 amino acids, preferably between 5 and 10 amino acids of the N- or C-terminal non- repetitive sequence. It is preferred that a polypeptide as described in the second and/or third aspect of the present invention is detected. Alternatively, the RNA encoding said polypeptide may be detected.
  • nucleotides comprising the hexanucleotide repeat the nucleotides 5' or 3' of the hexanucleotide repeats or both may be detected. It is particularly preferred that mRNA encoding by antisense strand of the respective hexanucleotide repeat region is detected.
  • mRNA comprising hexanucleotide repeats specifically interacts with certain nuclear proteins. Accordingly, the expression of one or more polypeptides in step (ii) may also be detected by binding of the mRNA to a RNA interacting factors.
  • RNA transcription products detected in this way preferably comprise GGGGCC-repeats, GGCCCC-repeats, GGCCUG-repeats or CAGGCC- repeats.
  • Preferred RNA interacting factors binding to the GGGGCC hexanucleotide repeats and which may be used for detection include but are not limited to hnRNP A2 B, p62, SFPQ, SF3B3, ELAV1, ILF3, NONO, HNRPR, ROA3, HNRPL, SAFB1, IF2B1, SAFB2, ROA1, DSRAD, DHX15, ILF2, DHX30, HNRPK, DDX21, or FUS, particularly preferred is hnRNP A3.
  • expression of one or more polypeptides in step (ii) is detected by detecting complexes, preferably agglomerates of hnRNP A3 and mRNA comprising hexanucleotide repeats, preferably GGGGCC-repeats, GGCCCC-repeats, GGCCUG-repeats or CAGGCC-repeats.
  • complexes preferably agglomerates of hnRNP A3 and mRNA comprising hexanucleotide repeats, preferably GGGGCC-repeats, GGCCCC-repeats, GGCCUG-repeats or CAGGCC-repeats.
  • the present invention provides a polypeptide comprising or consisting of dipeptide-repeats with a sequence selected from the group consisting of (Gly- Ala) a , (Gly-Pro) b , (Gly-Arg) c , (Ala-Pro) d , (Pro-Arg) e , (Gly-Leu) f , (Ala-Trp) g , (Pro-Gly ⁇ (Ala-Gln) j , (Gly-Pro) k , and (Pro-Arg)i wherein a is an integer of 16 or more, preferably 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500,
  • the polypeptide comprises further amino acids at its C-Terminus and/or at its N-Terminus.
  • the polypeptide comprises or consists of
  • At least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 3 are comprised at its C-terminus; or at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 14 are comprised at its N-terminus; or at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 3 are comprised at its C-terminus and at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 14 are comprised at its N-terminus;
  • (ii) (Gly-Pro) b dipeptide repeats, wherein b is an integer of 28 or more, preferably 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450,
  • (iii) (Gly-Arg)c dipeptide repeats, wherein c is an integer of 21 or more, preferably 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450,
  • At least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 5 are comprised at its C-terminus; or at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 16 are comprised at its N-terminus; at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 5 are comprised at its C-terminus and at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 16 are comprised at its N-terminus;
  • (iv) (Ala-Pro) d dipeptide repeats, wherein d is an integer of 17 or more, preferably 20 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more; at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 6 are comprised at its C-terminus; or at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 17 are comprised at its N-terminus; at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 6 are comprised at its C-terminus and at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 17 are comprised at least
  • (v) (Pro-Arg) e dipeptide repeats, wherein e is an integer of 24 or more, preferably 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more; at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 7 are comprised at its C-terminus; or at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 18 are comprised at its N-terminus; at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 7 are comprised at its C-terminus and at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 18 are comprised at its N-
  • (vi) (Gly-Leu) f dipeptide repeats, wherein f is an integer of 15 or more, preferably 20 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more; and at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 8 are comprised at its C-terminus; or at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 19 are comprised at its N-terminus; at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 8 are comprised at its C-terminus and at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 19 are comprised
  • h is an integer of 28 or more, preferably 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more; at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 10 are comprised at its C-terminus; or at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 21 are comprised at its N-terminus; at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 10 are comprised at its C-terminus and at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 21 are comprised at its N
  • K) (Ala-Gln) j dipeptide repeats, wherein j is an integer of 16 or more, preferably 20 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more; at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 11 are comprised at its C-terminus; or at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 22 are comprised at its N-terminus; at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 11 are comprised at its C-terminus and at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 22 are comprised at least
  • (Gly-Pro)k dipeptide repeats, wherein k is an integer of 28 or more, preferably 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more; at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 12 are comprised at its C-terminus; or at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 23 are comprised at its N-terminus; at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 12 are comprised at its C-terminus and at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 23 are comprised at its N-terminus
  • the N-terminus of the non-repetitive amino acid sequence according to SEQ ID NO: 3 to 13 is linked via a peptide bond to the C-terminus of the respectively indicated DPR.
  • the C-terminus of the non-repetitive amino acid sequence according to SEQ ID NO: 14 to 24 is linked via a peptide bond to the N-terminus of the respectively indicated DPR.
  • the length of the contiguous amino acid sequence is preferably 5 amino acids, 10 amino acids, 15 amino acids or the entire sequence according to SEQ ID NO: 3 to 24.
  • a detectable tag is attached, preferably covalently bound, to said polypeptide.
  • Suitable tags are preferably selected from the group consisting of a fluorophore, a chromophore, a radiolabel, a metal colloid, an enzyme, a chemiluminescent or a bioluminescent molecule. It is particularly preferred that the detectable tag is a fluorophor, preferably GFP, or an enzyme, preferably a luciferase.
  • the present invention provides a polypeptide comprising or consisting of
  • (i) (Gly-Ala) m dipeptide repeats wherein m is an integer of 10 or more, preferably 15, 20 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more; at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 3 at its C-terminus; or at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 14 at its N-terminus; or at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 3 at its C-terminus and at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 14 at its N-terminus;
  • o is an integer of 10 or more, preferably 15, 20 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350,
  • (iii) (Gly-Arg)p dipeptide repeats, wherein p is an integer of 10 or more, preferably 15, 20 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more; at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 5 at its C-terminus; or at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 16 at its N-terminus; or at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 5 at its C-terminus and at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 16 at its N-terminus;
  • (v) (Pro-Arg) r dipeptide repeats, wherein r is an integer of 10 or more, preferably 15, 20 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500,
  • (vi) (Gly-Leu) s dipeptide repeats, wherein s is an integer of 10 or more, preferably 15, 20 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more; at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 8 at its C-terminus; or at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 19 at its N-terminus; or at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 8 at its C-terminus, and/or at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 19 at its N-terminus;
  • (viii) (Pro-Gly) u dipeptide repeats, wherein u is an integer of 10 or more, preferably 15, 20 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more, at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 10 at its C-terminus, or at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 21 at its N-terminus or at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 10 at its C-terminus and at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 21 at its N-terminus;
  • (ix) (Ala-Gln)w dipeptide repeats, wherein w is an integer of 10 or more, preferably 15, 20 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more; at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 11 at its C-terminus; or at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 22 at its N-terminus; or at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 11 at its C-terminus and at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 22 at its N- terminus;
  • (x) (Gly-Pro) x dipeptide repeats, wherein x is an integer of 10 or more, preferably 15, 20 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more; at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 12 at its C-terminus; or at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 23 at its N-terminus; or at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 12 at its C-terminus, and at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 23 at its N-terminus; or
  • (xi) (Arg-Pro)y dipeptide repeats, wherein y is an integer of 10 or more, preferably 15, 20 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more; at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 13 at its C-terminus; or at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 24 at its N-terminus; or at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 13 at its C-terminus and at least three contiguous amino acids of the amino acid sequences according to SEQ ID NO: 24 at its N-terminus.
  • the N-terminus of the non-repetitive amino acid sequence according to SEQ ID NO: 3 to 13 is linked via a peptide bond to the C-terminus of the respectively indicated DPR.
  • the C-terminus of the non-repetitive amino acid sequence according to SEQ ID NO: 14 to 24 is linked via a peptide bond to the N-terminus of the respectively indicated DPR.
  • the length of the contiguous amino acid sequence is preferably 5 amino acids, 10 amino acids, 15 amino acids or the entire sequence according to SEQ ID NO: 3 to 24.
  • polypeptide comprises or consists of a sequence according any one of the following sequences:
  • SEQ ID NO: 27 greskeearspslvpappppppppgspgpgcrqfhqsleakarhpasvremrgkvkm rral ⁇ 'apastrassrqpnpkq parmppphsptrhrlrlrrrgrrhrnrspapgpppgpp ⁇ rpGP [GP] 0 GP
  • SEQ ID NO: 28 rltrrkqggkqpqpvassgtqesrarGR[GR] p GRgwgagpgagpgrgcgcgacarg gggagggewvseeaaswrvavwgsaagkrrg
  • SEQ ID NO: 29 geppllpaplpgsrtpnshppgcrllthplatacasaaagagtataappra rarpdh
  • SEQ ID NO: 30 sprrqgpsrvpseprlgpqkpraahppafpqarplstrgslfsspqrqrsqrvpgketarvl ragkqgrgqipipcpcaaaaaaagkpgarmqaippvargesptpsfgqrneresknassseesprfyprlfpaaepqt atrqdaasslthspppappppraqapqpqprpgpapgpapttPR[PR] r PRplards
  • SEQ ID NO: 32 qehqepdyyperafpsrfaacsarpgaarprqprppaqlqrpekarspalgttqpalraippwa appkagdgwpglrlvggagawplepgsarrsalaalraepgswrhgeewlrgagdatvgvsacvraadrAW[A W] t AWacacacacacpgngfrqtlrlr
  • SEQ ID NO: 33 phcepnpgagamvrsgcgaratrrwgf ⁇ afgpqtgPG[PG] u PGpapapapalgtgsg ⁇ SEQ ID NO: 34: gsagppkeptdlthlrlqqtdllhflqrqqrvadrvlkqhvqqhlerepgrerqrnlsvcrn p ⁇ gqAQ[AQ] w AQalsa rtqaetptvaspaprnhss wrqlpgsamaasalr ⁇ aepgsrghapapptn ⁇ rpgtt pspafggaaqggiarsagwvvpsagerafsglcscaggrgcrgraapgralqaakrlgkalsg
  • SEQ ID NO: 35 spplpsafarsrpraqtaratapgtgagarastqpqrlpepvpragagagagGP[GP] x G
  • SEQ ID NO: 36 ptacsnstcnstvmgsrgasvnatsawarrsqgrrrrrrrPR[PR] y PRpclrperrpkppps ITp ⁇ attphhgassrvrlamrlarsgamqapeatpppp ⁇ igg ⁇ g ⁇ hhl legpp ⁇ spgaragwc
  • m is an integer of 10 or more, preferably 15, 20 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 60 or more,
  • o is an integer of 10 or more, preferably 15, 20 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 60 or more,
  • p is an integer of 10 or more, preferably 15, 20 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 60 or more,
  • q is an integer of 10 or more, preferably 15, 20 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 60 or more,
  • r is an integer of 10 or more, preferably 15, 20 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 60 or more,
  • s is an integer of 10 or more, preferably 15, 20 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 60 or more,
  • t is an integer of 10 or more, preferably 15, 20 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 60 or more,
  • u is an integer of 10 or more, preferably 15, 20 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 60 or more,
