Diagnostic agents and methods for alzheimer's disease

Abstract

The invention relates to identification of an intron retention type Tau splicing isomer as a novel Alzheimer's disease biomarker. Provided herein are polypeptides that generate binding molecules, such as antibodies, oligonucleotides, and antibodies having specificity for a Tau11i isomer, methods for detecting a Tau11i isomer in a sample, and methods for diagnosing Alzheimer's disease.

Description

Technical Field

[0001] The present invention relates to intron-retaining Tau splicing isomers and methods for detecting the same. The present invention also relates to agents comprising oligonucleotides and antibodies and methods for diagnosing Alzheimer's disease. Background Art

[0002] Alzheimer's disease (AD) is a progressive dementia that begins with short-term memory decline and mild learning disabilities and progresses to more severe brain dysfunction, particularly visual-spatial agnosia, conceptual apraxia, constructional apraxia, etc., and ultimately reaches motor impairment and so-called personality disintegration. It is a familial and sporadic neurodegenerative disease that encompasses many risk factors such as genetic makeup and aging. Synaptic and neuronal loss in AD is caused by the interaction between β-amyloid plaque pathology and Tau neurofibrillary tangle pathology and impaired innate clearance pathways. The microtubule-associated protein Tau and its hyperphosphorylated forms form the major components of intracellular neurofibrillary tangles, which are hallmarks of multiple dementias including AD and frontotemporal dementia. This evidence forms the basis of the AD hypothesis, in which intracellular accumulation of Tau leads to microtubule disassembly, dendritic spine atrophy, and axonal degeneration, malfunction of communication between neurons, and cell death. Increasing evidence suggests that epigenetic alterations and abnormal RNA splicing events at multiple AD susceptibility loci during the aging process may render the healthy brain vulnerable to AD onset, and there are reports showing differential intron retention (IR) events in the Tau gene (Adusumalli et al., Aging Cell 18, e12928 (2019); Garcia-Escudero et al., Acta Neuropathol 142, 159-177 (2021); Trabzuni et al., Hum. Mol. Genet. 21, 4094-4103 (2012)); IR is associated with the course of complex diseases such as cancer; but its association with AD has not been explored.

[0003] Therefore, it is necessary to clarify the relationship between AD and intron-retaining Tau protein, identify novel epitopes of Tau pathological species, and provide agents and methods that can be used for diagnosing AD. Summary of the Invention

[0004] 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.

[0005] In a first aspect, there is provided an isolated or recombinant Tau11i-derived polypeptide comprising the following amino acid sequence:

[0006] (1) The amino acid sequence HKPGSPVEGEGWDGRVQGV, as shown in SEQ ID NO: 14 or an antigenic fragment thereof, or

[0007] (2) The amino acid sequence SPVEGEGWDGRVQG, as shown in SEQ ID NO: 15, or

[0008] (3) An amino acid sequence generated by substitution, deletion, addition, or insertion of one or two or more amino acids in the amino acid sequence shown in SEQ ID NO: 14 or SEQ ID NO: 15.

[0009] In some embodiments, the isolated or recombinant Tau11i-derived polypeptide may consist of the amino acid sequence shown in (1) or (2) and have a cysteine residue conjugated to its terminus. Adding a terminal cysteine to the peptide sequence facilitates antibody production by enabling conjugation to a carrier protein. In some embodiments, the isolated or recombinant Tau11i-derived polypeptide may consist of the amino acid sequence HKPGSPVEGEGWDGRVQGV-C shown in SEQ ID NO: 1 or the amino acid sequence C-SPVEGEGWDGRVQG shown in SEQ ID NO: 2.

[0010] In a second aspect, there is provided an isolated antibody or an antigen-binding fragment thereof that specifically binds to an isolated or recombinant Tau11i-derived polypeptide comprising: the amino acid sequence shown in SEQ ID NO: 14 or an antigenic fragment thereof, or SEQ ID NO: 15, or an amino acid sequence generated by substitution, deletion, addition, or insertion of one or two or more amino acids in the amino acid sequence shown in SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments, the polypeptide to which the antibody or its antigen-binding fragment binds consists of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0011] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region, the heavy chain variable region comprising the amino acid sequence as shown in residues 1 to 136 of SEQ ID NO:29, and the light chain variable region comprising the amino acid sequence as shown in residues 1 to 132 of SEQ ID NO:34. In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region, the heavy chain variable region comprising the following complementarity-determining regions: HCDR1 having the amino acid sequence as shown in SEQ ID NO:31, HCDR2 having the amino acid sequence as shown in SEQ ID NO:32, and HCDR3 having the amino acid sequence as shown in SEQ ID NO:33, and the light chain variable region comprising the following complementarity-determining regions: LCDR1 having the amino acid sequence as shown in SEQ ID NO:36, LCDR2 having the amino acid sequence as shown in SEQ ID NO:37, and LCDR3 having the amino acid sequence as shown in SEQ ID NO:38.

[0012] In a third aspect, oligonucleotides are provided for amplifying a polynucleotide sequence spanning the exon 11 and intron 11 junction of the Tau gene. In some embodiments, the oligonucleotides consist of the nucleotide sequences as shown in SEQ ID NO:7 and SEQ ID NO:8.

[0013] In a fourth aspect, a kit is provided which comprises the antibody or antigen-binding fragment thereof provided in the second aspect. In some embodiments, the kit further comprises the polypeptide of the first aspect.

[0014] In a fifth aspect, polynucleotide molecules are provided that encode the polypeptide according to the first aspect or the antibody or antigen-binding fragment thereof according to the second aspect. In some embodiments, the polynucleotide molecule is cDNA.

[0015] In a sixth aspect, a method for detecting the polypeptide according to the first aspect is provided, wherein the method comprises contacting a sample suspected of comprising the polypeptide with the antibody or antigen-binding fragment according to the second aspect. Preferably, the method is carried out in vitro.

[0016] In a seventh aspect, a method for diagnosing Alzheimer's disease or identifying a subject at risk of developing Alzheimer's disease is provided, wherein the method comprises:

[0017] (a) contacting a sample derived from the subject with the antibody or antigen-binding fragment thereof according to the second aspect; or

[0018] (b) performing a nucleic acid detection assay to evaluate the abundance of intron 11-retained mRNA in a sample derived from the subject; or

[0019] (c) Contacting a sample derived from the subject with a Tau11i epitope-specific aptamer that specifically binds to a polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO: 1, 2, 13, 14, or 15.

[0020] In some embodiments, the sample is a brain tissue. In some embodiments, the sample is cerebrospinal fluid (CSF). In some embodiments, the sample is plasma. In other embodiments, the sample is saliva, tears, or urine. In some embodiments, the nucleic acid detection assay comprises amplifying a polynucleotide sequence spanning the exon 11 and intron 11 junction of the Tau gene. In other embodiments, the amplification comprises using oligonucleotides having the nucleotide sequences shown in SEQ ID NO: 7 and 8. In other embodiments, the nucleic acid detection assay is a PCR-based assay.

[0021] In an eighth aspect, a method for screening candidate therapeutic or prophylactic drugs for Alzheimer's disease is provided, wherein the method comprises using any one of the following as an indicator: (a) reducing the amount of a polypeptide containing Tau (such as Tau11i described below) in a subject with Alzheimer's disease or at risk of developing Alzheimer's disease, or (b) inhibiting the production of a polypeptide containing Tau (such as Tau11i described below) in a subject with Alzheimer's disease or at risk of developing Alzheimer's disease; wherein the Tau protein comprises the amino acid sequence of the polypeptide according to the first aspect.

[0022] In a ninth aspect, a method for preparing a Tau11i-specific antibody is provided, wherein the method comprises: (a) injecting the polypeptide according to the first aspect into an animal to induce an immune response against the polypeptide; (b) isolating the antibody that binds to the polypeptide; (c) selecting the antibody of (b) that binds to Tau11i but not to full-length Tau, thereby generating a Tau11i-specific antibody.

