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WO2024049357A1 - Agent de diagnostic et procédé pour la maladie d'alzheimer - Google Patents

Agent de diagnostic et procédé pour la maladie d'alzheimer Download PDF

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Publication number
WO2024049357A1
WO2024049357A1 PCT/SG2023/050599 SG2023050599W WO2024049357A1 WO 2024049357 A1 WO2024049357 A1 WO 2024049357A1 SG 2023050599 W SG2023050599 W SG 2023050599W WO 2024049357 A1 WO2024049357 A1 WO 2024049357A1
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seq
amino acid
acid sequence
set forth
antibody
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Chin Tong ONG
Zhen Kai NGIAN
Yow Yong TAN
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Temasek Life Sciences Laboratory Ltd
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Temasek Life Sciences Laboratory Ltd
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Priority to AU2023334129A priority Critical patent/AU2023334129A1/en
Priority to CN202380073638.5A priority patent/CN120092179A/zh
Publication of WO2024049357A1 publication Critical patent/WO2024049357A1/fr
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    • 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
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
    • G01N2440/14—Post-translational modifications [PTMs] in chemical analysis of biological material phosphorylation
    • 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
    • G01N2800/2821—Alzheimer

Definitions

  • the present invention relates to an intron-retaining Tau splicing isoform and methods of detecting the same.
  • the invention also relates to agents, including oligonucleotides and antibodies, and methods of diagnosis for Alzheimer's disease.
  • AD Alzheimer’s disease
  • AD Alzheimer’s disease
  • a progressive dementia that starts with decrease of short-term memory and mild learning disability, develops higher brain dysfunction, particularly visuospatial agnosia, ideational apraxia, constructive apraxia and the like, and finally reaches movement disorder and so-called personality destruction.
  • It is a familial and sporadic neurodegenerative disease that encompasses many risk factors such as genetic makeup and ageing.
  • Synaptic and neuronal loss in AD is caused by interplay between the pathology of amyloid-p plaques and Tau neurofibrillary tangles with impairment of innate clearance pathways.
  • microtubule-associated protein Tau and its hyper-phosphorylated version form the main constituent of intracellular neurofibrillary tangles which is a hallmark of several dementias, including AD and frontotemporal dementia.
  • AD frontotemporal dementia.
  • This evidence forms the basis of a hypothesis for AD, wherein the intracellular accumulation of Tau leads to microtubule disassembly, dendritic spinal collapse, and degeneration of axons, malfunction in communication between neurons and cell death.
  • IR differential intron retention
  • AD intron-retaining Tau proteins
  • the present invention aims to provide agents and methods for diagnosing Alzheimer's disease or identifying a subject at risk of developing Alzheimer's disease.
  • an isolated or recombinant Tau11i-derived polypeptide comprising the following amino acid sequence:
  • the isolated or recombinant Tau11 i-derived polypeptide may consist of amino acid sequence set forth in (1) or (2) with a cysteine residue conjugated to an end. Terminal cysteine is added to the peptide sequence to allow its conjugation to carrier proteins, which helps antibody production.
  • the isolated or recombinant Tau11 i- derived polypeptide may consist of the amino acid sequence HKPGSPVEGEGWDGRVQGV- C set forth in SEQ ID NO: 1 or the amino acid sequence C-SPVEGEGWDGRVQG set forth in SEQ ID NO: 2.
  • an isolated antibody or antigen-binding fragment thereof that specifically binds to the isolated or recombinant Tau11 i-derived polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 14 or antigenic fragment thereof, or SEQ ID NO: 15, or an amino acid sequence resulting from substitution, deletion, addition or insertion of one or two or more amino acids in the amino acid sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15.
  • the polypeptide in which the antibody or the antigen binding fragment thereof binds to consists of the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO: 2.
  • the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence shown as residues 1 to 136 of SEQ ID NO: 29 and/or a light chain variable region comprising the amino acid sequence shown as residues 1 to 132 of SEQ ID NO: 34.
  • the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising complementarity determining regions HCDR1 having an amino acid sequence set forth in SEQ ID NO: 31 , HCDR2 having an amino acid sequence set forth in SEQ ID NO: 32, and HCDR3 having an amino acid sequence set forth in SEQ ID NO: 33, and/or a light chain variable region comprising complementarity determining regions LCDR1 having an amino acid sequence set forth in SEQ ID NO: 36, LCDR2 having an amino acid sequence set forth in SEQ ID NO:37, and LCDR3 having an amino acid sequence set forth in SEQ ID NO: 38.
  • oligonucleotides for amplifying a polynucleotide sequence spanning the junction of exon 11 and intron 11 of a Tau gene.
  • the oligonucleotides consist of the nucleotide sequences set forth in SEQ ID NO: 7 and SEQ ID NO: 8.
  • kits comprising the antibody or antigen binding fragment thereof provided in the second aspect.
  • the kit further comprises the polypeptide of the first aspect.
  • a polynucleic acid molecule encoding the polypeptide according to the first aspect, or the antibody or antigen binding fragment thereof according to the second aspect.
  • the polynucleic acid molecule is cDNA.
  • a method of detecting the polypeptide according to the first aspect comprising contacting a sample suspected of comprising the polypeptide with the antibody or the antigen-binding fragment according to the second aspect.
  • the method is in vitro.
  • a seventh aspect there is provided a method for diagnosing Alzheimer's disease or identifying a subject at risk of developing Alzheimer's disease, wherein the method comprises:
  • the sample is brain tissue. In some embodiments, the sample is cerebrospinal fluid (CSF). In some embodiments, the sample is blood plasma. In other embodiments, the sample is saliva, lacrimal fluid or urine.
  • the nucleic acid detection assay comprises amplifying a polynucleotide sequence spanning the junction of exon 11 and intron 11 of a Tau gene. In further embodiments, the amplification comprises using oligonucleotides having the nucleotide sequences as set forth in SEQ ID NOs: 7 and 8. In further embodiments, the nucleic acid detection assay is a PCR-based assay.
  • a method of screening a candidate therapeutic or prophylactic drug for Alzheimer's disease comprises using any of the following as an index: (a) reduction of the amount of polypeptide-containing Tau (e.g. Tau11i as hereinafter described) in the body of an Alzheimer's disease subject or a subject at risk of developing Alzheimer's disease, or (b) inhibition of production of polypeptide-containing Tau (e.g. Tau11i as hereinafter described) in the body of an Alzheimer's disease subject or a subject at risk of developing Alzheimer's disease; wherein the Tau protein comprises the amino acid sequence of the polypeptide according to the first aspect.