  • w is an integer of 10 or more, preferably 15, 20 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 60 or more,
  • x is an integer of 10 or more, preferably 15, 20 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 60 or more, and
  • y is an integer of 10 or more, preferably 15, 20 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 60 or more.
  • a detectable tag is attached, preferably covalently bound, to said polypeptide.
  • Suitable tags are preferably selected from the group consisting of a fluorophore, a chromophore, a radiolabel, a metal colloid, an enzyme, a chemiluminescent or a bioluminescent molecule. It is particularly preferred that the detectable tag is a fluorophor, preferably GFP, or an enzyme, preferably a luciferase.
  • the present invention provides a ligand which specifically binds to or forms a complex with a polypeptide according to the second and/or third aspect of the present invention or a RNA molecule encoding a polypeptide according to the second and/or third aspect of the present invention. Accordingly, in embodiments of the fourth aspect the ligand interacts with a polypeptide according to the second aspect of the invention, i.e.
  • a polypeptide comprising or consisting of dipeptide-repeats with a sequence selected from the group consisting of (Gly-Ala) a , (Gly-Pro) b , (Gly-Arg) c , (Ala-Pro) d , (Pro-Arg) e , (Gly-Leu) f , (Ala- Trp) g , (Pro-Gly) h , (Ala-Gln) j , (Gly-Pro) k , and (Pro-Arg)i wherein a is an integer of 16 or more, preferably 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more;
  • the ligand binds to an epitope formed by dipeptide repeat itself, to an epitope formed by the junction between the dipeptide repeat and the N- or C-terminal non-repetitive sequence or to an epitope in the N- or C-terminal non-repetitive sequence.
  • the ligand binds to a polypeptide according to the third aspect of the present invention, it may specifically bind to an epitope formed by dipeptide repeat itself, to an epitope formed by the junction between the dipeptide repeat and the N- or C- terminal non-repetitive sequence or to an epitope in the N- or C-terminal sequence.
  • the epitope is formed by the junction between the dipeptide repeats and the N- or C- terminal non-repetitive sequence it is preferred that the epitope comprises between 3 and 17 amino acids, preferably between 5 and 10 amino acids of the dipeptide repeat sequence and between 3 and 20 amino acids, preferably between 5 and 16 amino acids, more preferably between 7 and 10 amino acids, of the N- or C-terminal non-repetitive sequence.
  • the ligand is selected from the group consisting of
  • the antibody is selected from the group consisting of polyclonal, monoclonal, monovalent, bispecific, heteroconjugate, multispecific, recombinant, heterologous, heterohybrid, chimeric, humanized (in particular CDR-grafted), deimmunized, or human antibodies. More preferably the antibody is a humanized antibody. In further preferred embodiments the antibody is an IgG antibody.
  • the antigen-binding fragment is selected from the group consisting of Fc fragment, Fab fragments, Fab' fragment, F(ab') 2 fragments, single domain antibodies (sdAb), nanobodies, single chain Fv (scFv), tandem scFvs, diabodies, bispecific diabodies, single-chain diabodies (scDb), triabodies, bi-specific T-cell engagers (BiTEs), and dual affinity retargeting molecules (DART).
  • the ligand binds to a RNA molecule encoding a polypeptide according to the second and/or third aspect of the present invention it is preferred that the ligand is an oligonucleotide, preferably an oligonucleotide have a nucleotide sequence which is complementary to the sequence of the RNA molecule encoding a polypeptide according to the second and/or third aspect of the present invention.
  • a detectable tag is attached, preferably covalently bound, to the ligand.
  • Suitable tags are preferably selected from the group consisting of a fluorophore, a chromophore, a radiolabel, a metal colloid, an enzyme, a chemiluminescent or a bioluminescent molecule. It is particularly preferred that the detectable tag is a fluorophor, preferably GFP, or an enzyme, preferably a luciferase.
  • the present invention provides a method of identifying an inhibitor of expression of a polypeptide comprising or consisting of dipeptide-repeats, comprising the steps of:
  • the cell provided in step (i) is a prokaryotic or a eukaryotic cell, more preferably selected from the group consisting of a bacterial cell, yeast cell, isolated primary cell or cell line.
  • the isolated primary cell or the cell line is mammalian derived, more preferably human derived.
  • said polynucleotide comprises a hexanucleotide repeat, more preferably having a sequence selected from the group consisting of GGGGCC and GGCCTG with genomic flanking regions from human C9orf72 or NOP 56, respectively.
  • said cell comprises said polynucleotide, preferably comprising said hexanucleotide repeats, in its genome, preferably in the first intron of C9orf72 and/or NOP56.
  • said cell comprises said polynucleotide, preferably comprising said hexanucleotide repeats, outside its genome, preferably comprised in a vector, preferably comprised in an intron or exon of said vector.
  • said vector allows for the expression of the polynucleotide in the cell, i.e.
  • said vector may further comprise regulatory elements such as promoter, enhancer and the like, and optionally also one or more selection markers allowing for the selection, identification, and/or isolation of cells comprising the vector over cells not comprising the vector.
  • the hexanucleotide repeat is present in a number that corresponds to the number of hexanucleotide repeats characteristic of the diseased state.
  • the hexanucleotide is (GGGGCC)n or the reverse complementary hexanucleotide in the complementary strand (GGCCCC) n and the number "n" is at least 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, and 500 or more.
  • the hexanucleotide is (GGCCTG) n or the hexanucleotide in the complementary strand (CAGGCC)n and the number "n" is at least 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, and 500 or more.
  • the expression of the polypeptide is detected by a biotechnological method described above, preferably selected from the group consisting of RT-PCR, quantitative real-time PCR, competitive RT-PCR, southern blotting, NPA, Western Blot, ELISA, FACS, and immunocytochemistry (e.g. via fluorescence microscopy or electron microscopy), or any other method known to the skilled artisan.
  • a biotechnological method described above preferably selected from the group consisting of RT-PCR, quantitative real-time PCR, competitive RT-PCR, southern blotting, NPA, Western Blot, ELISA, FACS, and immunocytochemistry (e.g. via fluorescence microscopy or electron microscopy), or any other method known to the skilled artisan.
  • polypeptide used in this aspect of the invention is identical to the polypeptide of the second or third aspect described above.
  • the expression of the polypeptide is reduced by at least 10%, more preferably by at least 20%, more preferably by at least 30%, more preferably by at least 40% and even more preferably by at least 50% or more.
  • the present inventors have discovered that the following proteins specifically bind to RNA comprising (GGGGCC) n repeats. It is, therefore, credible that the binding of these proteins to the RNA comprising (GGGGCC) n repeats has a role in the expression of polypeptide comprising or consisting of dipeptide-repeats.
  • the inhibitor may interfere with the expression, preferably translation of hnRNP A2/B, hnRNP A3, p62, SFPQ, SF3B3, ELAV1, ILF3, NONO, HNRPR, ROA3, HNRPL, SAFB1, IF2B1, SAFB2, ROA1, DSRAD, DHX15, ILF2, DHX30, HNRPK, DDX21, or FUS or alternatively, with the interaction between these proteins and the RNA comprising (GGGGCC) n repeats or may be a ligand specifically binding to one of these proteins.
  • the present invention provides a method of identifying an inhibitor of the toxic effect of a polypeptide comprising or consisting of dipeptide-repeats, comprising the steps of: (i) providing a cell comprising a polynucleotide sequence encoding said polypeptide,
  • the cell provided in step (i) is a prokaryotic or a eukaryotic cell, more preferably selected from the group consisting of a bacterial cell, yeast cell, isolated primary cell or cell line.
  • the isolated primary cell or the cell line is mammalian derived, more preferably a human derived cell.
  • said polynucleotide comprises a hexanucleotide repeat, more preferably having a sequence selected from the group consisting of GGGGCC and GGCCTG.
  • said cell comprises said polynucleotide, preferably comprising said hexanucleotide repeats, in its genome, preferably in the first intron of C9orf72 and/or NOP56.
  • said cell comprises said polynucleotide, preferably comprising said hexanucleotide repeats, outside its genome, preferably comprised in a vector, preferably comprised in an intron or exon of said vector.
  • said vector allows for the expression of the polynucleotide in the cell, i.e.
  • said vector may further comprise regulatory elements such as promoter, enhancer and the like, and optionally also one or more selection markers allowing for the selection, identification, and/or isolation of cells comprising the vector over cells not comprising the vector.
  • the hexanucleotide repeat is present in a number that corresponds to the number of hexanucleotide repeats characteristic of the diseased state.
  • the hexanucleotide is (GGGGCC) n and the number "n" is at least 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, and 500 or more.
  • the hexanucleotide is (GGCCTG) n and the number V is at least 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, and 500 or more.
  • the toxic effect of the polypeptide is detected by a biotechnological method described above, preferably selected from the group consisting of MTT assay, LDH assay, or any other method known to the skilled artisan.
  • polypeptide used in this aspect of the invention is identical to the polypeptide of the second or third aspect described above.
  • the expression of the polypeptide is reduced by at least 10%, more preferably by at least 20%, more preferably by at least 30%, more preferably by at least 40% and even more preferably by at least 50% or more.
  • the present invention provides a method of identifying an inhibitor of the formation of aggregates comprising a polypeptide comprising or consisting of dipeptide-repeats, comprising the steps of:
  • the cell provided in step (i) is a prokaryotic or a eukaryotic cell, more preferably selected from the group consisting of a bacterial cell, yeast cell, isolated primary cell or cell line.
  • the isolated primary cell or the cell line is mammalian derived.
  • said polynucleotide comprises a hexanucleotide repeat, more preferably having a sequence selected from the group consisting of GGGGCC and GGCCTG.
  • the hexanucleotide repeat is present in a number that corresponds to the number of hexanucleotide repeats characteristic of the diseased state.
  • the hexanucleotide is (GGGGCC) n and the number "n" is at least 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, and 500 or more.
  • the hexanucleotide is (GGCCTG) n and the number "n" is at least 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, and 500 or more.
  • said cell comprises said polynucleotide, preferably comprising said hexanucleotide repeats, in its genome, preferably in the first intron of C9orf72 and/or NOP56.
  • said cell comprises said polynucleotide, preferably comprising said hexanucleotide repeats, outside its genome, preferably comprised in a vector, preferably comprised in an intron or exon of said vector.
  • said vector allows for the expression of the polynucleotide in the cell, i.e.
  • said vector may further comprise regulatory elements such as promoter, enhancer and the like, and optionally also one or more selection marker allowing for the selection, identification, and/or isolation of cells comprising the vector over cells not comprising the vector.
  • regulatory elements such as promoter, enhancer and the like
  • selection marker allowing for the selection, identification, and/or isolation of cells comprising the vector over cells not comprising the vector.
  • said cell-free system comprises one or more of the subcellular fractions selected from the list consisting of nucleoli, nucleus, ribosome, endoplasmatic reticulum, and Golgi apparatus.
  • a cell-free expression system is used to identifying an inhibitor of the formation of aggregates.
  • the formation of aggregates is detected by a biotechnological method, preferably selected from the group consisting of filter trap assay, Western Blot, size-exclusion chromatography (e.g. gel-filtration chromatography, or gel permeation chromatography), and immunocytochemistry (e.g. via fluorescence microscope or electron microscopy), or any other method known to the skilled artisan or described in the example section.
  • a biotechnological method preferably selected from the group consisting of filter trap assay, Western Blot, size-exclusion chromatography (e.g. gel-filtration chromatography, or gel permeation chromatography), and immunocytochemistry (e.g. via fluorescence microscope or electron microscopy), or any other method known to the skilled artisan or described in the example section.
  • formation of aggregates is reduced by at least 10%, more preferably by at least 20%, more preferably by at least 30%, more preferably by at least 40% and even more preferably by at least 50% or more.
  • the inhibitor is selected from the group consisting of hexanucleotide specific siRNA, the ligand of the fourth aspect of the present invention, hnRNP A3 and p62.