[0023] In a tenth aspect, a method for screening and / or selecting a Tau11i epitope-specific aptamer is provided, the method comprising: (a) performing systematic evolution of ligands by exponential enrichment (SELEX) technology to identify a Tau11i epitope-specific aptamer, wherein the SELEX technology comprises providing a polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO: 1, 2, 13, 14, or 15 as a target ligand.

[0024] In an eleventh aspect, an aptamer is provided that is capable of binding to a polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO: 1, 2, 13, 14, or 15. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings illustrate the disclosed embodiments and are used to explain the principles of the disclosed embodiments. However, it should be understood that the design drawings are for illustrative purposes only and not a definition of the limitations of the present invention.

[0026] Figure 1 An increase in the retention of intron 11 of the Tau gene in AD is shown. (A) Intron 11 contains a premature termination codon (line 2: gagtcctgg tga ggc…), and its typical polyadenylation site is located 1 kb downstream. The arrows indicate the qPCR primers. (B) Integrative Genomics Viewer of intron 11 from control (top two rows) and AD (bottom two rows) patients. (C) Left: Statistically significantly higher intron retention (IR) was observed only in the female AD dorsolateral prefrontal cortex (n = 34; right column) compared to controls (CT, n = 32; left column). Each point represents a single normalized IR ratio. Right: There was no difference in the expression of the Tau gene between the CT (left column) and AD (right column) cohorts. (D) qPCR validation of intron 11 (relative to Tau exon 1 / exon 2) and Tau expression (exon 1 / exon 2 and exon 5 / exon 6 relative to GAPDH) in three different pairs of brain tissues from control (left column) and AD (right column) cohorts. “FL” and “TL” represent frontal lobe and temporal lobe, respectively. Data are represented as the mean ± SD of triplicates, and p-values were calculated by two-tailed t-test.

[0027] Figure 2 It is shown that truncated Tau11i oligomerizes and is present in the Sarkosyl-insoluble fraction. (A) Schematic diagrams of Flag-tagged full-length and truncated Tau proteins with specific antibody recognition sites. Amino acids encoded by intron 11 are shown in light gray text. pT181: phosphorylated Tau (T181), Tau4R: 275-291, Tau46: 404-421, AT8: phosphorylated Tau (S202 / T205). (B-C) Immunoblotting and quantification of Tau11i HMW species in (B) 293 cells transfected with Tau441 (left well) or Tau11i (right well); and (C) neural stem cells (NSCs) (left lane) expressing Tau441 (middle lane) or Tau11i (right lane) proteins. Data are represented as the mean ± SEM. (D) Immunoblotting shows an increase in the Sarkosyl-insoluble fraction of Tau11i in 293T cells (n = 2; top blot, right lane) and day 44 neurons (bottom blot) (n = 3 each) expressing Flag-Tau441 or Flag-Tau11i. Quantification of the relative levels of Sarkosyl-insoluble Tau to total lysate is represented as the mean ± SD. All p-values were calculated by two-tailed t-test.

[0028] Figure 3 Elevated Tau11i levels are shown in different AD brains. (A) Immunoblots of the temporal lobe, amygdala (AMYDG.), parietal lobe, frontal lobe, and hippocampus of control ("C" lane) and AD ("A" lane) show enrichment of Tau11i (∼55 kDa white arrow) in AD samples. Samples were also immunoblotted with AT8 and actin antibodies. Each number represents a unique human subject. Seven pairs of control and AD samples were quantified, and the data are presented as mean ± SD. The Tau11i level in the control of each brain region was set to 1. Sample quantification is underlined. p values were calculated by paired two-tailed t tests. (B) Immunoblots show that Tau11i is present in the sarkosyl-insoluble fraction (white arrow) of AD hippocampus and temporal lobe. 10% of the total lysate (Lys), 15% of the soluble fraction (Sol.), and 50% of the sarkosyl-insoluble fraction (Insol.) were loaded. "Tech Rep" represents technical replicates. (C) Immunofluorescence shows weak co-localization of Tau11i aggregates with Tau4R fibrils in the AD temporal lobe. Scale bar represents 20 μm. Each circle represents a field of view. Data are presented as mean ± SD, and p values were calculated by two-tailed t tests.

[0029] Figure 4 It is shown that the Tau11i protein binds poorly to the microtubule network and is more stable. (A) Images of day 21 and day 44 neurons expressing Tau441 or Tau11i stained with α-tubulin and Flag antibodies. Scale bar represents 50 μm. Quantification shows lower co-localization of Tau11i with α-tubulin compared to Tau441. Each circle represents a field of view, and solid circles are from the second biological replicate. Data are presented as mean ± SD. (B) Immunoblots of 293T cells harvested on different days "D" after transfection using Flag and actin antibodies. Quantification of Tau levels (D7 relative to D3; right bar for Tau11i) is presented as mean ± SEM (n = 5). (C) Immunoblots of Tau levels after treating 293T cells with cycloheximide (CHX) on day 5 after transfection. "Rep" represents replicates. Quantification of Tau levels (8 h relative to 0 h) is presented as mean ± SD, where each circle represents a replicate (n = 12; right for Tau11i). p values were calculated by two-tailed t tests.

[0030] Figure 5Depicts the retention of intron 11 of the Tau gene in other neurodegenerations and the characterization of Tau11i protein. (A) Dot plot of the normalized intron retention (IR) ratio in PD and PSP cohorts. Each dot represents a single value. The CT column is the healthy control, and the PD or PSP column is labeled as diseased patients. Data are presented as mean ± SD, and p-values were calculated by two-tailed t-test. Related to Figure 1 . (B) Immunoblots of 293T cells (top) and NSCs (bottom) expressing Flag-Tau441 (left well) or Flag-Tau11i (right well). HMW species are marked with white arrows. "Rep" represents replicates. Related to Figure 2 .

[0031] Figure 6 Depicts immunofluorescence results showing higher levels of Tau11i in the AD temporal lobe (middle inset). (A) Images and quantification of Tau11i on DAPI-positive cells are presented as mean ± SD, where each circle represents a field of view. P-values were calculated by two-tailed t-test. (B) Immunoblots of control "C8" and AD "A1" temporal lobes. Related to Figure 3 C.

[0032] Figure 7 Shows lower co-localization levels of Tau11i with α-tubulin compared to Tau441 in mature neurons. Representative images from different biological replicates of neurons at (A) day 21 and (B) day 44. Scale bar is 100 μm. Quantification of Flag-Tau on α-tubulin is presented as mean ± SD, where each circle represents a field of view. P-values were calculated by two-tailed t-test. "Rep" represents replicates. Related to Figure 4 A.

[0033] Figure 8 Shows that Tau11i has weaker co-localization signals with α-tubulin and higher protein stability in mature neurons compared to Tau441. (A) Representative images of two biological replicates of day 44 neurons expressing Flag-Tau441 or Flag-Tau11i. Scale bar is 100 μm. Quantification of Flag-Tau on α-tubulin is presented as mean ± SD, where each circle represents a field of view. P-values were calculated by two-tailed t-test. Related to Figure 4 A. "Rep" represents replicates. (B) Representative immunoblots of 293T cells harvested at different days "D" after transfection with Flag and actin antibodies. Related to Figure 4 B.

[0034] Figure 9Detection of Tau intron 11 retention in total RNA extracted from human plasma is shown. (A) Schematic of the experiment. Total RNA was extracted from 0.5 ml of control human plasma (i-DNA Biotech) using RNAzol (Sigma). After treatment with DNase I to remove genomic DNA, reverse transcription was performed using random hexamers. The cDNA was subjected to PCR and analyzed by separation on an agarose gel. (B) The Tau gene was amplified using different primers and the intron retention events previously identified in AD brains were detected. The HBB gene is highly expressed, so we used it as a positive control and tested for the presence of genomic contamination. HADC2 is not present in plasma and was used as a negative control. Lane 3 shows the amplification of Tau intron 11 in plasma RNA.