  • a) reduction of the amount of polypeptide-containing Tau e.g. Tau11i as hereinafter described
  • the Tau protein comprises the amino acid sequence of the polypeptide according to the first aspect.
  • a method of preparing a Tau11 i-specific antibody comprising: (a) injecting the polypeptide according to the first aspect into an animal to induce an immune response to the polypeptide; (b) isolating antibodies that bind to the polypeptide; (c) selecting antibodies of (b) that binds to Tau11 i but do not bind to full length Tau thereby producing an Tau 11 i-specific antibody.
  • a method of screening and/or selecting a Tau11i epitopespecific aptamer comprises: (a) conducting systematic evolution of ligands by exponential enrichment (SELEX) technique to identify Tau11 i epitope-specific aptamers, wherein the SELEX technique comprises providing a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs. 1 , 2, 13, 14 or 15 as a target ligand.
  • SELEX systematic evolution of ligands by exponential enrichment
  • an aptamer capable of binding to a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs. 1 , 2, 13, 14 or 15.
  • FIG. 1 shows an increased retention of intron 11 of Tau gene in AD.
  • Intron 11 contains a premature stop codon (line 2: gagtcctgatgaaac... ) with a canonical polyadenylation site that is located 1 kb downstream. Arrows indicate qPCR primers.
  • B Integrative Genomic Viewer of intron 11 from control (top two rows) and AD (bottom two rows) patients.
  • FIG. 2 shows that truncated Tau11i oligomerizes and is found in a Sarkosyl-insoluble fraction.
  • A Schematic of Flag-tag full-length and truncated Tau proteins with specific antibody recognition sites. Amino acids encoded by intron 11 are represented in light grey text.
  • pT181 phosphor-Tau (T181), Tau4R: 275-291 , Tau46: 404-421 , AT8: phosphor-Tau (S202/T205).
  • B-C Immunoblot and quantification of Tau11i HMW species in (B) 293 cells transfected with Tau441 (left well) or Tau11i (right well); and (C) Neuronal stem cells (NSC) (left lane) that expressed eitherTau441 (middle lane) orTaul li (right lane) proteins. Data presented as mean ⁇ SEM.
  • FIG. 3 shows an elevated level of Tau11i in different AD brain tissues.
  • A Immunoblot of control (”C” lanes) and AD (”A” lanes) temporal lobe, amygdala (AMYDG.), parietal lobe (PARIETAL), frontal lobe and hippocampus showed enrichment of Tau11i ( ⁇ 55 kDa white arrowhead) in AD samples. Samples were also immunoblotted with AT8 and actin antibodies. Each number depicts a unique human subject. Quantification of 7 pairs of control and AD samples with data presented as mean ⁇ SD. The level of Tau11 i in control for each brain region was set as 1 . Sample quantified was underlined.
  • FIG. 4 shows that Tau11i protein associates poorly with the microtubule network and is more stable.
  • A Images of Day 21 and Day 44 neurons expressing Tau441 or Tau11i stained with a-tubulin and Flag antibodies. Scale bar represents 50 pm. Quantification showed lower colocalization of Tau11i with a-tubulin as compared to Tau441. Each circle represents one field with filled circle from second biological replicate. Data presented as mean ⁇ SD.
  • FIG. 5 depicts retention of intron 11 of the Tau gene in other neurodegeneration and characterization of Tau11 i protein.
  • A Dot plot of normalized intron retention (IR) ratio from PD and PSP cohorts. Each dot represents individual value. CT column is healthy control and PD or PSP column labels diseased patient. Data is presented as mean ⁇ SD, p-value is calculated by 2-tailed t-test. Related to FIG. 1.
  • B Immunoblot of 293T cells (top) and NSC (bottom) expressing either Flag-Tau441 (left well) or Flag-Tau11i (right well). HMW species is marked by white arrowhead. "Rep" stands for replicate.
  • FIG. 6 depicts the results of immunofluorescence which show a higher level of Tau11 i in AD temporal lobe (middle panels).
  • A Image and quantification of Tau11i over DAPI positive cells is presented as mean ⁇ SD where each circle represents one field. P-value was calculated by 2-tailed t-test.
  • B Immunoblot of control "C8" and AD "A1" temporal lobe. Related to FIG. 3C.
  • FIG. 7 shows that Tau11i has a lower level of co-localization with a-tubulin as compared to Tau441 in mature neurons.
  • FIG. 4A shows that Tau11i has a lower level of co-localization with a-tubulin as compared to Tau441 in mature neurons.
  • FIG. 8 shows that Tau11i has a weaker co-localization signal with a-tubulin in mature neurons and higher protein stability when compared to Tau441.
  • A Representative images from two biological replicates of Day 44 neurons expressing either Flag-Tau441 or Flag-Tau11i. Scale bar is 100 pm. Quantification of Flag-Tau over a-tubulin is presented as mean ⁇ SD where each circle represents one field. P-value was calculated by 2-tailed t-test. Related to FIG. 4A. "Rep" stands for replicate.
  • B Representative immuno-blots of 293T cells harvested on different day “D” after transfection with Flag and actin antibodies. Related to FIG. 4B.
  • FIG. 9 shows the detection of Tau intron 11 retention in total RNA extracted from human plasma.
  • A Schematic of experiment. Total RNA was extracted from 0.5 ml of control human plasma (i-DNA Biotech) using RNAzol (Sigma). After DNase I treatment to remove genomic DNA, reverse transcription was performed with random hexamer. cDNA was subjected to PCR and resolved on agarose gel.
  • B Different primers were used to amplify the Tau gene and detect intron retention events previously identified in AD brains. The HBB gene is highly expressed, therefore we used it as positive control and tested for the presence of genomic contamination. HADC2 is not present in blood plasma and was used as a negative control. Amplification of Tau intron 11 from plasma RNA is shown in lane 3.
  • FIG. 10 shows that Tau11i is detectable in human plasma with an anti-Tau11 i antibody.
  • A 293 cells or 293 cells transfected with Flag-Tau11i were lysed and Co-IP with rabbit anti- Tau11 i antibody, which was generated with antigen peptide consisting of the amino acid sequence set forth in SEQ ID NO: 15. The Co-IP beads were boiled and immunoblotted with Flag antibody. Arrow heads show immunoprecipitated Tau11i protein.