  • additional compound may be provided which may be involved in the formation of aggregates such as e.g. compounds binding to or forming complexes with said polypeptide.
  • the additional compound is selected from the group consisting of hippuristanol, and a small molecule inhibitor of the eIF4A helicase.
  • polypeptide used in this aspect of the invention is identical to the polypeptide of the second or third aspect described above.
  • the present invention provides an inhibitor of the expression of a polypeptide comprising dipeptide-repeats, an inhibitor of a toxic effect of a polypeptide comprising dipeptide-repeats, or an inhibitor of the formation of aggregates comprising a polypeptide comprising dipeptide-repeats for treating or preventing a disease characterized by an expansion of genomic hexanucleotide repeats.
  • the inhibitor reduces or completely abolishes the expression of the hexanucleotide into the polypeptide comprising dipeptide-repeats.
  • the inhibitor reduces or abolishes the transcription, RNA splicing, translation or protein folding into the polypeptide comprising dipeptide-repeats, preferably by binding to or forming a complex with a factor involved in the expression of said polypeptide, which is subsequently not able to fulfil its function anymore resulting the prevention of the expression of the polypeptide.
  • a factor involved in the expression of said polypeptide which is subsequently not able to fulfil its function anymore resulting the prevention of the expression of the polypeptide.
  • a factor is a transcription factor or a RNA interacting factor.
  • the inhibitor or a nucleic acid encoding said inhibitor is provided to a healthy individual before the onset of the disease or to a patient having or being suspected of having or being susceptible to said disease in order to treat said disease or to prevent the onset or the progression of the disease, i.e. for prophylactic reasons.
  • the inhibitor reduces the expression, i.e. the transcription, the RNA splicing and/or the translation, of the polypeptide by at least 10%, more preferably by at least 20%, more preferably by at least 30%, more preferably by at least 40% and even more preferably by at least 50% or more.
  • the inhibitor reduces or completely blocks the toxic effect of the polypeptide comprising dipeptide-repeats and thereby the damage caused by the expression of said polypeptide or the formation of aggregates of said polypeptide, to the cell.
  • the inhibitor reduces the toxic effect of the polypeptide by reducing the damaging effect the polypeptide has on the cell, by activating cellular processes restoring the cell, and/or by activating cellular processes prolonging the cell's viability which was reduced due to the toxic effect of the polypeptide.
  • the inhibitor reduces the toxic effect of the polypeptide by at least 10%, more preferably by at least 20%, more preferably by at least 30%, more preferably by at least 40% and even more preferably by at least 50% or more.
  • the inhibitor reduces or completely blocks the accumulation of the polypeptide comprising dipeptide-repeats and thereby the formation of aggregates.
  • the inhibitor reduces or completely blocks the accumulation of the polypeptide comprising dipeptide-repeats by effecting or altering the electrostatic interaction properties of said polypeptide, by altering the 3 -dimensional structure of the polypeptide or by altering or preventing the 3-dimensional folding of the polypeptide after translation.
  • the inhibitor reduces the formation of aggregates by at least 10%, more preferably by at least 20%, more preferably by at least 30%, more preferably by at least 40% and even more preferably by at least 50% or more.
  • the inhibitor is used to treat or prevent a disease characterized by an expansion of genomic hexanucleotide repeats through administration of said inhibitor or a nucleic acid encoding said inhibitor is administered to a patient having, suspected of having or being susceptible to said disease.
  • the inhibitor is selected from the group consisting of hexanucleotide specific siRNA, the ligand of the fourth aspect of the present invention, or siRNAs specifically inhibiting expression of hnRNP A2/B, hnRNP A3, p62, SFPQ, SF3B3, ELAV1 , ILF3, NONO, HNRPR, ROA3, HNRPL, SAFB1, IF2B1, SAFB2, ROA1, DSRAD, DHX15, ILF2, DHX30, HNRPK, DDX21, or FUS or a ligand specifically binding to these proteins.
  • siRNAs specifically inhibiting expression of hnRNP A2/B, hnRNP A3, p62, SFPQ, SF3B3, ELAV1 , ILF3, NONO, HNRPR, ROA3, HNRPL, SAFB1, IF2B1, SAFB2, ROA1, DSRAD, DHX15, ILF2, DHX30, HNRPK, DDX21,
  • a detectable tag is attached, preferably covalently bound, to the inhibitor.
  • Suitable tags are preferably selected from the group consisting of a fluorophore, a chromophore, a radiolabel, a metal colloid, an enzyme, a chemiluminescent or a bioluminescent molecule.
  • the inhibitor is used to treat or prevent a disease characterized by an expansion of genomic hexanucleotide repeats through administration of said inhibitor or a nucleic acid encoding said inhibitor to a patient having, suspected of having or being susceptible to said disease.
  • the patient susceptible to a disease characterized by the expansion of genomic hexanucleotide repeats does not show any symptoms of said disease yet but is vulnerable to the emergence of a disease due to genetic predisposition.
  • the patient suspected of having a disease characterized by the expansion of genomic hexanucleotide repeats shows early signs or symptoms of said disease.
  • said patient shows more signs or symptoms or more pronounced signs or symptoms of the disease.
  • the disease characterized by an expansion of hexanucleotide repeats is selected from amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), amyotrophic lateral sclerosis- frontotemporal dementia (ALS-FTD) and spinocerebellar ataxia (SCA36).
  • ALS amyotrophic lateral sclerosis
  • FDD frontotemporal dementia
  • ALS-FTD amyotrophic lateral sclerosis- frontotemporal dementia
  • SCA36 spinocerebellar ataxia
  • the present invention provides the polypeptide of the second or third aspect of the present invention, the ligand of the fourth aspect of the present invention, the inhibitor of the eighth aspect of the present invention and/or a nucleotide sequence encoding said polypeptide, said ligand or said inhibitor for use in diagnosis, prophylaxis, or treatment of a disease characterized by an expansion of genomic hexanucleotide repeats.
  • nucleic acid encoding said ligand or said inhibitor can only be used in this and the other aspects of the invention, if said ligand or inhibitor is a polypeptide. Accordingly, it is preferred that a nucleic acid is used, if the ligand or inhibitor is an antibody, antigen-binding fragment thereof or an antibody-like protein.
  • the polypeptide of the second or third aspect of the present invention is used for diagnosing a disease characterized by an expansion of genomic hexanucleotide repeats, preferably by detecting said polypeptide in an individual having, suspected of having, or being susceptible to said disease.
  • said polypeptide is detected by any of the above described methods, preferably by means of a Western Blot, ELISA, FACS, and immunocytochemistry (e.g. via fluorescence microscopy or electron microscopy).
  • the detection of the polypeptide of the second or third aspect of the present invention is an indicator for the presence of the disease.
  • the polypeptide of the second or third aspect of the present invention is used for prophylaxis or treatment of said disease, preferably via immunization, more preferably via passive immunization of the individual or patient.
  • the ligands of the invention preferably antibodies of the invention or nucleic acids encoding these are administered to a patient to bind the DPRs formed, to prevent aggregation and to remove the DPRs from the brain.
  • the immune system of the individual/patient is primed/activated/strengthened to react against the polypeptide, i.e. by activating the proliferation of B- or T-cells and/or the production of antibodies directed against said polypeptide.
  • said polypeptide or a nucleic acid encoding said polypeptide is provided to a healthy individual before the onset of the disease.
  • the polypeptide is provided to a patient having said disease, being suspected of having said disease or being susceptible to said disease in order to treat the disease.
  • the activation of the immune system may lead to a bettering or complete disappearance of the symptoms and/or cause of the disease.
  • the polypeptide is provided to the individual or patient in form of a vaccine.
  • the ligand of the fourth aspect of the present invention is used in diagnosing a disease characterized by an expansion of genomic hexanucleotide repeats.
  • the ligand itself may be detected in a sample provided by a patient by any of the methods described above.
  • the ligand preferably a ligand further comprising a detectable tag, may be used to detect the presence of the polypeptide of the second and third aspect of the present invention, or an mRNA molecule encoding said polypeptide, in the sample of the individual or patient.
  • Suitable tags are preferably selected from the group consisting of a fluorophore, a chromophore, a radiolabel, a metal colloid, an enzyme, a chemiluminescent or a bioluminescent molecule.
  • the ligand is for use in the passive and/or active immunization of an individual or a patient.
  • the ligand of the fourth aspect of the present invention may be used to treat a disease characterized by the expansion of genomic hexanucleotide repeats.
  • the ligand binds to or forms a complex with the polypeptide of the second or third aspect of the present invention, thereby preventing the formation of polypeptide aggregates.
  • the ligand binds to or forms a complex with a factor involved in the expression, i.e. the transcription, R A splicing or translation of said polypeptide.
  • said ligand or a nucleic acid encoding said ligand is provided to a healthy individual before the onset of the disease or to a patient having, being suspected of having, or being susceptible to said disease. It is preferred to use the ligand of the fourth aspect of the present invention for passive immunization of an individual or a patient.
  • the ligand of the fourth aspect of the present invention may be used for prophylactic purposes in that it is used to induce an immune response against said ligand thereby also inducing an immune response against the ligand in complex with the polypeptide or the ligand in complex with a mRNA molecule encoding said polypeptide. Accordingly, it is preferred that said ligand or a nucleic acid encoding said ligand is provided to a healthy individual before the onset of the disease or to a patient having or being suspected of having said disease. It is preferred to use the ligand of the fourth aspect of the present invention for active immunization of an individual or a patient.
  • the inhibitor of the eighth aspect of the present invention is for use in treating a patient having or suspected of having a disease characterized by an expansion of genomic hexanucleotide repeats, preferably by passive immunization.
  • the inhibitor binds to or forms a complex with a factor involved in the expression, i.e. the transcription, R A splicing or translation of said polypeptide, and thereby prevents the expression of the polypeptide.
  • the inhibitor or a nucleic acid encoding said inhibitor is provided to a healthy individual before the onset of the disease or to a patient having or being suspected of having said disease. It is preferred to use the inhibitor of the eighth aspect of the present invention for immunization, preferably passive immunization, of an individual or a patient.
  • the individual/patient is provided with a vaccine.
  • the vaccine comprises a vector, preferably comprising one or more nucleic acid sequences encoding said polypeptide, said ligand and/or said inhibitor.
  • Said vector preferably comprises further nucleic acid sequences allowing for the expression of the polypeptide, ligand or inhibitor in a cell, preferably in a cell of the individual or patient.
  • the present invention provides a kit of parts for diagnosing, treating or preventing a disease characterized by an expansion of genomic hexanucleotide repeats comprising the polypeptide of the second aspect of the present invention, ligand of the fourth aspect of the present invention, the inhibitor of the eighth aspect of the present invention and/or nucleotide sequence encoding said polypeptide, said ligand or said inhibitor, and optionally further comprising a container, and/or a data carrier, preferably comprising instructions for one or more of the methods of the first, fifth, to seventh sixth aspect of the present invention.
  • the present invention provides a pharmaceutical comprising the polypeptide of the second aspect of the present invention, the ligand of the fourth aspect of the present invention, the inhibitor of the eighth aspect of the present invention and/or nucleotide sequence encoding said polypeptide, said ligand or said inhibitor.
  • said pharmaceutical is a vaccine, preferably comprising a vector.
  • said vector comprises one or more nucleic acid sequences encoding said polypeptide, said ligand and/or said inhibitor.
  • Said vector preferably comprises further nucleic acid sequences allowing for the expression of the polypeptide, ligand or inhibitor in a cell, preferably in a cell of the individual or patient.
  • the pharmaceutical further comprises a pharmaceutically acceptable carrier, excipient and/or adjuvant and optionally one or more additional active substances.