[0035] Figure 10 Detection of Tau11i in human plasma by the anti-Tau11i antibody is shown. (A) 293 cells or 293 cells transfected with Flag-Tau11i were lysed and Co-IP was performed with a rabbit anti-Tau11i antibody generated from an antigenic peptide consisting of the amino acid sequence shown in SEQ ID NO: 15. The Co-IP beads were boiled and immunoblotted with a Flag antibody. The arrow shows the immunoprecipitated Tau11i protein. (B) Pre-cleared control human plasma (i-DNA Biotech) was subjected to Co-IP with a Tau11i antibody, separated and analyzed by SDS-PAGE, and analyzed by LC-MS / MS after in-gel digestion and column purification.

[0036] Figure 11 Detection of Tau11i by ELISA is depicted. (A) Schematic of Flag-Tau441 and Flag-Tau11i peptides (purified from 293T cells). (B) Optimization of ELISA. In Experiment 1, wells were coated with Tau11i or Tau441 peptide and probed with a Tau11i antibody. In Experiment 2, wells coated with Tau peptide were probed with Tau11i, IgG, or a phosphorylated Tau181 antibody. In Experiment 3, the Tau11i antibody was coated on the plate and Flag-Tau11i or Flag-Tau441 peptide was added. Detection was performed using an anti-Flag antibody. The results show that the anti-Tau11i antibody is much more specific for Tau11i compared to Tau441, while Tau441 is at approximately the negative control level.

[0037] Figure 12Depicts the detection of Tau11i by monoclonal antibody 8A12. (A) Schematic diagrams of Flag-Tau441 and Flag-Tau11i peptides. (B) Immunoblotting of total lysates of 293 cells and neural stem cells (NSCs) expressing Flag-Tau441 or Flag-Tau11i proteins with Flag or monoclonal Tau11i antibody (8A12). PICO indicates a lower exposure, while DURA indicates a higher exposure. Detailed Description of the Invention

[0038] The following detailed description refers to specific details and embodiments in which the present invention may be implemented by way of illustration. These embodiments are described in sufficient detail to enable those skilled in the art to implement the present invention. Other embodiments may be utilized, and structural and logical changes may be made without departing from the scope of the present invention. The various embodiments are not necessarily mutually exclusive, as some embodiments may be combined with one or more other embodiments to form new embodiments.

[0039] References mentioned in this specification are listed in the form of a reference list for convenience and are attached at the end of the examples. The entire content of such references is incorporated herein by reference, but the mention of them in the specification does not imply that they form part of the common general knowledge.

[0040] Definition

[0041] For convenience, certain terms used in the specification, examples, and appended claims are collected herein.

[0042] Generally, the technical, scientific, and medical terms used herein have the same meanings as understood by those skilled in the art to which the present invention pertains. In addition, the following technical remarks and definitions are provided. These definitions should not in any way limit the scope of the present invention to these terms only, but are presented for a better understanding of the following description.

[0043] As used herein, the term "a" or "an" entity refers to one or more of that entity; for example, "an antibody" is understood to represent one or more antibodies. Thus, the terms "a" (or "an"), "one or more" and "at least one" may be used interchangeably herein.

[0044] As used herein, "Alzheimer's disease" or "AD" includes both familial Alzheimer's disease and sporadic Alzheimer's disease.

[0045] As used herein, the term "comprising" or "including" shall be construed to specify the presence of the stated feature, integer, step or component, but not to preclude the presence or addition of one or more other features, integers, steps or components or groups thereof. However, in the context of the present disclosure, the term "comprising" or "including" also includes "consisting of". Variants of the word "comprising", such as "comprise" and "comprises", and variants of "including", such as "include" and "includes", have corresponding varying meanings.

[0046] As used herein, the terms "subject", "individual" or "patient" are used interchangeably and refer to any subject, particularly a mammalian subject such as a human patient, who is the subject of a desired diagnosis, prognosis, prevention or treatment.

[0047] As used herein, the term "sample" refers to any biological material obtained from a subject or patient. For example, a sample can include blood, cerebrospinal fluid ("CSF") and / or urine. In other instances, a sample can include whole blood or plasma. A sample can also include a biopsy or tissue sample, including neural tissue. In still other instances, a sample can include whole cells and / or cell lysates. Blood samples can be collected by methods known in the art.

[0048] As used herein, "Tau" or "tau" refers to the native monomeric form of Tau and is also used to generally identify other conformational isomers of Tau, such as oligomers or aggregates of Tau. It is also used to collectively refer to all types and forms of Tau.

[0049] As used herein, "monomeric Tau" or "Tau monomer" refers to Tau protein that is completely soluble in an aqueous medium without aggregated complexes. "Aggregated Tau", "oligomeric Tau" and "Tau oligomers" refer to multiple aggregated monomers of Tau peptides or proteins, or multiple aggregated monomers of Tau-like peptides / proteins, or multiple aggregated monomers of modified or truncated Tau peptides / proteins, or multiple aggregated monomers of other derivatives of Tau peptides / proteins, forming oligomeric or polymeric structures that are insoluble or soluble in vitro in an aqueous medium and in vivo in a mammalian or human body (more particularly in the brain), but specifically refer to multiple aggregated monomers of Tau or multiple aggregated monomers of modified or truncated Tau peptides / proteins or their derivatives that are insoluble or soluble in a mammalian or human body (more particularly in the brain), respectively.

[0050] Polynucleotide, Gene and Nucleic Acid Detection

[0051] As used herein, the terms "polynucleotide" or "polynucleic acid" are used interchangeably and refer 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. The term "polynucleotide" is intended to encompass both single nucleic acids and plural nucleic acids and refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA) or plasmid DNA (pDNA), or a PCR amplicon. A polynucleotide can include conventional phosphodiester bonds or non-conventional bonds (e.g., amide bonds such as those found in peptide nucleic acids (PNAs)). By "isolated" polynucleotide is meant a nucleic acid molecule, DNA, or RNA that has been removed from its natural environment.

[0052] As used herein, the term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that includes the coding sequence necessary to produce an RNA or polypeptide or its precursor. A functional polypeptide can be encoded by the full-length coding sequence or any portion of the coding sequence, so long as the desired activity or functional property of the polypeptide (e.g., enzymatic activity, ligand binding, signal transduction, etc.) is retained. The term "portion" when used in reference to a gene may refer to a fragment of the gene. The size of the fragment can range from a few nucleotides to the entire gene sequence minus one nucleotide.

[0053] The term "gene" also encompasses the coding region of a structural gene and includes sequences located adjacent to the coding region at both the 5' end and the 3' end, e.g., at a distance of about 1 kb from either end, such that the gene corresponds to the length of a full-length mRNA (e.g., including the coding sequence, regulatory sequences, structural sequences, and other sequences). The sequence located 5' to the coding region and present on the mRNA is called the 5' untranslated or non-translated sequence. The sequence located 3' or downstream of the coding region and present on the mRNA is called the 3' untranslated sequence or 3' non-translated sequence. The term "gene" encompasses both the cDNA and genomic forms of a gene. In some organisms (e.g., eukaryotes), the genomic form or clone of a gene contains coding regions interrupted by non-coding sequences called "introns" or "intervening regions" or "intervening sequences". Introns are gene segments that are transcribed into nuclear RNA; introns can contain regulatory elements such as enhancers. Introns are removed or "spliced out" from the nuclear transcript or primary transcript; thus, introns are not present in the mRNA transcript. mRNA serves to specify the amino acid sequence or order in the nascent polypeptide during translation.