  • Pre-cleared control human plasma i-DNA Biotech
  • FIG. 11 depicts the detection of Tau11i by ELIZA.
  • A Schematic of Flag-Tau441 & Flag- Tau11 i peptide (which were purified from 293T cells).
  • B Optimization of ELISA.
  • wells were coated with Tau11 i or Tau441 peptide & probed with Tau11i antibody.
  • Tau peptide-coated wells were probed with either Tau11i, IgG or phospho- Tau181 antibodies.
  • Tau11i antibody was coated onto plates with the addition of either Flag-Tau11 i or Flag-Tau441 peptides.
  • An anti-Flag antibody was used for detection.
  • Results show the anti-Tau11 i antibody having much higher specificity for Tau11i compared with Tau441 , which was around negative control level.
  • FIG.12 depicts the detection of Tau11i by monoclonal antibody 8A12.
  • A Schematic of Flag- Tau441 & Flag-Tau11i peptide.
  • B Immunoblot of total lysates from 293 cells and neuronal stem cells (NSC) that expressed either Flag-Tau441 or Flag-Tau11i protein with Flag or monoclonal Tau11i antibody (8A12).
  • NSC neuronal stem cells
  • PICO indicates lower exposure whereas DURA indicates higher exposure.
  • a or “an” entity refers to one or more of that entity; for example, “an antibody,” is understood to represent one or more antibodies.
  • the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
  • Alzheimer's disease or “AD” includes both familial Alzheimer's disease and sporadic Alzheimer's disease.
  • the term “comprising” or “including” is to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps or components, or groups thereof.
  • the term “comprising” or “including” also includes “consisting of”.
  • the variations of the word “comprising”, such as “comprise” and “comprises”, and “including”, such as “include” and “includes”, have correspondingly varied meanings.
  • the term "subject” or “individual” or “patient” may be used interchangeably and refers to any subject, particularly a mammalian subject, e.g., a human patient, for whom diagnosis, prognosis, prevention, or therapy is desired.
  • sample refers to any biological material obtained from a subject or patient.
  • a sample can comprise blood, cerebrospinal fluid ("CSF"), and/or urine.
  • CSF cerebrospinal fluid
  • a sample can comprise whole blood or plasma.
  • a sample can also include a biopsy or tissue sample including neural tissue.
  • a sample can comprise whole cells and/or a lysate of the cells. Blood samples can be collected by methods known in the art.
  • Tau or “tau” refers to the native monomer form of Tau and is also used to generally identify other conformers of Tau, for example, oligomers or aggregates of Tau. It is also used to refer collectively to all types and forms of Tau.
  • “monomeric Tau” or “Tau monomer” refers to completely solubilized Tau proteins without aggregated complexes in aqueous medium.
  • “Aggregated Tau”, “oligomeric Tau” and “Tau oligomer” refers to multiple aggregated monomers of Tau peptides or proteins, or of Tau-like peptides/proteins, or of modified or truncated Tau peptides/proteins or of other derivates of Tau peptides/proteins forming oligomeric or polymeric structures which are insoluble or soluble both in vitro in aqueous medium and in vivo in the mammalian or human body more particularly in the brain, but particularly to multiple aggregated monomers of Tau or of modified or truncated Tau peptides/proteins or of derivatives thereof, which are insoluble or soluble in the mammalian or human body more particularly in the brain, respectively.
  • polynucleotide or “polynucleic acid” may be used interchangeably and refers to a polymer comprising multiple nucleotide monomers (e.g., ribonucleotide monomers or deoxyribonucleotide monomers).
  • the term includes, for example, genomic DNA, cDNA, RNA and DNA-RNA hybrid molecules.
  • Polynucleic acid molecules/polynucleotides can be naturally occurring, recombinant, or synthetic.
  • polynucleotide is intended to encompass a singular nucleic acid as well as plural nucleic acids, and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA (pDNA), or PCR amplicons.
  • a polynucleotide can comprise a conventional phosphodiester bond or a non- conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)).
  • isolated polynucleotide is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment.
  • the term "gene” refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises coding sequences necessary for the production of an RNA, or of a polypeptide or its precursor.
  • a functional polypeptide may be encoded by a full-length coding sequence or by any portion of the coding sequence as long as the desired activity or functional properties (e.g., enzymatic activity, ligand binding, signal transduction, etc.) of the polypeptide are retained.
  • portion when used in reference to a gene may refer to fragments of that gene. The fragments may range in size from a few nucleotides to the entire gene sequence minus one nucleotide.
  • the term "gene” also encompasses the coding regions of a structural gene and includes sequences located adjacent to the coding region on both the 5' and 3' ends, e.g. , for a distance of about 1 kb on either end, such that the gene corresponds to the length of the full-length mRNA (e.g., comprising coding, regulatory, structural and other sequences).
  • the sequences that are located 5' of the coding region and that are present on the mRNA are referred to as 5' non-translated or untranslated sequences.
  • the sequences that are located 3' or downstream of the coding region and that are present on the mRNA are referred to as 3' nontranslated or 3' untranslated sequences.
  • genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed "introns” or “intervening regions” or “intervening sequences.”
  • Introns are segments of a gene that are transcribed into nuclear RNA; introns may contain regulatory elements such as enhancers. Introns are removed or “spliced out” from the nuclear or primary transcript; introns therefore are absent in the mRNA transcript.
  • the mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.
  • nucleic acid detection assay refers to any method of determining the nucleotide composition of a nucleic acid of interest.
  • Nucleic acid detection assay may include, but are not limited to, DNA/RNA sequencing methods, probe hybridization methods, structure specific cleavage assays (e.g., the INVADER assay, Hologic, Inc.; and are described, e.g., in U.S. Pat. Nos. 5846717, 5985557, 5994069, 6001567, 6090543, and 6872816; Lyamichev et al., Nat. Biotech., 17:292 (1999), Hall et al., Proc. Natl. Acad. Sci.
  • enzyme mismatch cleavage methods e.g., Variagenics, U.S. Pat. Nos. 6110684, 5958692, 5851770
  • polymerase chain reaction PCR
  • branched hybridization methods e.g., Chiron, U.S. Pat. Nos. 5849481 , 5710264, 5124246, and 5624802
  • rolling circle replication e.g., U.S. Pat. Nos. 6210884, 6183960 and 6235502
  • NASBA e.g., U.S. Pat. No. 5,409,818)
  • molecular beacon technology e.g., U.S. Pat. No.