  • the additional active substances may be effective against the same or against a different disease or disorder. Accordingly, additional active substances comprise those having a beneficial effect in the treatment of a disease characterized by an expansion of genomic hexanucleotide repeats.
  • additional active substances may be directed at the treatment of a different diseases or disorder or conditions suitable to be administered in combination with the active ingredient for the treatment of a disease characterized by an expansion of genomic hexanucleotide repeats.
  • the pharmaceutical of the eleventh aspect contains a therapeutically effective amount of said polypeptide, ligand, inhibitor or nucleic acid encoding said polypeptide, ligand, or inhibitor, preferably in purified form, together with a suitable amount of carrier and/or excipient so as to provide the form for proper administration to the patient.
  • the formulation should suit the mode of administration.
  • the pharmaceutical can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
  • the pharmaceutical can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • pharmaceutically acceptable carriers can be either solid or liquid.
  • Solid form compositions include powders, tablets, pills, capsules, lozenges, cachets, suppositories, and dispersible granules.
  • a solid excipient can be one or more substances, which may also act as diluents, flavouring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.
  • the excipient is preferably a finely divided solid, which is in a mixture with the finely divided inhibitor of the present invention.
  • the active ingredient is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.
  • Suitable excipients are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like.
  • a low melting wax such as a mixture of fatty acid glycerides or cocoa butter
  • the active component is dispersed homogeneously therein, as by stirring.
  • the molten homogeneous mixture is then poured into convenient sized moulds, allowed to cool, and thereby to solidify. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.
  • Liquid form compositions include solutions, suspensions, and emulsions, for example, water, saline solutions, aqueous dextrose, glycerol solutions or water/propylene glycol solutions.
  • solutions for parenteral injections (e.g. intravenous, intraarterial, intraosseous infusion, intramuscular, subcutaneous, intraperitoneal, intradermal, and intrathecal injections)
  • liquid preparations can be formulated in solution in, e.g. aqueous polyethylene glycol solution.
  • a saline solution is a preferred carrier when the pharmaceutical composition is administered intravenously.
  • the pharmaceutical composition is in unit dosage form.
  • the composition may be subdivided into unit doses containing appropriate quantities of the active component.
  • the unit dosage form can be a packaged composition, the package containing discrete quantities of the composition, such as packaged tablets, capsules, and powders in vials or ampoules.
  • the unit dosage form can be a capsule, an injection vial, a tablet, a cachet, or a lozenge itself, or it can be the appropriate number of any of these in packaged form.
  • composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the pharmaceutical is administered via an administration route selected from the group consisting of intranasal administration, intramuscular administration, subcutaneous administration, oral administration, and topical administration.
  • the present invention provides the pharmaceutical of the tenth aspect of the present invention for use in diagnosis, prophylaxis, or treatment of a disease characterized by an expansion of genomic hexanucleotide repeats.
  • the disease characterized by an expansion of hexanucleotide repeats is selected from amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), amyotrophic lateral sclerosis- frontotemporal dementia (ALS-FTD) and spinocerebellar ataxia (SCA36).
  • ALS amyotrophic lateral sclerosis
  • FDD frontotemporal dementia
  • ALS-FTD amyotrophic lateral sclerosis- frontotemporal dementia
  • SCA36 spinocerebellar ataxia
  • the hexanucleotide repeats have a sequence selected from the group consisting of GGGGCC and GGCCTG.
  • the hexanucleotide (GGGGCC)n is responsibly for the formation of aggregates responsible for ALS, FTD or ALS-FTD, and the number "n" is at least 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, and 500 or more.
  • the hexanucleotide is (GGCCTG)n is responsibly for the formation of aggregates responsible for SCA36, and the number "n" is at least 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, and 500 or more.
  • the pharmaceutical is administered to a healthy individual in order to prevent the onset of the disease or to a patient having, being suspected of having or being susceptible for said disease in order to prevent the progression of the disease or in order o treat said disease.
  • the pharmaceutical reduces the expression, i.e. the transcription, the RNA splicing and/or the translation, of the polypeptide by at least 10%, more preferably by at least 20%, more preferably by at least 30%, more preferably by at least 40% and even more preferably by at least 50% or more and thereby treats said disease or prevents the progression or onset of said disease.
  • the pharmaceutical reduces the toxic effect of the polypeptide by at least 10%, more preferably by at least 20%, more preferably by at least 30%, more preferably by at least 40% and even more preferably by at least 50% or more, and thereby treats said disease or prevents the progression or onset of said disease.
  • the pharmaceutical reduces the formation of aggregates by at least 10%, more preferably by at least 20%, more preferably by at least 30%, more preferably by at least 40% and even more preferably by at least 50% or more, and thereby treats said disease or prevents the progression or onset of said disease.
  • Rat monoclonal poly-GR-repeat-specific antibody 5A2 was originally generated against peptide antigen (GQSRGQSRGRGRGRGRGRGKGK; SEQ ID NO: 47) from EBNA2 and detects specifically GST-(GR) 15 ( Figure 1A) (Kremmer et al., 1995).
  • p62 mouse, BD Transduction
  • rabbit TDP Proteintech
  • phospho-TDP-43 Ser409/Ser410, clone 1D3
  • rat GST antibody (6G9, Sigma) are commercially available.
  • the hexanucleotide repeat was amplified by repeat primed PCR (6) from patient lymphoblasts (AGTACTCGCTGAGGGTGAAC; SEQ ID NO: 48; and CGTACGC ATCCCAGTTTGAGAGCCCCGGCCCCGGCCCCGGCCCC; SEQ ID NO: 49) and topo-cloned into pEF6-V5/His (Invitrogen). Neither the multiple cloning site nor the C9orfi2 genomic sequence contain a ATG start codons. Additionally multiple stop codons are present in all reading frames upstream of the GGGGCC repeat. Plasmids were transformed into Stbl2 bacteria (Invitrogen) that were grown at 32C to avoid recombination. Because of the extreme GC-content only ⁇ 20 repeats could be sequenced. Higher repeat numbers were therefore estimated by restriction digest (Figure IB).
  • Paraffin-embedded sections were first deparaffinized with xylene and ethanol. After brief wash with deionized water, antigen retrieval was performed by microwaving sections six times 3 min in 100 mM citrate buffer (pH 6.0). Sections were allowed to settle for 20 min followed by a brief wash with deionized water. Endogenous peroxidase was blocked with 5% H202 in methanol for 16 min. After rinse with deionized water, sections were settled in PBS with 0.05% Brij35. Blocking was performed with 2% FCS in PBS for 5 min. Primary antibodies were diluted with blocking solution and incubated overnight at 4°C.
  • rat monoclonal antibodies 5A2 and 1D3 rabbit anti-rat secondary antibody (PARIS Anticorps) was additionally used at 1 :2000 dilution for 1 h. Sections were washed two times with PBS with 0.05% Brij35 for 5 min and developed with IHC-Kit DCS Supervision 2 HRP (DCS) or NovoLink Polymer Detection System for rat antibodies (Leica) using DAB as chromogen. Microscopic images were obtained with a BX50 microscope and Cell-D software (Olympus). For immunofluorescence species specific secondary antibodies conjugated with Alexa-488 or Alexa-544 (Invitrogen) were used for double staining. TOPRO-3 (Invitrogen) was used as nuclear stain.
  • Example 5 Cellular distribution patterns of the DPR proteins To determine the cellular distribution patterns of these DPR proteins in patients with C9orf72 mutations, we focused on the cerebellum and hippocampus in the immunohistochemical analysis as these brain regions contain abundant inclusions pathology (Al-Sarraj et al., 201 1 ; Mahoney et al., 2012; Bigio et al., 2012) (Fig. 3A and 4A/B). Strikingly, in all cases with C9orf72 repeat expansion, poly-GA specific antibodies detected dot-like neuronal cytoplasmic inclusions (Fig. 3B and 4C).
  • poly-GA aggregates were not detectable in FTLD-TDP cases without C9orf72 repeat expansions or in cases with Huntington's disease, which features expanded poly-Q stretches (Fig. 7A-B).
  • Fig. 7A-B In total we identified poly-GA, poly-GP and poly-GR containing intraneuronal aggregates in all seven patients analyzed with genetically confirmed C9orp2 repeat expansion, but not in nine other cases with normal repeat length (see Fig. 2).
  • some patients with C9orf72 mutations show remarkably few phospho-TDP-43 inclusions throughout the brain.
  • RNA was extracted using R easy kit (Qiagen) including a DNase I step to remove genomic DNA. Sufficient RNA quality was confirmed using the Agilent RNA 6000 Nano kit on the Agilent 2100 Bioanalyzer. The resulting RIN values (4.7-7.2) were not statistically different between C9orf72 cases and controls (p 0.25, Student's t-test).
  • Quantitative PCR was performed using SsoFast EvaGreen (Biorad) using C9orf72 specific intronic primers (AAGAGGCGCGGGTAGAAG; SEQ ID NO: 39; and AGTCGCTAGAGGCGAAAGC; SEQ ID NO: 40) and intron-spanning exonic primers for C9orf72 (all isoforms: ACTGGAAT GGGGATCGCAGCA; SEQ ID NO: 41; and ACCCTGATCTTCCATTCTCTCTCTCTGTGCC; SEQ ID NO: 42; isoforms containing alternative exon lb: CTGCGGTTGCGGTGCCTGC; SEQ ID NO: 43; and AGCTGGAG ATGGCGGTGGGC ; SEQ ID NO: 44) and the house- keeping gene YWHAZ (TGAACAAAAGACGGAAGGTGCTG; SEQ ID NO: 45; and TCTGATAGGATGTGTTGGTTGCA; SEQ ID NO: 46).
  • C9orf72 specific intronic primers AAGA
  • poly-GA In the cerebellum in total 370 to 822 (poly-GA analysis), 123 to 566 (poly-GP analysis) and 211 to 596 (poly-GR analysis) inclusions were counted per patient. In CA4 in total 48-73 inclusions were analyzed per patient for each DPR species. In the cerebellum more than 90%of p62-positive inclusions were also positive for poly-GA. In the hippocampus this number was even higher: 98%. Additionally we analyzed colocalization of all poly-GP and poly-GR in CA4. Consistent with the impression from the immunohistochemical analysis (see above), poly-GA is the predominant DPR species. This strongly suggests that poly-GA is the main component in p62-positive and TDP-43 negative inclusions and thus, an important new disease protein in C9orf72 patients allowing for the diagnosis, prophylaxis and treatment of the disease.
  • RNA interacting factors such as heterogenous ribonucleoproteins (hnR Ps), splicing factors and mRNA binding proteins (Figure 10).
  • hnR Ps heterogenous ribonucleoproteins
  • Figure 10 mRNA binding proteins
  • DPR pathology is predominant in clinically relevant brain regions (hippocampus and frontotemporal neocortex) and likely precedes TDP-43 pathology, because we occasionally observed poly-GA in the core of TDP-43 inclusions, but never found phospho-TDP-43 staining inside of DPR inclusions.
  • C9orf72 patients show cerebellar atrophy that does not occur in the other genetic or sporadic variants of FTLD/ALS lacking cerebellar DPR inclusions.
  • DPR pathology is a direct consequence of the hexanucleotide expansion, the most common genetic cause of FTLD/ALS.
  • C9orf72 expansion carrier had abundant DPR pathology and behavioral variant clinical FTLD, but no detectable TDP-43 inclusion pathology.
  • DPR-specific antibodies will be a valuable tool to further dissect the role of the abundant TDP-43 negative inclusions in different brain regions.
  • the dipeptide-repeat proteins are the major disease protein in the cortical and cerebellar signature inclusions in FTLD/ALS patients with C9orf72 hexanucleotide expansion and are directly linked to the predominant pathology.
  • the unusual translation mechanism and the highly abnormal product facilitate more selective diagnostic and/or therapeutic approaches than possible for other neurodegenerative diseases.
  • Suitable diagnostic and/or therapeutic approaches include the application and/or administration of oligonucleotides, antibodies or antigen-binding fragments thereof, antibody-like proteins, and or peptidomimetics directed against the hexanucleotide repeats or the DPR-protein.
  • TDP-43 is a component of ubiquitin- positive tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Biochem Biophys Res Commun 351 (3):602-611.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Toxicology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Neurology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biophysics (AREA)
  • Neurosurgery (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé de détection d'une maladie caractérisé par une expansion de répétitions hexanucléotidiques dans le génome ainsi que les polypeptides desdites répétitions hexanucléotidiques, les ligands se liant spécifiquement au polypeptide, des procédés permettant d'identifier un inhibiteur empêchant l'expression et/ou l'agrégation dudit polypeptide.
PCT/EP2013/000190 2013-01-22 2013-01-22 Protéines à répétitions de dipeptides comme cible thérapeutique dans des maladies neurodégénératives avec expansion de répétitions hexanucléotidiques WO2014114303A1 (fr)