[0054] As used herein, the term "nucleic acid detection assay" refers to any method for determining the nucleotide composition of a nucleic acid of interest. Nucleic acid detection assays may include, but are not limited to, DNA / RNA sequencing methods, probe hybridization methods, structure-specific cleavage assays (e.g., INVADER assays, Hologic, Inc.; and as described, for example, in U.S. Patent Nos. 5,846,717, 5,985,557, 5,994,069, 6,001,567, 6,090,543, and 6,872,816; Lyamichev et al., Nat. Biotech., 17:292 (1999), Hall et al., Proc. Natl. Acad. Sci. USA, 97:8272 (2000), and US2009 / 0253142); enzyme mismatch cleavage methods (e.g., Variagenics, U.S. Patent Nos. 6,110,684, 5,958,692, 5,851,770); polymerase chain reaction (PCR); branched hybridization methods (e.g., Chiron, U.S. Patent Nos. 5,849,481, 5,710,264, 5,124,246, and 5,624,802); rolling circle replication (e.g., U.S. Patent Nos. 6,210,884, 6,183,960, and 6,235,502); NASBA (e.g., U.S. Patent No. 5,409,818); molecular beacon technology (e.g., U.S. Patent No. 6,150,097); electronic sensor technology (Motorola, U.S. Patent Nos. 6,248,229, 6,221,583, 6,013,170, and 6,063,573); cycling probe technology (e.g., U.S. Patent Nos. 5,403,711, 5,011,769, and 5,660,988); Dade Behring signal amplification methods (e.g., U.S. Patent Nos. 6,121,001, 6,110,677, 5,914,230, 5,882,867, and 5,792,614); ligase chain reaction (e.g., Barany, F., Proc. Natl. Acad. Sci. USA 88, 189-93 (1991)); sandwich hybridization methods (e.g., U.S. Patent No. 5,288,609); and specific high-sensitivity enzymatic reporter unlocking (e.g., Gootenberg J.S. et al., Science 356, 438-442 (2017)).

[0055] As used herein, the term "amplification of a nucleic acid" generally refers to the production of multiple copies of a polynucleotide or a portion of a polynucleotide, typically starting from a small number of polynucleotides (e.g., a single polynucleotide molecule, 10 to 100 copies of a polynucleotide molecule, which may be identical or may not be identical), wherein the amplification product or amplicon is typically detectable. Amplification of a polynucleotide encompasses a variety of chemical and enzymatic processes. During polymerase chain reaction (PCR) or ligase chain reaction (LCR; see, e.g., U.S. Patent No. 5,494,810), the generation of multiple DNA copies from one or a few copies of a target or template DNA molecule is a form of amplification.Additional types of amplification may include, but are not limited to, allele-specific PCR (see, e.g., U.S. Patent No. 5,639,611), assembly PCR (see, e.g., U.S. Patent No. 5,965,408), helicase-dependent amplification (see, e.g., U.S. Patent No. 7,662,594), hot start PCR (see, e.g., U.S. Patent Nos. 5,773,258 and 5,338,671), intersequence-specific PCR, inverse 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. Patent No. 5,508,169), methylation-specific PCR (see, e.g., Herman, et al., Proc. Natl. Acad. Sci. USA 93(18)9821-9826 (1996)), 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, et al., BMC Genom. 9:80 (2008)), nested PCR, overlap extension PCR (see, e.g., Higuchi, et al., Nucleic Acids Res. 16(15)7351-7367 (1998)), real-time PCR (see, e.g., Higuchi, et al., Biotechnol. 10:413-417 (1992); Higuchi, et al., Biotechnol. 11:1026-1030 (1993)), reverse transcription PCR (see, e.g., Bustin, S.A., Hum. Genet. 84, 571-573 (2000)), solid-phase PCR, thermal asymmetric interlaced PCR, and touchdown PCR (see, e.g., Don et al., Nucleic Acids Res. 19(14)4008 (1991); Roux, K.H., BioTechniques 16(5)812-814 (1994); Hecker, K.H., & Roux, K.H., BioTechniques 20(3)478-485 (1996)).Polynucleotide amplification can also be achieved 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. WO05023091A2; U.S. Patent Application Publication No. 20070202525).

[0056] As used herein, the term "oligo" or "oligonucleotide" refers to a short - length single - stranded or double - stranded polydeoxynucleotide that is chemically synthesized and then purified by known methods. Oligonucleotides can be used in nucleic acid amplification techniques, where such oligonucleotides can alternatively be referred to as primers.

[0057] Polypeptide of the Present Invention

[0058] As used herein, the term "polypeptide" is intended to cover both the singular "polypeptide" and the plural "polypeptides", and refers to a polymeric molecule of at least two amino acids (monomers) covalently linked by an amide bond (also called a peptide bond). The term "polypeptide" refers to any one or more chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains", or any other term used to refer to one or more chains of two or more amino acids are included within the definition of "polypeptide", and the term "polypeptide" can be used in place of or interchangeably with any of these terms. "Polypeptide" also means a product that has been modified after the expression of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / capping groups, proteolytic cleavage, or by non - naturally occurring amino groups. The term "polypeptide" also encompasses both naturally occurring and artificial (e.g., engineered or variant) full - length proteins and functional fragments of proteins.

[0059] As used herein, 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, for example, the ability to bind a specific epitope. The functional fragment can be of any size, as long as the fragment retains the activity / function of the full - length protein.

[0060] Antibody

[0061] The term "antibody" is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, multispecific antibodies (e.g., bispecific antibodies), and their antigen-binding fragments and similar antigen-binding molecules, provided that they exhibit the desired antigen-binding activity. An "antibody" as used herein can be of any type (IgG, IgM, IgD, IgE, IgA, and IgY) or immunoglobulin (Ig) class (IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass according to the invention.

[0062] As used herein, the terms "Tau11i antibody", "Tau11i-specific antibody", "anti-Tau11i antibody", and "antibody that specifically recognizes Tau11i" refer to an antibody that can bind to Tau11i with sufficient affinity and specificity to be used as a diagnostic and / or therapeutic agent targeting Tau11i. In some embodiments, the anti-Tau11i antibody binds to an unrelated non-Tau11i protein to an extent less than about 10% of the binding of the antibody to Tau11i, as measured by methods known in the art. In certain embodiments, the antibody that binds to Tau11i has a dissociation constant (K D ) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM. Unless otherwise expressly stated, the terms "Tau11i-specific antibody", "anti-Tau11i antibody", and "antibody that specifically recognizes Tau11i" as used herein refer to an antibody that binds to monomeric Tau11i, oligomeric Tau11i, and / or phosphorylated Tau11i.

[0063] As used herein, the term "chimeric antibody" refers to at least one antibody molecule in which the amino acid sequence in the constant region has been altered such that the antibody is more similar to a human antibody and still retains its original binding ability.

[0064] As used herein, the term "humanized antibody" refers to at least one antibody molecule in which the amino acid sequences in the variable and constant regions have been altered such that the antibody is more similar to a human antibody and still retains its original binding ability.

[0065] As used herein, an "antigen-binding fragment" or "antibody fragment" of an antibody refers to an incomplete or isolated portion of the full sequence of the antibody that retains the antigen-binding function of the parental antibody. Examples of 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 multispecific antibodies formed from antibody fragments. Fragments of the 8A12 antibody are also encompassed by the present invention so long as they retain the desired affinity of the full-length antibody. Specifically, it can be at least one amino acid shorter.

[0066] As used herein, an "antibody that binds the same epitope" as a reference antibody refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay, and conversely, the reference antibody blocks the binding of this antibody to its antigen by 50% or more in a competition assay.

[0067] The term "isolated" as used herein is defined as a biological component, such as a nucleic acid, peptide, or protein, that is substantially separated, produced separately, or purified from other biological components, namely other chromosomes and extrachromosomal DNA and RNA, and proteins, in the cells of the organism in which the component naturally occurs. Thus, isolated nucleic acids, peptides, and proteins include nucleic acids and proteins purified by standard purification methods. The term also includes nucleic acids, peptides, and proteins prepared by recombinant expression in host cells and chemically synthesized nucleic acids.

[0068] The following is a description of exemplary, non-limiting embodiments of the present invention.

[0069] This disclosure is in part based on the inventors' discovery that the retention of intron 11 in the Tau gene introduces a premature termination codon, resulting in the production of a truncated Tau11i polypeptide. In this regard, the inventors have successfully identified that this novel C-terminal truncated Tau-11i species not only exhibits biochemical properties similar to pathological Tau, but is also more abundantly found in the 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 may be downstream of these events. In view of the above, the polypeptides disclosed herein can thus be advantageously used as biomarkers for AD and useful diagnostic methods.