  • the term “amplification of nucleic acids” generally refers to the production of multiple copies of a polynucleotide, or a portion of the polynucleotide, typically starting from a small amount of the polynucleotide (e.g., a single polynucleotide molecule, 10 to 100 copies of a polynucleotide molecule, which may or may not be exactly the same), where the amplification products or amplicons are generally detectable.
  • Amplification of polynucleotides encompasses a variety of chemical and enzymatic processes.
  • the generation of multiple DNA copies from one or a few copies of a target or template DNA molecule during a polymerase chain reaction (PCR) or a ligase chain reaction (LCR; see, e.g., U.S. Pat. No. 5494810) are forms of amplification.
  • Additional types of amplification may include, but are not limited to, allele-specific PCR (see, e.g., U.S. Pat. No. 5639611), assembly PCR (see, e.g., U.S. Pat. No. 5965408), helicase-dependent amplification (see, e.g., U.S. Pat. No.
  • Hot-start PCR see, e.g., U.S. Pat. Nos. 5773258 and 5338671
  • intersequence-specific PCR see, e.g., Triglia, et al., Nucleic Acids Res., 16:8186 (1988)
  • ligation-mediated PCR see, e.g., Guilfoyle, R. et al., Nucleic Acids Res., 25:1854-1858 (1997); U.S. Pat. No. 5508169
  • methylation-specific PCR see, e.g., Herman, et al., Proc. Natl. Acad. Sci.
  • miniprimer PCR multiplex ligation-dependent probe amplification (see, e.g., Schouten, et al. Nucleic Acids Res. 30 (12):e57 (2002)), multiplex PCR (see, e.g., Chamberlain et al., Nucleic Acids Res. 16(23) 11141-11156 (1998); Ballabio, et al., Hum. Genet. 84(6) 571-573 (1990); Hayden, etal., BMC Genom. 9:80 (2008), nested PCR, overlapextension PCR (see, e.g., Higuchi, et al., Nucleic Acids Res.
  • Polynucleotide amplification also can be accomplished using digital PCR (see, e.g., Kalinina, et al., Nucleic Acids Res. 25; 1999-2004, (1997); Vogelstein B. and Kinzler K. W., Proc. Natl. Acad. Sci. USA. 96; 9236-41 , (1999); International Patent Publication No. W005023091A2; US Patent Application Publication No. 20070202525).
  • oligo or "oligonucleotide” refers to short length single or double stranded polydeoxynucleotides which are chemically synthesized and then purified by known methods. Oligos may be used in nucleic acid amplification techniques where such oligos may alternatively be termed as primers. of the invention
  • polypeptide is intended to encompass a singular "polypeptide” as well as plural “polypeptides,” and refers to a polymer molecule of at least two amino acids (monomers) covalently linked by amide bonds (also known as peptide bonds).
  • polypeptide refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product.
  • polypeptides include peptides, dipeptides, tripeptides, oligopeptides, "protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of “polypeptide,” and the term “polypeptide” can be used instead of, or interchangeably with any of these terms.
  • the “polypeptide” is also intended to the product modified after the expression for referring to polypeptide, including but not limited to, glycosylation, acetylation, phosphorylation, amidation, by known protection/end- capping group derivatization, proteolytic cleavage or pass through non-naturally occurring amino.
  • polypeptide also encompasses a naturally-occurring as well as artificial (e.g., engineered or variant) full-length protein as well as a functional fragment of the protein.
  • the term “functional fragment” refers to a portion of a polypeptide/protein that retains some or all of the activity or function (e.g., biological activity or function, such as enzymatic activity or antigen-binding function) of the full-length polypeptide/protein, such as, e.g., the ability to bind to a specific epitope.
  • the functional fragment can be any size, provided that the fragment retains the activity/functionality of the full-length protein.
  • antibody is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, chimeric, humanized antibodies, multi-specific antibodies (e.g., bispecific antibodies), and antigenbinding fragments thereof and similar antigen-binding molecules so long as thy exhibit the desired antigen-binding activity.
  • the "antibody” as used herein can be any type (IgG, IgM, IgD, IgE, IgA and IgY) or the immunoglobulin molecules of classification (lgG1 , lgG2, lgG3, I gG4, lgA1 and lgA2) or sub-class according to immunoglobulin (Ig) of the present invention.
  • Tau11 i antibody As used herein, the terms “Tau11 i antibody”, “Tau11i-specific antibody”, “anti-Tau11i antibody”, and “an antibody that specifically recognises Tau11 i” refer to an antibody that is capable of binding Tau11 i with sufficient affinity and specificity to be useful as a diagnostic and/or therapeutic agent in targeting Tau11 i. In some embodiments, the extent of binding of an anti-Tau11 i antibody to an unrelated, non-Tau11i protein is less than about 10% of the binding of the antibody to Tau11i as measured by known methods in the art.
  • an antibody that binds to Tau11i has a dissociation constant (KD) of ⁇ 1 pM, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM.
  • KD dissociation constant
  • chimeric antibody refers to at least one antibody molecule in which the amino acid sequence in the constant regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
  • humanized antibody refers to at least one antibody molecule in which the amino acid sequence within the variable and constant regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
  • an "antigen-binding fragment” or “antibody fragment” of an antibody refers to an incomplete or isolated portion of the full sequence of the antibody which retains the antigen binding function of the parent antibody.
  • antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multi-specific antibodies formed from antibody fragments. Fragments of the 8A12 antibody are encompassed by the invention so long as they retain the desired affinity of the full-length antibody. In particular, it may be shorter by at least one amino acid.
  • an “antibody that binds to the same epitope” as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by 50% or more.
  • isolated is herein defined as a biological component (such as a nucleic acid, peptide or protein) that has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extra-chromosomal DNA and RNA, and proteins.
  • Nucleic acids, peptides and proteins which have been isolated thus include nucleic acids and proteins purified by standard purification methods.
  • the term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
  • the present disclosure is based, in part, on the inventors’ discovery that the retention of intron 11 in the Tau gene introduces a premature stop codon, which results in the production of truncated Tau11i polypeptide.
  • the inventors have successfully identified that this new C-terminal truncated Tau-11i species not only exhibits biochemical properties that resemble pathological Tau, it is also more abundantly found in AD brain tissues tested. Without being bound by theory, it is believed that the dysregulation of RNA splicing and intron retention is a hallmark of AD pathogenesis, suggesting that Tau11i might be downstream of these events.
  • the polypeptides disclosed herein therefore may advantageously serve as a biomarker and a useful diagnostic for AD.