Priority Applications (15)

Application Number Priority Date Filing Date Title
PCT/EP2013/000190 WO2014114303A1 (fr) 2013-01-22 2013-01-22 Protéines à répétitions de dipeptides comme cible thérapeutique dans des maladies neurodégénératives avec expansion de répétitions hexanucléotidiques
SI201431324T SI2948777T1 (sl) 2013-01-22 2014-01-22 Dipeptidno ponavljajoči se proteini kot terapevtski cilj pri nevrodegenerativnih boleznih s ponavljajočo ekspanzijo heksanukleotida
PL14701089T PL2948777T3 (pl) 2013-01-22 2014-01-22 Białka z powtórzeniami dipeptydowymi jako cel terapeutyczny w chorobach neurodegeneracyjnych z ekspansją powtórzeń heksanukleotydowych
DK14701089.6T DK2948777T3 (da) 2013-01-22 2014-01-22 Dipeptid-repeat-proteiner som terapeutisk mål i neurodegenerative sygdomme med hexanukleotid-repeat-udvidelse
RSP20191145 RS59283B1 (sr) 2013-01-22 2014-01-22 Proteini sa dipeptidnim ponovkom kao terapeutsko ciljno mesto kod neurodegenerativnih bolesti sa ekspanzijom heksanukleotidnog ponovka
HUE14701089A HUE046434T2 (hu) 2013-01-22 2014-01-22 Dipeptid-ismétlõdõ fehérjék terápiás célpontként neurodegeneratív betegségekben, hexanukleotid ismétlõdõ expanzióval
LTEP14701089.6T LT2948777T (lt) 2013-01-22 2014-01-22 Baltymai su pasikartojančiais dipeptidais, kaip terapinis taikinys neurodegeneracinių ligų su padidėjusiu heksanukleotidų pasikartojimu atveju
US14/762,623 US10066007B2 (en) 2013-01-22 2014-01-22 Dipeptide-repeat proteins as therapeutic target in neurodegenerative diseases with hexanucleotide repeat expansion
PT14701089T PT2948777T (pt) 2013-01-22 2014-01-22 Proteínas de repetição por dipeptídeos como alvo terapêutico em doenças neurodegenerativas com expansão de hexanucleotídico repetido
ES14701089T ES2744804T3 (es) 2013-01-22 2014-01-22 Proteínas de repetición de dipéptidos como objetivo terapéutico en enfermedades neurodegenerativas con expansión de repetición de hexanucleótidos
PCT/EP2014/051204 WO2014114660A1 (fr) 2013-01-22 2014-01-22 Protéines à répétition dipeptidique en tant que cible thérapeutique dans des maladies neurodégénératives ayant une expansion de répétition hexanucléotidique
EP14701089.6A EP2948777B1 (fr) 2013-01-22 2014-01-22 Protéines à répétition dipeptidique en tant que cible thérapeutique dans des maladies neurodégénératives ayant une expansion de répétition hexanucléotidique
EP19180100.0A EP3588091A1 (fr) 2013-01-22 2014-01-22 Protéines à répétitions de dipeptides en tant que cible thérapeutique dans des maladies neurodégénératives avec expansion de répétition hexanucléotidique
HRP20191608 HRP20191608T1 (hr) 2013-01-22 2019-09-05 Proteini s ponavljajućim dipeptidom kao terapeutsko ciljno mjesto kod neurodegenerativnih bolesti s ekspanzijom ponavljajućeg heksanukleotida
CY20191100935T CY1122079T1 (el) 2013-01-22 2019-09-05 Πρωτεϊνες επαναληψης διπεπτιδιου ως θεραπευτικος στοχος στις νευροεκφυλιστικες νοσους με επεκταση επαναληψης των εξανουκλεοτιδιων