[0070] To this end, in one aspect of the present disclosure, there is provided an isolated or recombinant polypeptide comprising the following amino acid sequence:

[0071] (1) The amino acid sequence HKPGSPVEGEGWDGRVQGV, as shown in SEQ ID NO:14 or an antigenic fragment thereof, or

[0072] (2) The amino acid sequence SPVEGEGWDGRVQG, as shown in SEQ ID NO:15, or

[0073] (3) An amino acid sequence resulting from substitution, deletion, addition, or insertion of one or two or more amino acids in the amino acid sequence shown in SEQ ID NO:14 or SEQ ID NO:15.

[0074] In some embodiments, the isolated or recombinant Tau11i-derived polypeptide may consist of the amino acid sequence shown in (1) or (2), and have a cysteine residue conjugated to its terminus. In this regard, adding a terminal cysteine to the polypeptide sequence enables its conjugation to a carrier protein, which aids in antibody production. In some embodiments, the isolated or recombinant Tau11i-derived polypeptide may consist of the amino acid sequence HKPGSPVEGEGWDGRVQGV-C shown in SEQ ID NO:1 or C-SPVEGEGWDGRVQG shown in SEQ ID NO:2.

[0075] In some embodiments, the amino acid sequence of the isolated or recombinant polypeptide may include 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 shown 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.

[0076] Those skilled in the art will understand that the amino acid sequence of the polypeptide disclosed herein may vary sufficiently as long as the polypeptide retains its functionality and can exhibit the desired activity (e.g., the polypeptide serves as a binding epitope for Tau11i-specific antibodies and / or aptamers).

[0077] Table 1. Amino Acid Sequences of the Polypeptides of the Present Disclosure and Nucleotide Sequences of the Primers Used in the Present Invention

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087] It should be understood that the polypeptides of the present invention can be produced according to known peptide synthesis methods, such as solid-phase synthesis methods, liquid-phase synthesis methods, etc. The obtained polypeptides can be purified and separated by known purification methods, such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, combinations of these, etc. The polypeptides of the present invention can also be produced by culturing transformants containing nucleic acids encoding the polypeptides and separating and purifying the polypeptides from the resulting culture.

[0088] The polypeptides disclosed herein are suitable for or can be used for the following: preparing epitope-specific antibodies, localizing antibody epitopes and enzyme binding sites, and designing novel enzymes, drugs, and vaccines.

[0089] Thus, in another aspect, antibodies or antigen-binding fragments thereof that are capable of binding to the polypeptides of the present disclosure are provided. Preferably, the antibodies or antigen-binding fragments of the present invention are capable of binding to the Tau11i-specific epitope with sufficient affinity and specificity such that the antibody or its antigen-binding fragment can be used as a diagnostic agent and / or therapeutic agent targeting Tau11i. In some embodiments, the antibodies described herein can be polyclonal antibodies or monoclonal antibodies.

[0090] In some embodiments, the isolated antibody or its antigen-binding fragment specifically binds to a polypeptide comprising the amino acid sequence HKPGSPVEGEGWDGRVQGV (SEQ ID NO:14) or an 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 shown by SEQ ID NO:14 or SEQ ID NO:15.

[0091] In some embodiments, the isolated antibody or its antigen-binding fragment specifically binds to a polypeptide consisting of the amino acid sequence shown by SEQ ID NO:1 or SEQ ID NO:2.

[0092] In some embodiments, the isolated antibody or its antigen-binding fragment specifically binds to a 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 shown by SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:13.

[0093] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region, the heavy chain variable region comprising the following complementarity-determining regions: HCDR1 having the amino acid sequence shown in SEQ ID NO:31, HCDR2 having the amino acid sequence shown in SEQ ID NO:32, and HCDR3 having the amino acid sequence shown in SEQ ID NO:33, and the light chain variable region comprising the following complementarity-determining regions: LCDR1 having the amino acid sequence shown in SEQ ID NO:36, LCDR2 having the amino acid sequence shown in SEQ ID NO:37, and LCDR3 having the amino acid sequence shown in SEQ ID NO:38. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprising the amino acid sequence shown by residues 1 to 132 of SEQ ID NO:34, and the heavy chain variable region comprising the amino acid sequence shown by residues 1 to 136 of SEQ ID NO:29.

[0094] In another aspect, provided is a method of preparing a Tau11i-specific antibody using a polypeptide disclosed herein, the method comprising

[0095] (1) injecting the polypeptide of the present disclosure into an animal to induce an immune response to the polypeptide;

[0096] (2) isolating the antibody that binds to the polypeptide;

[0097] (3) selecting the antibody of (2) that binds to Tau11i but not to full-length Tau, thereby generating a Tau11i-specific antibody.

[0098] In some embodiments, the method may comprise (1) injecting a Tau11i-derived polypeptide of the present disclosure into a non-human mammal to form B cells specific for Tau11i; (2) selecting B cells specific for Tau11i; (3) fusing the selected B cells with immortalized cells to generate hybridomas, wherein the hybridomas are capable of producing an antibody or a fragment thereof specific for Tau11i; and (4) optionally isolating the antibody from the hybridomas and sequencing the variable heavy and light chains.

[0099] In some embodiments, the non-human mammal may be a mouse, rabbit, cow, goat, or sheep.

[0100] In another aspect, provided are polynucleotide molecules encoding a polypeptide, an antibody, or an antigen-binding fragment thereof as disclosed herein. In some embodiments, the polynucleotide may be cDNA.

[0101] In another aspect, oligonucleotides are also provided for amplifying a polynucleotide sequence spanning the exon 11 and intron 11 junction of the Tau gene. In some embodiments, the oligonucleotides consist of the nucleotide sequences shown in SEQ ID NO:7 and / or SEQ ID NO:8.

[0102] In another aspect, a method for detecting the polypeptide of the present disclosure is provided, the method comprising: contacting a sample suspected of comprising the polypeptide disclosed herein with an antibody or antigen-binding fragment of the present disclosure. For the purposes of the present invention, the method is preferably carried out in vitro.

[0103] In another aspect, a method for diagnosing Alzheimer's disease or identifying a subject at risk of developing Alzheimer's disease is also provided, wherein the method comprises: contacting a sample derived from the subject with an antibody or an antigen-binding fragment thereof as described herein.

[0104] In this regard, it should be understood that the antibody or antigen-binding fragment of the present disclosure can specifically bind to the polypeptide disclosed herein to form an antibody-polypeptide complex. Accordingly, the methods disclosed herein may further comprise the step of detecting the antibody-polypeptide complex, wherein the presence of the antibody-polypeptide complex can be used to inform the diagnosis and / or assessment of the risk of the subject having AD.

[0105] In a further aspect, a method for diagnosing Alzheimer's disease or identifying a subject at risk of developing Alzheimer's disease is also provided, wherein the method comprises: performing a nucleic acid detection assay to assess the abundance of intron 11-retained mRNA in a sample from the subject.

[0106] In some embodiments, the nucleic acid detection assay may comprise amplifying a polynucleotide sequence spanning the exon 11 and intron 11 junction of the Tau gene. In various embodiments, the amplification involves using oligonucleotides having the nucleotide sequences shown in SEQ ID NO:7 and / or 8.

[0107] In this regard, it can be understood that the abundance of intron 11-retained mRNA in the sample can be compared with that in a normal control to determine the diagnosis or risk factor of the subject having AD. As described herein, the presence of the Tau11i polypeptide is generally significantly higher in AD patients. Accordingly, it can be understood that a standard value for normal individuals with appropriate error bars can be generated by averaging the results of statistically significant samples of individuals known not to be affected by this disorder. Accordingly, determining and / or analyzing the degree of difference in the abundance of intron 11-retained mRNA in the sample compared to the control can be used to inform the diagnosis and / or assessment of the risk of the subject having AD.

[0108] In some embodiments, the sample can be a biological fluid or tissue. For example, the biological fluid can include, but is not limited to, blood and its fractions, such as plasma or serum, cerebrospinal fluid, urine, lymph fluid, synovial fluid, pericardial fluid, peritoneal fluid, amniotic fluid, saliva, nasal fluid, etc.