  • an isolated or recombinant polypeptide comprising the following amino acid sequence:
  • the isolated or recombinant Tau11 i-derived polypeptide may consist of amino acid sequence set forth in (1) or (2) with a cysteine residue conjugated to an end.
  • the addition of terminal cysteine to the polypeptide sequence may allow its conjugation to carrier proteins, which helps in antibody production.
  • the isolated or recombinant Tau11i-derived polypeptide may consist of the amino acid sequence HKPGSPVEGEGWDGRVQGV-C set forth in SEQ ID NO: 1 or C-SPVEGEGWDGRVQG set forth in SEQ ID NO: 2.
  • the amino acid sequence of the isolated or recombinant polypeptide may comprise or consist of an amino acid sequence resulting from substitution, deletion, addition or insertion of one or two or more amino acids in the amino acid sequence set forth in SEQ ID NO: 1 , SEQ ID NO: 2 or SEQ ID NO: 13. In some embodiments, the amino acid sequence may consist of SEQ ID NO: 13.
  • the amino acid sequences of the polypeptides disclosed herein may be sufficiently varied so long as the polypeptides maintain their functionality and can exhibit the required activity (for example, the polypeptide serving as a binding epitope for Tau11i-specific antibodies and/or aptamers). Table 1. Amino acid sequences of polypeptides of the disclosure and nucleotide sequences of primers used in the present invention
  • polypeptides of the present invention can be produced according to a known peptide synthesis method, for example, solid phase synthesis process, liquid phase synthesis process and the like.
  • the obtained polypeptides can be purified and isolated by a known purification method, for example, solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, a combination of these, and the like.
  • the polypeptides of the present invention can also be produced by culturing a transformant containing the nucleic acids encoding the polypeptides and separating and purifying the polypeptides from the resulting culture.
  • the polypeptides disclosed herein are suitable for or can be used for preparing epitopespecific antibodies, mapping antibody epitopes and enzyme binding sites and designing novel enzymes, drugs and vaccines.
  • an antibody or antigen-binding fragment thereof capable of binding to the polypeptides of the present disclosure.
  • the antibody or the antigen-binding fragment of the present invention is capable of binding to Tau11 i-specific epitopes with sufficient affinity and specificity such that the antibody or the antigen-binding fragment thereof is useful as a diagnostic and/or therapeutic agent in targeting Tail'l l i.
  • the antibody described herein may be a polyclonal antibody or a monoclonal antibody.
  • the isolated antibody or antigen-binding fragment thereof specifically binds to the polypeptide comprising the amino acid sequence HKPGSPVEGEGWDGRVQGV (SEQ ID NO: 14) or antigenic fragment thereof, or the amino acid sequence SPVEGEGWDGRVQG, (SEQ ID NO: 15), or an amino acid sequence resulting from substitution, deletion, addition or insertion of one or two or more amino acids in the amino acid sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15.
  • the isolated antibody or antigen-binding fragment thereof specifically binds to the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO: 2.
  • the isolated antibody or antigen-binding fragment thereof specifically binds to the polypeptide consisting of an amino acid sequence resulting from substitution, deletion, addition or insertion of one or two or more amino acids in the amino acid sequence set forth in SEQ ID NO: 1 , SEQ ID NO: 2 or SEQ ID NO: 13.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising complementarity determining regions HCDR1 having an amino acid sequence set forth in SEQ ID NO: 31 , HCDR2 having an amino acid sequence set forth in SEQ ID NO: 32, and HCDR3 having an amino acid sequence set forth in SEQ ID NO: 33, and/or a light chain variable region comprising complementarity determining regions LCDR1 having an amino acid sequence set forth in SEQ ID NO: 36, LCDR2 having an amino acid sequence set forth in SEQ ID NO:37, and LCDR3 having an amino acid sequence set forth in SEQ ID NO: 38.
  • the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence shown as residues 1 to 132 of SEQ ID NO: 34 and a heavy chain variable region comprising the amino acid sequence shown as residues 1 to 136 of SEQ ID NO: 29.
  • the method may comprise (1) injecting a non-human mammal with a Tau11 i-derived polypeptide of the present disclosure to form a B cell specific for Tau11i; (2) selecting a B cell specific to the Tau11i; (3) fusing the selected B cell with an immortalized cell to produce a hybridoma, wherein the hybridoma is capable of producing an antibody or a fragment thereof specific for the Tau11i; and (4) optionally isolating the antibody from the hybridoma and sequencing the variable heavy and light chains.
  • the non-human mammal may be a mouse, rabbit, cow, goat or sheep.
  • polynucleic acid molecule encoding the polypeptide, the antibody or antigen binding fragment thereof as disclosed herein.
  • the polynucleic acid may be a cDNA.
  • oligonucleotides for amplifying a polynucleotide sequence spanning the junction of exon 11 and intron 11 of a Tau gene.
  • the oligonucleotides consist of the nucleotide sequences set forth in SEQ ID NO: 7 and/or SEQ ID NO: 8.
  • a method for detecting the polypeptide of the present disclosure comprises: a step of contacting a sample suspected of comprising the polypeptide disclosed herein with the antibody or antigen-binding fragment of the present disclosure.
  • the method is preferably in vitro.
  • a method for diagnosing Alzheimer's disease or identifying a subject at risk of developing Alzheimer's disease comprising: contacting a sample derived from the subject with the antibody or antigen binding fragment thereof as described herein.
  • the antibody or antigen-binding fragment of the present disclosure may bind specifically to the polypeptides disclosed herein to form an antibody-polypeptide complex.
  • the methods disclosed herein may further comprise the step of detecting the antibody-polypeptide complex, wherein the presence of said antibody-polypeptide complex may be used to inform the diagnosis and/or assessment of the risk of said subject in developing AD.
  • a method for diagnosing Alzheimer's disease or identifying a subject at risk of developing Alzheimer's disease comprising: conducting a nucleic acid detection assay to assess the abundance of intron 11 retaining mRNA in a sample from the subject.
  • the nucleic acid detection assay may comprise amplifying a polynucleotide sequence spanning the junction of exon 11 and intron 11 of a Tau gene.
  • the amplification involves using oligonucleotides having the nucleotide sequences set forth in SEQ ID NOs: 7 and/or 8.
  • the abundance of intron 11 retaining mRNA in said sample may be compared with the abundance in normal controls to establish the diagnosis or risk factor of the subject in developing AD.