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/EP2013/000190 WO2014114303A1 (fr) 2013-01-22 2013-01-22 Protéines à répétitions de dipeptides comme cible thérapeutique dans des maladies neurodégénératives avec expansion de répétitions hexanucléotidiques

Publications (1)

Publication Number Publication Date
WO2014114303A1 true WO2014114303A1 (fr) 2014-07-31

Family

ID=47632965

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2013/000190 WO2014114303A1 (fr) 2013-01-22 2013-01-22 Protéines à répétitions de dipeptides comme cible thérapeutique dans des maladies neurodégénératives avec expansion de répétitions hexanucléotidiques

Country Status (1)

Country Link
WO (1) WO2014114303A1 (fr)

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2948471A1 (fr) 2013-01-24 2015-12-02 Mayo Foundation for Medical Education and Research Procédés et matériaux de détection d'une dégénérescence du lobe frontotemporal positive aux expansions de répétitions hexanucléotidiques du gène c9orf72 ou d'une sclérose latérale amyotrophique positive aux expansions de répétitions hexanucléotidiques du gène c9orf72
WO2016050822A2 (fr) 2014-09-30 2016-04-07 Neurimmune Holding Ag Anticorps dirigés contre des répétitions dipeptidiques (dpr) d'origine humaine
WO2016196324A1 (fr) * 2015-05-29 2016-12-08 University Of Floridia Research Foundation, Inc. Méthodes de diagnostic de la maladie de huntington
WO2016210372A3 (fr) * 2015-06-25 2017-02-02 University Of Southern California Procédés de traitement de maladies neurologiques
CN108795935A (zh) * 2018-05-23 2018-11-13 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) 日本血吸虫SjELAV-like 1基因的siRNA及其应用
WO2018208972A1 (fr) * 2017-05-09 2018-11-15 University Of Massachusetts Méthodes de traitement de la sclérose latérale amyotrophique (sla)
US10295547B2 (en) 2013-03-14 2019-05-21 University Of Florida Research Foundation, Incorporated Use and treatment of di-amino acid repeat-containing proteins associated with ALS
WO2019210054A1 (fr) * 2018-04-27 2019-10-31 Biogen Ma Inc. Anticorps anti-(poly-ga) dirigé contre des répétitions dipeptidiques (dpr) d'origine humaine
US10711274B2 (en) 2014-03-18 2020-07-14 University Of Massachusetts RAAV-based compositions and methods for treating amyotrophic lateral sclerosis
US10940161B2 (en) 2016-04-04 2021-03-09 University Of Florida Research Foundation, Incorporated Manipulation of EIF3 to modulate repeat associated non-ATG (RAN) translation
EA038285B1 (ru) * 2015-08-07 2021-08-04 Нейриммьюн Холдинг Аг Происходящее от человека антитело к дипептидным повторам (dpr)
US11345911B2 (en) 2017-04-17 2022-05-31 University Of Florida Research Foundation, Incorporated Regulation of RAN translation by PKR and eIF2A-P pathways
US11739330B2 (en) 2017-09-22 2023-08-29 University Of Massachusetts SOD1 dual expression vectors and uses thereof
US11903910B2 (en) 2017-09-26 2024-02-20 University Of Florida Research Foundation, Incorporated Use of metformin and analogs thereof to reduce RAN protein levels in the treatment of neurological disorders

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1270724A2 (fr) * 2001-06-18 2003-01-02 National Institute of Advanced Industrial Science and Technology Récepteurs couplés aux protéines de liaison guanosin triphosphate
US20040102608A1 (en) * 2002-05-13 2004-05-27 Cornell Research Foundation, Inc. Multiblock copolymers having improved mechanical properties
WO2010115033A2 (fr) 2009-04-02 2010-10-07 Regents Of The University Of Minnesota Polypeptides associés à des expansions de répétitions nucléotidiques et leurs utilisations

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1270724A2 (fr) * 2001-06-18 2003-01-02 National Institute of Advanced Industrial Science and Technology Récepteurs couplés aux protéines de liaison guanosin triphosphate
US20040102608A1 (en) * 2002-05-13 2004-05-27 Cornell Research Foundation, Inc. Multiblock copolymers having improved mechanical properties
US20050261425A1 (en) * 2002-05-13 2005-11-24 Cornell Research Foundation, Inc. Multiblock copolymers having improved mechanical properties
WO2010115033A2 (fr) 2009-04-02 2010-10-07 Regents Of The University Of Minnesota Polypeptides associés à des expansions de répétitions nucléotidiques et leurs utilisations

Non-Patent Citations (40)