[0109] In some embodiments, the sample can be brain tissue, cerebrospinal fluid, plasma, saliva, tear fluid, or urine.

[0110] In another aspect, a method for screening candidate therapeutic or prophylactic agents for Alzheimer's disease is provided, comprising using any one of the following as an indicator: (1) reducing the amount of Tau protein in a subject with Alzheimer's disease or at risk of developing Alzheimer's disease, or (2) inhibiting the production of Tau protein in a subject with Alzheimer's disease or at risk of developing Alzheimer's disease; wherein the Tau protein comprises the amino acid sequence of the polypeptide disclosed herein.

[0111] In another aspect of the present disclosure, a kit is also provided, which comprises the antibody or antigen-binding fragment of the present invention. In some embodiments, the kit may further comprise the polypeptide of the present disclosure.

[0112] For the purposes of the present invention, the kit as described herein is preferably equipped for performing one or more of the methods described above. In some embodiments, the kit may comprise reagents for detecting the antibody or antigen-binding fragment of the present invention. In other embodiments, the kit may comprise reagents for detecting a complex that may form between the antibody or its antigen-binding fragment and a test sample. Reagents suitable for detecting the complex may include reagents that can incorporate a detectable label, such as a fluorophore, a radioactive moiety, an enzyme, a biotin / avidin label, a chromophore, a chemiluminescent label, etc.

[0113] The kit may also optionally comprise other reagents required for performing the diagnostic or prognostic assays described herein, such as buffers, salts, enzymes, enzyme cofactors, substrates, detection reagents, etc. Other components may also be included in the kit, such as buffers and solutions for separating and / or processing the test sample (e.g., pretreatment reagents). The kit may additionally comprise one or more other controls. One or more components of the kit may be lyophilized, and the kit may further comprise reagents suitable for reconstituting the lyophilized components.

[0114] In addition to the antibodies or antigen-binding fragments disclosed herein, it is contemplated that aptamers may be suitably applicable to the practice of the present invention. Aptamers are nucleic acid probes that form a specific three-dimensional structure and are capable of binding to a defined target such as a polypeptide with high affinity and specificity. In this regard, those skilled in the art will understand that methods for screening epitope-specific aptamers are known in the art, for example, via the systematic evolution of ligands by exponential enrichment (SELEX) process (e.g., as described in Teng et al., J. Am. Chem. Soc., 140(43), 14314-14323 (2018), incorporated herein by reference).

[0115] Accordingly, in another aspect herein, provided is a method for screening and / or selecting a Tau11i epitope-specific aptamer, the method comprising: (a) performing the systematic evolution of ligands by exponential enrichment (SELEX) technique to identify a Tau11i epitope-specific aptamer, wherein the SELEX technique comprises providing a polypeptide comprising the amino acid sequence shown in any one of SEQ ID NOs. 1, 2, 13, 14, or 15 as a target ligand.

[0116] Accordingly, in another aspect, provided is an aptamer capable of binding to a polypeptide comprising the amino acid sequence shown in any one of SEQ ID NOs. 1, 2, 13, 14, or 15. In some embodiments, the aptamer may be a DNA aptamer or an RNA aptamer.

[0117] In a further aspect, provided is also a method for diagnosing Alzheimer's disease or identifying a subject at risk of developing Alzheimer's disease, wherein the method comprises: contacting a sample derived from the subject with a Tau11i epitope-specific aptamer as provided herein.

[0118] Unless otherwise specified or apparent from the context and the understanding of one of ordinary skill in the art, values expressed as ranges can assume any specific value or sub-range within the stated range in various embodiments, to one tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. Unless otherwise specified or apparent from the context, "about" with respect to a numerical value generally refers to a range of values that fall within ±10% of that value, in some embodiments ±5%, in some embodiments ±1%, and in some embodiments ±0.5% of that value. In any embodiment of a numerical value preceded by "about", an embodiment in which the exact value is recited is provided. When an embodiment is provided in which a numerical value is not preceded by "about", an embodiment in which that value is preceded by "about" is also provided. When a range is preceded by "about", unless the context clearly dictates otherwise, the "about" in the provided embodiments applies to both the lower and upper limits of the range, or to the lower or upper limit. When phrases such as "at least", "up to", "not exceeding" or similar phrases appear before a series of numbers, unless the context clearly dictates otherwise, it should be understood that the phrase applies to each number in the list in various embodiments (it should be understood that, depending on the context, 100% of a value, e.g., a value expressed as a percentage, may be the upper limit). For example, "at least 1, 2 or 3" should be understood to mean "at least 1, at least 2 or at least 3" in various embodiments. It should also be understood that any and all reasonable lower and upper limits are explicitly contemplated.

[0119] The present invention has now been generally described, and the present invention will be more readily understood by reference to the following examples, which are provided only by way of illustration and are not intended to limit the present invention.

[0120] Examples

[0121] As described in Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (2012), standard molecular biology techniques known in the art and not specifically described are generally followed.

[0122] Example 1: Materials and General Methods

[0123] High-throughput analysis of intron retention

[0124] DLPFC datasets of healthy control subjects and AD patients (syn21088596) in the Religious Orders Study / Memory and Aging Project (ROSMAP) were downloaded from the AD Knowledge Portal (de Jager et al., Sci.Data, 5(1), 1 - 13(2018)). RNA-seq reads were first mapped to the human reference genome (GRCh38.100), and intron retention (IR) was quantified using the IRFinder pipeline software with default settings (Middleton et al., Genome Biol. 18, 1 - 11(2017)). The IR ratio was calculated by dividing the intron abundance by the sum of the intron and exon abundances. Intron 11 of the Tau (MAPT) gene (MAPT / ENSG00000186868 / clean / 17:46014324 - 46018617:+) investigated in this study had a minimum splicing sequencing depth of greater than five reads. The generalized linear model in the 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 a p-value < 0.05 was considered significant. Dot plots were generated using the "ggplot" function in the "ggplot2" package (https: / / ggplot2.tidyverse.org) with the normalized IR ratios from individual human subjects determined by DESeq2. Counts for the Tau gene were generated using the featureCounts (v.2.0.1.) software, and differential expression was calculated using the DESeq2 (v.1.30.1) software.

[0125] Cell lines and human brain tissues

[0126] Human NSCs (XCL-1, SC-001-1V) were purchased from XCell Science Inc. and differentiated according to the manufacturer's protocol. Total brain RNA, protein lysates, and frozen tissue sections were purchased from MYBIOSOURCE and BIOCHAIN. Parietal (BA40) samples C6, C7, A6, and A7 have been previously described (e.g., Chua, et al., Neurochem.Int. 152, 105251(2022)). Human brain samples and tissue sections (donor information)

[0127] “C” represents control, and “A” represents Alzheimer's disease.

[0128] Total protein lysates from different brain regions were purchased from:

[0129] (a) MYBIOSOURCE

[0130] 1) C1: Normal adult, brain, temporal lobe lysate (MBS657128) - male, 77 years old.

[0131] 2) A1: AD adult, brain, temporal lobe lysate (MBS657032) - male, 87 years old.

[0132] 3) C4: Normal adult, brain, frontal lobe lysate (MBS657031) - male, 60 years old.

[0133] 4) A4: AD adult, brain, frontal lobe lysate (MBS657343) - female, 85 years old.

[0134] 5) C5: Normal adult, brain, hippocampal lysate (MBS657489) - male, 60 years old.

[0135] 6) A5: AD adult, brain, hippocampal lysate (MBS657034) - female, 93 years old.

[0136] (b) BIOCHAIN

[0137] 1) C8: Normal adult, brain, temporal lobe lysate (P1234078) - female, 70 years old.

[0138] 2) A1: AD adult, brain, temporal lobe lysate (P1236978Alz) - male, 87 years old.

[0139] 3) C9: Normal adult, brain, frontal lobe lysate (P1234051) - male, 82 years old.

[0140] 4) C2: Normal adult, brain, amygdala lysate (P1234036) - male, 22 years old.

[0141] 5) A2: AD adult, brain, amygdala lysate (P1236036Alz) - female, 65 years old.