  • the presence of Tau11i polypeptides are typically significantly higher in AD patients.
  • a standard value for normals with appropriate error bars may be generated by averaging the results from a statistically meaningful sample of individuals known not to be afflicted by this condition. Accordingly, determining and/or analyzing the degree of difference in abundance of intron 11 retaining mRNA in said sample against a control may thus be used to inform the diagnosis and/or assessment of the risk of said subject in developing AD.
  • the sample may be a biological fluid or tissue.
  • a biological fluid may include but not limited to, blood and fractions thereof such as plasma or serum, cerebrospinal fluid, urine, lymphatic fluids, synovial fluid, pericardial fluid, peritoneal fluid, amniotic fluid, saliva, nasal fluid etc.
  • the sample may be brain tissue, cerebrospinal fluid, blood plasma, saliva, lacrimal fluid or urine.
  • a method for screening a candidate therapeutic or prophylactic drug for Alzheimer's disease comprising using any of the following as an index: (1) reduction of the amount of Tau protein in the body of an Alzheimer's disease subject or a subject at risk of developing Alzheimer's disease, or (2) inhibition of production of Tau protein in the body of an Alzheimer's disease subject or a subject at risk of developing Alzheimer's disease; wherein the Tau protein comprises the amino acid sequence of the polypeptide as disclosed herein.
  • kits comprising the antibody or antigen-binding fragment of the present invention.
  • the kit may further comprise the polypeptide of the present disclosure.
  • the kit as herein described is preferably equipped to conduct the one or more methods as described above.
  • the kit may comprise a reagent to detect the antibody or the antigen-binding fragment of the present invention.
  • the kit may comprise a reagent to detect a complex that may have been formed between the antibody or the antigen-binding fragment thereof with a test sample.
  • Reagents that are suitable for detecting the complex may include reagents that may incorporate a detectable label, such as a fluorophore, radioactive moiety, enzyme, biotin/avidin label, chromophore, chemiluminescent label, or the like.
  • a detectable label such as a fluorophore, radioactive moiety, enzyme, biotin/avidin label, chromophore, chemiluminescent label, or the like.
  • kits may also optionally include other reagents required to conduct a diagnostic or prognostic assay as described herein, such as buffers, salts, enzymes, enzyme co-factors, substrates, detection reagents, and the like.
  • Other components such as buffers and solutions for the isolation and/or treatment of a test sample (e.g. pretreatment reagents), may also be included in the kit.
  • the kit may additionally include one or more other controls.
  • One or more of the components of the kit may be lyophilized and the kit may further comprise reagents suitable for the reconstitution of the lyophilized components.
  • aptamers may be suitably adapted for use in the practice of the present invention.
  • Aptamers are nucleic acid probes that form specific three dimensional formations, and are capable of binding to defined targets such as polypeptides with high affinity and specificity.
  • methods for screening epitopespecific aptamers are known in the art, for example, via the systematic evolution of ligands by exponential enrichment (SELEX) process (for example, as described in Teng et al., J. Am. Chem. Soc., 140(43), 14314-14323 (2016), incorporated herein by reference).
  • a method of screening and/or selecting a Tau11 i epitope-specific aptamer comprises: (a) conducting systematic evolution of ligands by exponential enrichment (SELEX) technique to identify Tau11 i epitope-specific aptamers, wherein the SELEX technique comprises providing a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs. 1 , 2, 13, 14 or 15 as a target ligand.
  • SELEX systematic evolution of ligands by exponential enrichment
  • an aptamer capable of binding to a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs. 1 , 2, 13, 14 or 15.
  • the aptamer may be a DNA aptamer or RNA aptamer.
  • a method for diagnosing Alzheimer's disease or identifying a subject at risk of developing Alzheimer's disease comprising: contacting a sample derived from the subject with the Tau11 i epitope-specific aptamer as provided herein.
  • Intron 11 of the Tau (MAPT) gene (MAPT/ENSG00000186868/clean/17:46014324-46018617: +) investigated in this study has a minimal splicing sequencing depth of greater than five reads.
  • Generalized Linear Model from DESeq2 software (Love et al., Genome Biol. 15(12), 1-21. (2014)) was applied to measure the differential IR ratio between control subjects and AD patients, where p-value ⁇ 0.05 was considered significant.
  • a normalized IR ratio from an individual human subject determined from DESeq2 was used to generate a dot plot with “ggplot” function in “ggplot2” package software (https://ggplot2.tidyverse.org).
  • Counts for Tau gene were generated using featureCounts (v.2.0.1.) software and differential expression was calculated by DESeq2 (v.1.30.1) software.
  • Human NSC (XCL-1 , SC-001-1 V) were purchased from XCell Science Inc. and differentiated according to manufacturer’s protocol. Brain total RNA, protein lysates and frozen tissue sections were purchased from MYBIOSOURCE and BIOCHAIN. Parietal lobe (BA40) samples C6, C7, A6, and A7 were previously described (e.g., Chua, etal., Neurochem. Int. 152, 105251 (2022)).
  • Total protein lysates from different brain regions were purchased from:
  • C1 Adult Normal, Brain, Temporal Lobe Lysate (MBS657128) - male, 77 years old.
  • A1 Adult AD, Brain, Temporal Lobe Lysate (MBS657032) - male, 87 years old.
  • C4 Adult Normal, Brain, Frontal Lobe Lysate (MBS657031) - male, 60 years old.
  • A4 Adult AD, Brain, Frontal Lobe Lysate (MBS657343) - female, 85 years old.
  • C5 Adult Normal, Brain, Hippocampus Lysate (M BS657489) - male, 60 years old.
  • A5 Adult AD, Brain, Hippocampus Lysate (MBS657034) - female, 93 years old.
  • C8 Adult Normal, Brain, Temporal Lobe Lysate (P1234078) - female, 70 years old.
  • A1 Adult AD, Brain, Temporal Lobe Lysate (P1236978Alz) - male, 87 years old.
  • C9 Adult Normal, Brain, Frontal Lobe Lysate (P1234051) - male, 82 years old.
  • C2 Adult Normal, Brain, Amygdala Lysate (P1234036) - male, 22 years old.
  • A2 Adult AD, Brain, Amygdala Lysate (P1236036Alz) - female, 65 years old.
  • C3 Adult Normal, Brain, Parietal lobe Lysate (P1234066) - male, 66 years old.
  • A3 Adult AD, Brain, Parietal lobe Lysate (P1236066Alz) - male, 73 years old.