* Cited by examiner, † Cited by third party
Title
"Current Protocols in Molecular Biology", 1991, JOHN WILEY & SONS, pages: 6.3.1 - 6.3.6
AL-SARRAJ S; KING A; TROAKES C ET AL.: "TDP-43 negative, neuronal cytoplasmic and intranuclear inclusions in the cerebellum and hippocampus define the pathology of C9orf72-linked FTLD and MND/ALS", ACTA NEUROPATHOL, vol. 122, no. 6, 2011, pages 691 - 702, XP019984649, DOI: doi:10.1007/s00401-011-0911-2
ARAI T; HASEGAWA M; AKIYAMA H ET AL.: "TDP-43 is a component of ubiquitin- positive tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis", BIOCHEM BIOPHYS RES COMMUN, vol. 351, no. 3, 2006, pages 602 - 611, XP024925727, DOI: doi:10.1016/j.bbrc.2006.10.093
ASHIZAWA T.; RANUN L.P.: "GGCCTG repeats put a hex on Purkinje cells and motor neurons in SCA36", NEUROLOGY, vol. 79, no. 4, 2012, pages 302 - 303
BENAJIBA L; LE BER I; CAMUZAT A ET AL.: "TARDBP mutations in motoneuron disease with frontotemporal lobar degeneration", ANN NEUROL, vol. 65, no. 4, 2009, pages 470 - 473
BIGIO EH; WEINTRAUB S; RADEMAKERS R; BAKER M; AHMADIAN SS; RADEMAKER A; WEITNER BB; MAO Q; LEE KH; MISHRA M: "Frontotemporal lobar degeneration with TDP-43 proteinopathy and chromosome 9p repeat expansion in C90RF72: clinicopathologic correlation", NEUROPATHOLOGY, 2012
BOXER AL; MACKENZIE IR; BOEVE BF; BAKER M; SEELEY WW; CROOK R; FELDMAN H; HSIUNG GY; RUTHERFORD N; LALUZ V: "Clinical, neuroimaging and neuropathological features of a new chromosome 9p-linked FTD-ALS family", J NEUROL NEUROSURG PSYCHIATRY, vol. 82, no. 2, 2011, pages 196 - 203
DEJESUS-HERNANDEZ M; MACKENZIE IR; BOEVE BF ET AL.: "Expanded GGGGCC hexanucleotide repeat in noncoding region of C90RF72 causes chromosome 9p-linked FTD and ALS", NEURON, vol. 72, no. 2, 2011, pages 245 - 256, XP028322560, DOI: doi:10.1016/j.neuron.2011.09.011
DESMYTER ET AL.: "Crystal structure of a camel single-domain VH antibody fragment in complex with lysozyme", NAT. STRUCTURE BIOL., vol. 3, 1996, pages 803 - 811, XP002518002, DOI: doi:10.1038/nsb0996-803
DIGNAM JD; LEBOVITZ RM; ROEDER RG: "Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei", NUCLEIC ACIDS RES, vol. 11, no. 5, 1983, pages 1475 - 1489
DILLNER J ET AL: "ANTIBODIES AGAINST A SYNTHETIC PEPTIDE IDENTIFY THE EPSTEIN-BARR VIRUS-DETERMINED NUCLEAR ANTIGEN", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 81, no. 15, 1984, pages 4652 - 4656, XP002699796, ISSN: 0027-8424 *
GASSER T; HARDY J; MIZUNO Y: "Milestones in PD genetics", MOV DISORD, vol. 26, no. 6, 2011, pages 1042 - 1048
GEALL ET AL.: "Non-viral delivery of self-amplifying RNA vaccines", PROC NATL ACAD SCI USA, vol. 109, 2012, pages 14604 - 14609
GIJSELINCK I; VAN LANGENHOVE T; VAN DER ZEE J ET AL.: "A C9orf72 promoter repeat expansion in a Flanders-Belgian cohort with disorders of the frontotemporal lobar degeneration-amyotrophic lateral sclerosis spectrum: a gene identification study", LANCETNEUROL, vol. 11, no. 1, 2012, pages 54 - 65, XP055044218, DOI: doi:10.1016/S1474-4422(11)70261-7
HAASS C; SELKOE DJ: "Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid beta-peptide", NAT REV MOL CELL BIOL, vol. 8, no. 2, 2007, pages 101 - 112, XP002545389, DOI: doi:10.1038/NRM2101
HATASU KOBAYASHI ET AL: "Expansion of Intronic GGCCTG Hexanucleotide Repeat inCauses SCA36, a Type of Spinocerebellar Ataxia Accompanied by Motor Neuron Involvement", AMERICAN JOURNAL OF HUMAN GENETICS, AMERICAN SOCIETY OF HUMAN GENETICS, CHICAGO, IL, US, vol. 89, no. 1, 18 May 2011 (2011-05-18), pages 121 - 130, XP028102426, ISSN: 0002-9297, [retrieved on 20110525], DOI: 10.1016/J.AJHG.2011.05.015 *
HUSTON ET AL.: "Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli", PROC. NATL. ACAD. SCI. USA, vol. 85, 1988, pages 5879 - 5883, XP000872837, DOI: doi:10.1073/pnas.85.16.5879
IVANOV IP; FIRTH AE; MICHEL AM; ATKINS JF; BARANOV PV: "Identification of evolutionarily conserved non-AUG-initiated N-terminal extensions in human coding sequences", NUCLEIC ACIDS RES, vol. 39, 2011, pages 4220
JANEWAY ET AL., IMMUNOBIOLOGY, GARLAND SCIENCE, 2001
JOSEPHS KA; HODGES JR; SNOWDEN JS; MACKENZIE IR; NEUMANN M; MANN DM; DICKSON DW: "Neuropathological background of phenotypical variability in frontotemporal dementia", ACTA NEUROPATHOL, vol. 122, no. 2, 2011, pages 137 - 153, XP019927310, DOI: doi:10.1007/s00401-011-0839-6
KREMMER E; KRANZ BR; HILLE A; KLEIN K; EULITZ M; HOFFINANN-FEZER G; FEIDEN W; HERRMANN K; DELECLUSE HJ; DELSOL G: "Rat monoclonal antibodies differentiating between the Epstein-Barr virus nuclear antigens 2A (EBNA2A) and 2B (EBNA2B", VIROLOGY, vol. 208, no. 1, 1995, pages 336 - 42
KUFER P.; LUTTERBTISE R.; BAEUERLE P.A.: "A revival of bispecific antibodies", TRENDS BIOTECHNOL., vol. 22, 2004, pages 238 - 244, XP004504363, DOI: doi:10.1016/j.tibtech.2004.03.006
LI H; WYMAN T; YU ZX; LI SH; LI XJ: "Abnormal association of mutant huntingtin with synaptic vesicles inhibits glutamate release", HUM MOL GENET, vol. 12, 2003, pages 2021
MA AS; MORAN-JONES K; SHAN J ET AL.: "Heterogeneous nuclear ribonucleoprotein A3, a novel RNA trafficking response element-binding protein", J BIOL CHEM, vol. 277, no. 20, 2002, pages 18010 - 18020
MACKENZIE IR; RADEMAKERS R; NEUMANN M.: "TDP-43 and FUS in amyotrophic lateral sclerosis and frontotemporal dementia", LANCET NEUROL., vol. 9, no. 10, 2010, pages 995 - 1007, XP027599046, DOI: doi:10.1016/S1474-4422(10)70195-2
MAHONEY CJ; DOWNEY LE; RIDGWAY GR; BECK J; CLEGG S; BLAIR M; FINNEGAN S; LEUNG KK; YEATMAN T; GOLDEN H: "Longitudinal neuroimaging and neuropsychological profiles of frontotemporal dementia with C90RF72 expansions", ALZHEIMERS RES THER., vol. 4, no. 5, 2012, pages 41, XP021123317, DOI: doi:10.1186/alzrt144
NAGAFUCHI SHIGEO ET AL: "Structure and expression of the gene responsible for the triplet repeat disorder, dentatorubral and pallidoluysian atrophy (DRPLA)", NATURE GENETICS, vol. 8, no. 2, 1994, pages 177 - 182, XP002699797, ISSN: 1061-4036 *
NEUMANN M; SAMPATHU DM; KWONG LK ET AL.: "Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis", SCIENCE, vol. 314, no. 5796, 2006, pages 130 - 133, XP002534603, DOI: doi:10.1126/SCIENCE.1134108
PEABODY DS: "Translation initiation at non-AUG triplets in mammalian cells", J BIOLCHEM, vol. 264, 1989, pages 5031
RADEMAKERS R; NEUMANN M; MACKENZIE IR: "Advances in understanding the molecular basis of frontotemporal dementia", NAT REV NEUROL, vol. 8, no. 8, 2012, pages 423 - 434
RANUM LP; COOPER TA: "RNA-mediated neuromuscular disorders", ANNU REV NEUROSCI, vol. 29, 2006, pages 259 - 277
RENTON AE; MAJOUNIE E; WAITE A ET AL.: "A Hexanucleotide Repeat Expansion in C90RF72 Is the Cause of Chromosome 9p21-Linked ALS-FTD", NEURON, vol. 72, no. 2, 2011, pages 257 - 268, XP028322561, DOI: doi:10.1016/j.neuron.2011.09.010
SIEBEN A; VAN LANGENHOVE T; ENGELBORGHS S ET AL.: "The genetics and neuropathology of frontotemporal lobar degeneration", ACTA NEUROPATHOL, vol. 124, no. 3, 2012, pages 353 - 372, XP035100879, DOI: doi:10.1007/s00401-012-1029-x
SREEDHARAN J; BLAIR IP; TRIPATHI VB ET AL.: "TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis", SCIENCE, vol. 319, no. 5870, 2008, pages 1668 - 1672, XP008153967, DOI: doi:10.1126/science.1154584
TOURIOL C. ET AL.: "Generation of protein isoform diversity by alternative initiation of translation at non-AUG codons", BIOL CELL, vol. 95, 2003, pages 169
VAN DER ZEE ET AL., HUMAN MUTATION, 2012
VAN DER ZEE J; GIJSELINCK I; DILLEN L ET AL.: "A Pan-European study of the C9orf72 Repeat Associated with FTLD: Geographic Prevalence", GENOMIC INSTABILITY AND INTERMEDIATE REPEATS, 2012
VANCE C; ROGELJ B; HORTOBAGYI T ET AL.: "Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis type 6", SCIENCE, vol. 323, no. 5918, 2009, pages 1208 - 1211, XP008153960, DOI: doi:10.1126/science.1165942
WHITWELL JL; WEIGAND SD; BOEVE BF; SENJEM ML; GUNTER JL; DEJESUS-HERNANDEZ M; RUTHERFORD NJ; BAKER M; KNOPMAN DS; WSZOLEK ZK: "Neuroimaging signatures of frontotemporal dementia genetics: C90RF72, tau, progranulin and sporadics", BRAIN, vol. 135, 2012, pages 794 - 806
ZU T; GIBBENS B; DOTY NS ET AL.: "Non-ATG-initiated translation directed by microsatellite expansions", PROC NATL ACAD SCI USA, vol. 108, no. 1, 2011, pages 260 - 265, XP055105616, DOI: doi:10.1073/pnas.1013343108