[0142] 6) C3: Normal adult, brain, parietal lobe lysate (P1234066) - male, 66 years old.

[0143] 7) A3: AD adult, brain, parietal lobe lysate (P1236066Alz) - male, 73 years old.

[0144] The following samples were obtained from the Newcastle Brain Tissue Resource, which is part of the Dementia Research Brain Network.

[0145] 1) C6: Normal adult, brain, parietal lobe lysate (283 / 96) - male, 77 years old.

[0146] 2) A6: AD adult, brain, parietal lobe lysate (120 / 09) - female, 85 years old.

[0147] 3) C7: Normal adult, brain, parietal lobe lysate (308 / 09) - male, 66 years old.

[0148] 4) A7: AD adult, brain, parietal lobe lysate (350 / 09) - female, 98 years old.

[0149] Frozen tissue sections were purchased from BIOCHAIN (BioChain Institute Inc., CA, USA).

[0150] 1) C10: Normal adult, brain, temporal lobe ((T1234078) - male, 26 years old.

[0151] 2) A10: AD adult, brain, temporal lobe (T1236078Alz) - male, 73 years old.

[0152] Total RNA from different brain regions (AMSBIO) was purchased from BIOCHAIN.

[0153] 1. AD brain, R1236035Alz - 50 (Lot#: A507294) - male, 87 years old.

[0154] 2. Normal brain R1234035 - P (Lot#: C404081) - 5 males, pooled, 21 - 29 years old.

[0155] 3. AD frontal lobe, R1236051Alz - 10 (Lot#: B204064) - male, 87 years old.

[0156] 4. Normal frontal lobe, R1234051 - P (Lot#: B804011) - 5 males, pooled, 22 - 29 years old.

[0157] 5. AD temporal lobe, R1236078Alz - 10 (Lot#: B408032) - male, 80 years old.

[0158] 6. Normal temporal lobe, R1234078 - P (Lot#: B411025) - 5 males, pooled, 23 - 27 years old.

[0159] Cell culture

[0160] Maintain HEK 293T cells (Clontech, 632180) in Dulbecco's Modified Eagle Medium (DMEM) GlutaMAX supplemented with 10% FBS, 1× non-essential amino acids (NEAA), and 1.0 mM sodium pyruvate TM (Gibco).

[0161] Seed human neural stem cells (NSCs) (XCL-1, SC-001-1V, XCell Science Inc.) onto Matrigel (Corning, 354277) and culture them in neural expansion medium [DMEM GlutaMAX TM at a ratio of 1:1 with Ham's F-12 nutrient mixture (both from Gibco), 1× NEAA, 1× N-2 supplement (Gibco, 17502048), 1× B-27 supplement (without vitamin A) (Gibco, 12587010), 0.5X GlutaMAX (Gibco, 35050061), 20 ng / ml human basic FGF (Gibco, PHG0261), 2 μg / ml heparin sulfate (Sigma, H4784)]. Differentiate virus-infected NSCs according to the manufacturer's protocol. Briefly, seed the cells at a density of 0.04 × 106 cells / cm 2 in NSC maintenance medium on polyornithine (20 μg / mL) and laminin (10 μg / mL)-coated plates (day 0). Then supplement the cells with neuron induction medium on days 1, 3, and 5. On day 6, harvest the formed neuronal progenitor cells (NPCs) by StemPro TM Accutase TM (Gibco, A1110501) and replate them at a density of 0.04 × 106 cells / cm 2 on polyornithine / laminin-coated wells in neuron maturation medium. Culture the neurons for the specified duration and change the medium every other day.

[0162] Transient transfection and lentiviral transduction

[0163] According to the manufacturer's protocol, transiently transfect full-length Tau and Tau-11i expression constructs into HEK 293T cells using 3000 reagent (Invitrogen, L3000008). Harvest the cells 48 h after transfection for analysis.

[0164] The Lenti-X lentiviral expression system (Clontech) was used to generate stable NSC lines expressing full-length (FL) Tau or Tau-11i protein. Briefly, lentiviruses were prepared by transfecting 293T cells with a mixture containing 7 μg of lentiviral plasmid (pLVX-Flag-Tau-FL or pLVX-Flag-Tau-11i) and the Lenti-X packaging single reagent (VSV-G) (Clontech, 631275). The media harvested at 24 h and 72 h post-transfection were combined and concentrated using a Lenti-X concentrator (Clontech, 631231) to generate the lentiviral supernatant. NSCs were infected with the lentiviral supernatant in the presence of polybrene (12 μg / ml). Forty-eight hours after viral transduction, stable clones were selected with 0.1 μg / ml puromycin. Western blotting and immunofluorescence were used to verify the expression of Tau-FL and Tau-11i in the stable lines.

[0165] Cloning of Tau constructs

[0166] The full-length (FL) Tau gene was PCR amplified from the VN-Tau plasmid (Addgene 87368) using the XbaI-Tau-F and SalI-Tau-R primers and blunt-cloned into the EcoRV site in the pBlueScript II SK+ vector to obtain PBS-Tau-FL. After digestion of PBS-Tau-FL with XbaI / SalI restriction enzymes, the full-length Tau fragment was cloned into the XbaI / PspXI sites in the pLVX-Flag-IRES-puro lentiviral vector to obtain pLVX-Flag-Tau-FL.

[0167] Three steps were taken to prepare the Tau-11i construct. (i) A partial Tau exon and intron 11 region was PCR amplified from human genomic DNA using Tau-11e-F and SalI-Tau-11i-R primers and cloned into the pGEMT vector (Promega) to obtain pGEMT-Tau-ex / in-11. (ii) BstEII makes a unique cut within exon 11. Then, the fragment of Tau exon and intron 11 released by BstEII / SalI restriction digestion of pGEMT-Tau-ex / in-11 was used to replace the 3'-end of the full-length Tau in PBSTau-FL to obtain PBS-Tau-11i. (iii) After XbaI / SalI restriction digestion of PBS-Tau-11i, the Tau-11i fragment was cloned into the XbaI / PspXI site in the pLVX-Flag-IRES-puro vector to obtain pLVX-Flag-Tau-11i. DNA sequencing was performed at each step to verify sequence integrity.

[0168] XbaI-Tau-F 5'-CTA TCTAGA GCTGAGCCCCGCCA-3'(SEQ ID NO:3)

[0169] SalI-Tau-R 5'-AAC GTCGA CTCACAAACCCTGCTTG-3'(SEQ ID NO:4)

[0170] Tau-11e-F 5'-GTGCAAATAGTCTACAAAC-3'(SEQ ID NO:5)

[0171] SalI-Tau-11i-R 5'- GTCGAC TCACCAGGACTCCTCC-3'(SEQ ID NO:6)

[0172] Preparation of custom Tau11i antibodies

[0173] GenScript (GenScript Biotech Corporation, NJ, USA) generated two custom anti-Tau11i polyclonal antibodies using the peptides HKPGSPVEGEGWDGRVQGV-C (SEQ ID NO:1) and C-SPVEGEGWDGRVQG (SEQ ID NO:2) as antigens, where C is the conjugated cysteine.

[0174] GenScript also generated a customized anti-Tau11i monoclonal antibody 8A12 using a peptide with the amino acid sequence shown in SEQ ID NO:15 as an antigen. Monoclonal antibody 8A12 includes a heavy chain variable region with the amino acid sequence shown in SEQ ID NO:29 and a light chain variable region with the amino acid sequence shown in SEQ ID NO:34 (Table 1). Monoclonal antibody 8A12 includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes the following complementary determining regions: HCDR1 with the amino acid sequence shown in SEQ ID NO:31, HCDR2 with the amino acid sequence shown in SEQ ID NO:32, and HCDR3 with the amino acid sequence shown in SEQ ID NO:33. The light chain variable region includes the following complementary determining regions: LCDR1 with the amino acid sequence shown in SEQ ID NO:36, LCDR2 with the amino acid sequence shown in SEQ ID NO:37, and LCDR3 with the amino acid sequence shown in SEQ ID NO:38 (Table 1).