  • C6 Adult Normal, Brain, Parietal Lobe Lysate (283/96) - male, 77 years old.
  • A6 Adult AD, Brain, Parietal Lobe Lysate (120/09) - female, 85 years old.
  • C7 Adult Normal, Brain, Parietal lobe Lysate (308/09) - male, 66 years old.
  • A7 Adult AD, Brain, Parietal lobe Lysate (350/09) - female, 98 years old.
  • Frozen tissue sections were purchased from BIOCHAIN (BioChain Institute Inc., CA, USA) .
  • C10 Adult Normal, Brain, Temporal Lobe ((T1234078) - male, 26 years old.
  • A10 Adult AD, Brain, Temporal Lobe (T1236078Alz) - male, 73 years old.
  • RNA from different brain regions were purchased from BIOCHAIN.
  • AD Brain R1236035Alz-50 (Lot#: A507294) - male, 87 years old.
  • HEK 293T cells (Clontech, 632180) were maintained in Dulbecco's modified Eagle's medium (DMEM) GlutaMAXTM (Gibco) supplemented with 10% FBS, 1 x nonessential amino acids (NEAA) and 1.0 mM sodium pyruvate.
  • DMEM Dulbecco's modified Eagle's medium
  • GlutaMAXTM GlutaMAXTM
  • FBS FBS
  • NEAA 1 x nonessential amino acids
  • NSC Human neural stem cells
  • XCL-1 Human neural stem cells
  • SC-001-1 V Human neural stem cells
  • XCell Science Inc. Human neural stem cells
  • matrigel Corning, 354277
  • neural expansion medium [1 :1 ratio of DMEM GlutaMAXTM to Ham's F-12 Nutrient Mix (both Gibco), 1x NEAA, 1x N-2 supplement (Gibco, 17502048), 1x B-27 supplement minus vitamin A (Gibco, 12587010), 0.5X GlutaMAX (Gibco, 35050061), 20 ng/ml human FGF-basic (Gibco, PHG0261), 2 pg/ml Heparin Sulfate (Sigma, H4784)].
  • NSCs Viral infected NSCs were differentiated according to manufacturer's protocols. Briefly, cells were seeded in NSC maintenance media at a density of 0.04 * 106 cells/cm 2 on poly-ornithine (20 pg/mL) & laminin (10 pg/mL)-coated plates (Day 0). Cells were then replenished with neuronal induction media on Day 1 , 3 and 5. On Day 6, neuronal precursor cells formed (NPCs) were harvested by StemProTM AccutaseTM (Gibco, A1110501) and replated on poly-ornithine/laminin-coated wells at 0.04 * 106 cells/cm 2 in neuronal maturation medium. Neurons were cultured for designated duration with media change every alternate day.
  • Lenti-X lentiviral expression system (Clontech) was used to generate stable NSC lines that expressed either full-length (FL) Tau or Tau-11i proteins. Briefly, lentivirus was prepared by transfecting 293T cells with a mixture that contained 7 pg of lentiviral plasmids (pLVX- Flag- Tau-FL or pLVX-Flag-Tau-11 i) and Lenti-X packaging single shot reagent (VSV-G) (Clontech, 631275). Media harvested 24 h and 72 h post transfection was combined and concentrated using Lenti-X concentrator (Clontech, 631231) to produce lentiviral supernatant.
  • Lenti-X concentrator (Clontech, 631231)
  • NSCs were infected with lentiviral supernatant in the presence of polybrene (12 pg/ml). After 48 h of viral transduction, stable clones were selected with 0.1 pg/ml of puromycin. Expression of Tau-FL and Tau-11i in stable lines were validated using western blot and immunofluorescence.
  • Full-length (FL) Tau gene was PCR amplified from VN-Tau plasmid (Addgene 87368) using Xbal-Tau-F and Sall-Tau-R primers and blunt-end cloned into pBlueScript II SK+ vector at EcoRV site to get PBS-Tau-FL. Following Xbal/Sall restriction of PBS-Tau-FL, full-length Tau fragment was cloned into pLVX-Flag-IRES-puro lentiviral vector at Xbal/PspXI sites to get pLVX-Flag-Tau-FL.
  • Sall-Tau-R 5'-AACGTCGACTCACAAACCCTGCTTG-3' SEQ ID NO: 4
  • Sall-Tau-11i-R 5'-GTCGACTCACCAGGACTCCTCC-3' (SEQ ID NO: 6)
  • Two custom anti-Tau11i polyclonal antibodies were generated by GenScript (GenScript Biotech Corporation, NJ, USA) using peptides HKPGSPVEGEGWDGRVQGV-C (SEQ ID NO: 1) and C-SPVEGEGWDGRVQG (SEQ ID NO: 2) as antigens, where C is a conjugated cysteine.
  • a custom anti-Tau11i monoclonal antibody, 8A12 was also generated by GenScript using a peptide with the amino acid sequence set forth in SEQ ID NO: 15 as the antigen.
  • the monoclonal antibody 8A12 comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 29 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 34 (Table 1).
  • the monoclonal antibody 8A12 comprises a heavy chain variable region comprising complementarity determining regions HCDR1 having an amino acid sequence set forth in SEQ ID NO: 31 , HCDR2 having an amino acid sequence set forth in SEQ ID NO: 32, and HCDR3 having an amino acid sequence set forth in SEQ ID NO: 33, and a light chain variable region comprising complementarity determining regions LCDR1 having an amino acid sequence set forth in SEQ ID NO: 36, LCDR2 having an amino acid sequence set forth in SEQ ID NO: 37, and LCDR3 having an amino acid sequence set forth in SEQ ID NO: 38 (Table 1).
  • Sarkosyl soluble and insoluble fractions were prepared accordingly published protocol (Smith et al., Neuropathol. 46(7), 641-653 (2020)). Tau expressing 293T cells and NPC stable lines were resuspended with 320 pl per 3e6 cells of low salt buffer (50 mM HEPES pH 7.6, 250 mM sucrose, 1 mM EDTA pH 8.0) with protease inhibitors cocktail (Roche), followed by addition of 40 pl of 5 M NaCI and 40 pl of 10 % sarkosyl.
  • low salt buffer 50 mM HEPES pH 7.6, 250 mM sucrose, 1 mM EDTA pH 8.0
  • protease inhibitors cocktail (Roche)
  • RNA of different brain tissues from healthy control subjects and AD patients were purchased from Biochain.