Cited By (38)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2948471A4 (fr) * 2013-01-24 2016-08-10 Mayo Foundation Procédés et matériaux de détection d'une dégénérescence du lobe frontotemporal positive aux expansions de répétitions hexanucléotidiques du gène c9orf72 ou d'une sclérose latérale amyotrophique positive aux expansions de répétitions hexanucléotidiques du gène c9orf72
US9448232B2 (en) 2013-01-24 2016-09-20 Mayo Foundation For Medical Education And Research Methods and materials for detecting C9ORF72 hexanucleotide repeat expansion positive frontotemporal lobar degeneration or C9ORF72 hexanucleotide repeat expansion positive amyotrophic lateral sclerosis
EP2948471A1 (fr) 2013-01-24 2015-12-02 Mayo Foundation for Medical Education and Research Procédés et matériaux de détection d'une dégénérescence du lobe frontotemporal positive aux expansions de répétitions hexanucléotidiques du gène c9orf72 ou d'une sclérose latérale amyotrophique positive aux expansions de répétitions hexanucléotidiques du gène c9orf72
US10295547B2 (en) 2013-03-14 2019-05-21 University Of Florida Research Foundation, Incorporated Use and treatment of di-amino acid repeat-containing proteins associated with ALS
US11035867B2 (en) 2013-03-14 2021-06-15 University Of Florida Research Foundation, Incorporated Use and treatment of di-amino acid repeat-containing proteins associated with ALS
US10663475B2 (en) 2013-03-14 2020-05-26 University Of Florida Research Foundation, Incorporated Use and treatment of di-amino acid repeat-containing proteins associated with ALS
US11760999B2 (en) 2014-03-18 2023-09-19 University Of Massachusetts RAAV-based compositions and methods for treating amyotrophic lateral sclerosis
US10954518B2 (en) 2014-03-18 2021-03-23 University Of Massachusetts RAAV-based compositions and methods for treating amyotrophic lateral sclerosis
US10851375B2 (en) 2014-03-18 2020-12-01 University Of Massachusetts RAAV-based compositions and methods for treating amyotrophic lateral sclerosis
US10711274B2 (en) 2014-03-18 2020-07-14 University Of Massachusetts RAAV-based compositions and methods for treating amyotrophic lateral sclerosis
JP2017538665A (ja) * 2014-09-30 2017-12-28 ニューリミューン ホールディング エイジー ヒト由来の抗ジペプチドリピート（ｄｐｒ）抗体
US10961322B2 (en) 2014-09-30 2021-03-30 Neurimmune Holding Ag Human-derived anti-dipeptide repeats (DPRs) antibody
KR102594327B1 (ko) 2014-09-30 2023-10-27 뉴리뮨 홀딩 아게 인간-유래의 항-디펩티드 반복체(dpr) 항체
CN108064248A (zh) * 2014-09-30 2018-05-22 生物控股有限公司 人源抗二肽重复（dpr）抗体
US10392447B2 (en) 2014-09-30 2019-08-27 Neurimmune Holding Ag Human-derived anti-dipeptide repeats (DPRs) antibody
WO2016050822A2 (fr) 2014-09-30 2016-04-07 Neurimmune Holding Ag Anticorps dirigés contre des répétitions dipeptidiques (dpr) d'origine humaine
IL278941B (en) * 2014-09-30 2022-07-01 Neurimmune Holding Ag dprs antibodies which originate in the human body
EP3201228A2 (fr) * 2014-09-30 2017-08-09 Neurimmune Holding AG Anticorps dirigés contre des répétitions dipeptidiques (dpr) d'origine humaine
KR20170061702A (ko) * 2014-09-30 2017-06-05 뉴리뮨 홀딩 아게 인간-유래의 항-디펩티드 반복체(dpr) 항체
CN108064248B (zh) * 2014-09-30 2022-03-15 生物控股有限公司 人源抗二肽重复(dpr)抗体
JP2021177788A (ja) * 2014-09-30 2021-11-18 ニューリミューン ホールディング エイジー ヒト由来の抗ジペプチドリピート（ｄｐｒ）抗体
WO2016050822A3 (fr) * 2014-09-30 2016-06-02 Neurimmune Holding Ag Anticorps dirigés contre des répétitions dipeptidiques (dpr) d'origine humaine
US10509045B2 (en) 2015-05-29 2019-12-17 University Of Florida Research Foundation, Incorporated Methods for diagnosing Huntington's disease
WO2016196324A1 (fr) * 2015-05-29 2016-12-08 University Of Floridia Research Foundation, Inc. Méthodes de diagnostic de la maladie de huntington
US10758545B2 (en) 2015-06-25 2020-09-01 University Of Southern California Methods to treat neurological diseases
WO2016210372A3 (fr) * 2015-06-25 2017-02-02 University Of Southern California Procédés de traitement de maladies neurologiques
EA038285B1 (ru) * 2015-08-07 2021-08-04 Нейриммьюн Холдинг Аг Происходящее от человека антитело к дипептидным повторам (dpr)
US10940161B2 (en) 2016-04-04 2021-03-09 University Of Florida Research Foundation, Incorporated Manipulation of EIF3 to modulate repeat associated non-ATG (RAN) translation
US11345911B2 (en) 2017-04-17 2022-05-31 University Of Florida Research Foundation, Incorporated Regulation of RAN translation by PKR and eIF2A-P pathways
WO2018208972A1 (fr) * 2017-05-09 2018-11-15 University Of Massachusetts Méthodes de traitement de la sclérose latérale amyotrophique (sla)
US11859179B2 (en) 2017-05-09 2024-01-02 University Of Massachusetts Methods of treating amyotrophic lateral sclerosis (ALS)
US11739330B2 (en) 2017-09-22 2023-08-29 University Of Massachusetts SOD1 dual expression vectors and uses thereof
US11903910B2 (en) 2017-09-26 2024-02-20 University Of Florida Research Foundation, Incorporated Use of metformin and analogs thereof to reduce RAN protein levels in the treatment of neurological disorders
EP3797114A4 (fr) * 2018-04-27 2022-05-11 Biogen MA Inc. Anticorps anti-(poly-ga) dirigé contre des répétitions dipeptidiques (dpr) d'origine humaine
CN112638932A (zh) * 2018-04-27 2021-04-09 比奥根Ma公司 人源性抗(聚-ga)二肽重复序列(dpr)抗体
WO2019210054A1 (fr) * 2018-04-27 2019-10-31 Biogen Ma Inc. Anticorps anti-(poly-ga) dirigé contre des répétitions dipeptidiques (dpr) d'origine humaine
CN108795935B (zh) * 2018-05-23 2022-06-21 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) 日本血吸虫SjELAV-like 1基因的siRNA及其应用
CN108795935A (zh) * 2018-05-23 2018-11-13 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) 日本血吸虫SjELAV-like 1基因的siRNA及其应用

Similar Documents

Publication Publication Date Title
US10066007B2 (en) Dipeptide-repeat proteins as therapeutic target in neurodegenerative diseases with hexanucleotide repeat expansion
WO2014114303A1 (fr) Protéines à répétitions de dipeptides comme cible thérapeutique dans des maladies neurodégénératives avec expansion de répétitions hexanucléotidiques
Dreuillet et al. In vivo and in vitro interaction between human transcription factor MOK2 and nuclear lamin A/C
JP2023524733A (ja) 遺伝子発現を活性化及びサイレンシングするためのエフェクタードメインの作出、同定及び性質決定のための組成物、システム及び方法
AU2018214601A1 (en) Targeted oligonucleotides
de Planell-Saguer et al. Biochemical and genetic evidence for a role of IGHMBP2 in the translational machinery
JP2021167353A (ja) 自己免疫疾患およびがんを処置するための組成物および方法
JP7271446B2 (ja) Ｈｌａ－ｇ転写物およびアイソフォームおよびそれらの使用
Wright et al. CGG repeats trigger translational frameshifts that generate aggregation-prone chimeric proteins
Hayer et al. Aberrant expression of the autoantigen heterogeneous nuclear ribonucleoprotein-A2 (RA33) and spontaneous formation of rheumatoid arthritis-associated anti-RA33 autoantibodies in TNF-α transgenic mice
US20230277618A1 (en) Therapeutic targeting of phosphate dysregulation in cancer via the xpr1:kidins220 protein complex
EP2076776A2 (fr) Autoantigènes utilisés dans le diagnosic, le pronostic et le traitement améliorés de maladies neurologiques inflammatoires
KR102309517B1 (ko) 표적 항원 발견, 표현형 선별 및 표적 세포 특이성 표적 에피토프를 규명하기 위한 그의 용도
US20220202935A1 (en) Immunogen for Preventing or Treating Familial Frontotemporal Dementia (FTD) and/or Amyotrophic Lateral Sclerosis (ALS)
JP7008328B2 (ja) 免疫制御剤
Kong et al. The cohesin loader NIPBL interacts with pre-ribosomal RNA and treacle to regulate ribosomal RNA synthesis
Wang et al. Subtractive phage display technology identifies zebrafish marcksb that is required for gastrulation
EP3937969A1 (fr) Compositions et procédés de modulation de la signalisation du cgrp pour réguler des cellules lymphoïdes innées intestinales
Damboriena Functional analysis of ANKRD55, a multiple sclerosis risk gene with unknown function
Ugidos Damboriena Functional analysis of ankrd55, a multiple sclerosis risk gene with unknown function.
Price Characterizing Motor Neuron-Specific mRNA Processing Defects in an Intermediate Mouse Model of Spinal Muscular Atrophy
WO2023178241A2 (fr) Procédés et compositions pour perturber des agrégats de tau à l'aide de séquences de répétition de polysérine ciblant des protéines exogènes
Das et al. Flanking sequences regulate the toxicity, non-ATG translation, and aggregation of RNA with tandem CAG Repeats
WO2024049357A1 (fr) Agent de diagnostic et procédé pour la maladie d'alzheimer
Tseng Repeat-associated non-AUG (RAN) Translation in GC-rich Repeats of C9ALS/FTD and FXTAS

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: 13702335

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 13702335

Country of ref document: EP

Kind code of ref document: A1