  • High-capacity cDNA reverse transcription kit (Applied Biosystems) was used to generate cDNA libraries from 1 pg of total RNA pre-treated with DNase I (Thermo Scientific).
  • Real-time quantitative PCR was carried out in triplicates (6 pl/reaction) with 0.2 pM of primers and 2X SYBR green master mix (Thermo Scientific) using 7900HT Fast Real-Time PCR machine (Applied Biosystems).
  • Relative level of Tau-11i was calculated using 2' ACT method where threshold cycle (CT) values were normalized to either Tau exon1/exon2 (Tau ex-1-F/ex-2-R) or Gapdh expression.
  • CT threshold cycle
  • Gapd/7-F 5 -CAATGACCCCTTCATTGACC-3' (SEQ ID NO: 11)
  • Gapdh-R 5'-GACAAGCTTCCCGTTCTCAG-3' (SEQ ID NO: 12)
  • RNA from different brain regions were purchased from Biochain.
  • AD Brain R1236035Alz-50 (Lot#: A507294) - male, 87 years old.
  • Flag-Tau11 i protein was specifically recognized by two different Tau11 i antibodies (Tau11i-A and Tau11 i-B) but not Tau46 (FIG. 2C).
  • Tau11 i also consistently oligomerizes to form HMW species in NSC, albeit at a lower level, as detected by Tau4R and pT181 antibodies (FIGS. 2C and 5B).
  • As formation of HMW species is one of the characteristics of pathological Tau in AD, other biochemical properties of Tau11 i were further examined in these cells.
  • Tau11i has lower binding efficiency to microtubules.
  • the stability of Tau441 and Tau11 i proteins was next investigated by time-course monitoring and cycloheximide assay of transiently transfected Tau protein.
  • the level of Tau11 i protein on Day 7 was around ⁇ 5% of that at Day 3 whereas Tau441 level dropped to about 0.4% (FIG. 4B and FIG. 8B).
  • Tau11 i was also more stable and exhibited less protein degradation than Tau441 following 8 h of cycloheximide treatment (FIG. 4C).
  • Tau11i Characterization of Tau11i in different cell types revealed several biochemical properties that resemble pathological Tau (Gotz et al., Annu Rev Pathol, 14, 239-261 (2019); Iqbal et al., Nat. Rev. Neurol., 12(1), 15-27. (2016)).
  • truncated Tau11i oligomerizes to form HMW species and is enriched in Sarkosyl-insoluble fractions (Example 3), has reduced association with microtubule networks in neurons and exhibits lower rate of protein turnover (Example 5).
  • Tau11i protein was significantly elevated in AD tissues as compared to healthy control.
  • endogenous Tau11i is also observed in Sarkosyl-insoluble fraction, consistent with its association with protein aggregates. Examples in this disclosure indicate that Tau11 i is a new biomarker for AD.
  • Antibodies and nuclear acid detection methods demonstrated herein are therefore suitable for use in diagnostic purposes or assessing an individual at risk of developing AD.
  • RNAzol RT RNAzol RT
  • Applied Biosystems was used to generate cDNA libraries from 1 pg of total RNA pre-treated with DNase I (Thermo Scientific). PCR was performed with Phusion DNA polymerase (NEB) with the primer pairs shown in FIG. 9C. Lane 3 of FIG. 9B shows a positive band representing an amplicon spanning the junction of Exon 11 and Intron 11 , which indicates that retention of Tau intron 11 is detectable in human plasma.
  • Tau11 i is also successfully detected in human plasma (FIGS. 10A-B).
  • the fractions pulled down by the Tau11 i-specific antibody, Tau11 i-B was then in-gel trypsin digested and subsequently subjected to LC-MS/MS. Mass spectrometry data confirmed that the pull-down fractions correspond to Tau species.
  • the reference database exclusively includes known variants of Tau.
  • Tau isoform F is Tau441 (441 amino acids) which contains 2N4R; isoform D has 383 amino acids which contains 0N4R.
  • intron 11-retained Tau and Tau11i protein circulating in the bloodstream offers a unique opportunity for early and fast detection of such Tau abnormality.
  • the accessibility of human plasma as a sample source greatly expedites the diagnostic process, eliminating the need for more invasive procedures that often pose risks and discomfort to patients.
  • the identification and quantification of intron 11-retained Tau or Tau11i protein within human plasma can establish a direct link between their levels and the progression of AD pathologies, thereby enabling precise and prompt intervention.
  • Adusumalli S., Ngian, Z. K., Lin, W. Q., Benoukraf, T., & Ong, C. T. (2019). Increased intron retention is a post - transcriptional signature associated with progressive aging and Alzheimer’ s disease. Aging cell, 18(3), e12928.
  • Garcia-Escudero V., Ruiz-Gabarre, D., Gargini, R., Perez, M., Garcia, E., Cuadros, R., Hernandez I.H., Cabrera J.R., Garcia-Escudero R., Lucas J. J., Hernandez F., & Avila, J. (2021).
  • a new non-aggregative splicing isoform of human Tau is decreased in Alzheimer’s disease. Acta Neuropathologica, 142, 159-177.
  • Methylation-specific PCR a novel PCR assay for methylation status of CpG islands. Proceedings of the national academy of sciences, 93(18), 9821-9826.
  • IRFinder assessing the impact of intron retention on mammalian gene expression. Genome biology, 18, 1-11.
  • MAPT expression and splicing is differentially regulated by brain region: relation to genotype and implication for tauopathies. Human molecular genetics, 27(18), 4094-4103.

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Abstract

L'invention concerne l'identification d'un isoforme d'épissage de Tau retenant un intron en tant que nouveau biomarqueur de la maladie d'Alzheimer. L'invention concerne également des polypeptides pour générer des molécules de liaison, telles que des anticorps spécifiques de l'isoforme Tau11i, des oligonucléotides et des anticorps destinés à être utilisés dans des procédés de détection de l'isoforme Tau11i dans un échantillon et des procédés destinés à être utilisés dans le diagnostic de la maladie d'Alzheimer.
PCT/SG2023/050599 2022-09-02 2023-08-31 Agent de diagnostic et procédé pour la maladie d'alzheimer Ceased WO2024049357A1 (fr)

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NGIAN Z.-K: "Truncated Tau caused by intron retention is enriched in Alzheimer's disease cortex and exhibits altered biochemical properties", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 119, no. 37, 6 September 2022 (2022-09-06), pages 1 - 7, XP093147973, ISSN: 0027-8424 *

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