[0175] Sarkosyl extraction

[0176] Sarkosyl soluble and insoluble fractions were prepared according to the published protocol (Smith et al., Neuropathol. 46(7), 641-653 (2020)). 293T cells and NPC stable lines expressing Tau were resuspended in low salt buffer (50 mM HEPES pH 7.6, 250 mM sucrose, 1 mM EDTA pH 8.0) with protease inhibitor mixture (Roche) at 320 μl per 3e6 cells, followed by the addition of 40 μl of 5M NaCl and 40 μl of 10% sarkosyl. The cell lysates were thoroughly mixed by pipetting, incubated on ice for 15 min with stirring, and then briefly sonicated (10 s on / 30 s off, high setting, 3 cycles, Bioruptor UCD-200TO). After complete resuspension, 40 μl of the cell lysate was harvested as the "total" lysate. After centrifugation at maximum speed for 1 h at 4°C, the supernatant was carefully collected as the "soluble" extract. The insoluble pellet was gently washed with low salt buffer (with 0.5M NaCl and 1% sarkosyl) and centrifuged at maximum speed for 10 min at 4°C to remove the wash buffer. Laemmli buffer was added to the "total" lysate, "soluble" fraction, and "insoluble" pellet, boiled for 10 min, and immunoblotting assays were performed.

[0177] Immunoblotting and antibodies

[0178] Different Syncria fractions, brain tissues, and cell lysates were boiled in 1X Laemmli buffer, separated and analyzed in SDS-PAGE gels, and transferred to PVDF membranes by standard Western blotting. The antibodies used were anti-Flag (Sigma, F1804), polyclonal anti-Tau11i (GenScript), monoclonal anti-Tau-11i antibody 8A12, anti-Tau46 (CST, 4019S), anti-Tau 4-repeat isoform RD4 (4R, Millipore, 05-804), anti-pTau-S202 / T205 (AT8, Invitrogen, MN1020), anti-pTau-T181 (CST, 12885S), anti-pTau-S396 (PHF1, Santa Cruz, sc-32275), and anti-β-actin (Sigma, A2228).

[0179] Immunofluorescence

[0180] Immunostaining of neurons and image quantification were performed as previously described (Rao et al., Nucleic Acids Res., 48(3), 1225-1238 (2020)). Briefly, mature neurons cultured in 12-well plates were fixed with 4% paraformaldehyde in PBS for 20 min, followed by two rounds of PBS washes and 1 h blocking (10% normal goat serum, 0.1% triton-X 100 in PBS) at room temperature. Neurons were then stained overnight at 4 °C with anti-MAP2 (Millipore, MAB3418), anti-α-tubulin (Santa Cruz, sc-53029), and anti-Flag (Sigma, F1804) antibodies (5 μg / ml in blocking buffer). After three rounds of PBS washes the next day, neurons were incubated with anti-mouse 594 and anti-rabbit FITC secondary antibodies (1:500, Invitrogen) for 2 h at room temperature. Cells were washed three times with PBS and mounted in VECTASHIELD with DAPI (Vector labs). At least three image fields per condition were captured using a confocal microscope (FV3000, 10× objective). The signal threshold was adjusted to obtain optimal coverage of Flag and α-tubulin staining. The relative fluorescence intensity of FLAG relative to α-tubulin was quantified using ImageJ software.

[0181] IR was verified by quantitative PCR (qPCR)

[0182] Total RNA from different brain tissues of healthy control subjects and AD patients (Amsbio) was purchased from Biochain. cDNA libraries were generated from 1 μg of total RNA pretreated with DNase I (Thermo Scientific) using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Real-time quantitative PCR was performed in triplicate (6 μl / reaction) using a 7900HT Fast Real-Time PCR System (Applied Biosystems) with 0.2 μM primers and 2X SYBR Green Master Mix (Thermo Scientific). The relative levels of Tau-11i (Tau ex-in11-F / R) were calculated using the 2 -ΔCT method, where the threshold cycle (CT) values were normalized to Tau exon 1 / exon 2 (Tau ex-1-F / ex-2-R) or Gapdh expression. Tau ex-in11-F 5'-ACCAGGTAGCCCTGTGGAAG-3' (SEQ ID NO:7)

[0183] Tau ex-in11-R 5'-CCGCAAGTTTCACACTCAAC-3' (SEQ ID NO:8)

[0184] Tau ex-1-F 5'-AAGACCAAGAGGGTGACACG-3' (SEQ ID NO:9)

[0185] Tau ex-2-R 5'-ACATCTTCCGCTGTTGGAGT-3' (SEQ ID NO:10)

[0186] Gapdh-F 5'-CAATGACCCCTTCATTGACC-3' (SEQ ID NO:11)

[0187] Gapdh-R 5'-GACAAGCTTCCCGTTCTCAG-3' (SEQ ID NO:12)

[0188] Human total RNA and protein lysates.

[0189] Total protein lysates from different brain regions were purchased from MyBiosource, Inc. (San Diego, USA).

[0190] 1. Normal adult, brain, temporal lobe lysate (MBS657128) - male, 77 years old.

[0191] 2. AD adult, brain, temporal lobe lysate (MBS657032) - male, 87 years old.

[0192] 3. Normal adult, brain, frontal lobe lysate (MBS657031) - male, 60 years old.

[0193] 4. AD adult, brain, frontal lobe lysate (MBS657343) - female, 85 years old.

[0194] 5. Normal adult, brain, hippocampal lysate (MBS657489) - male, 60 years old.

[0195] 6. AD adult, brain, hippocampal lysate (MBS657034) - female, 93 years old.

[0196] Total RNA from different brain regions (Amsbio) was purchased from Biochain.

[0197] 1. AD brain, R1236035Alz-50 (Lot#: A507294) - male, 87 years old.

[0198] 2. Normal brain R1234035-P (Lot#: C404081) - 5 males, pooled, 21 - 29 years old.

[0199] 3. AD frontal lobe, R1236051Alz-10 (Lot#: B204064) - male, 87 years old.

[0200] 4. Normal frontal lobe, R1234051-P (Lot#: B804011) - 5 males, pooled, 22 - 29 years old.

[0201] 5. AD temporal lobe, R1236078Alz-10 (Lot#: B408032) - male, 80 years old.

[0202] 6. Normal temporal lobe, R1234078-P (Lot#: B411025) - 5 males, pooled, 23 - 27 years old.

[0203] Example 2: Increased Retention of Tau Intron 11 in the AD Cohort

[0204] More than 30 pairs of healthy controls and AD dorsolateral prefrontal cortex (DLPFC) (de Jager et al., Scientific data, 5(1), 1 - 13(2018)) were analyzed using IRFinder software (Middleton et al., Genome Biol., 18, 1 - 11(2017)), showing increased retention of Tau intron 11 ( Figure 1A). These changes appear to be specific to AD, as no significant differences in the IR ratio were observed in the PD (Dumitriu et al., BMC Genom., 9(1), 1-17(2016)) and PSP (Allen et al., Sci.Data, 3(1), 1-10(2016)) datasets ( Figure 5 A). Next, the increase in IR in AD samples was verified by performing qPCR on the brains, frontal lobes, and temporal lobes from three different pairs of control and AD patients ( Figure 1 D). Compared to RNA sequencing of the entire transcriptome, the significant differences observed in qPCR could be explained by the targeted amplification of intron 11, while in RNA sequencing of the entire transcriptome, low-abundance intronic read counts may not have been fully represented. Although qPCR detected elevated levels of Tau mRNA in three AD brain tissue samples compared to controls ( Figure 1 D), no differential expression of the Tau gene was observed in the DLPFC ( Figure 1 C).

[0205] Example 3: Truncated Tau11i Oligomerizes and Is More Enriched in the Sarkosyl Insoluble Fraction

[0206] In the brain, full-length Tau encodes 441 amino acids (Tau441 in this article), while the premature stop codon in intron 11 ( Figure 1 A) may lead to protein truncation (Tau11i in this article, amino acids encoded by the retained intron ( Figure 2 A, light gray text)). The presence of a canonical polyadenylation site 1 kb downstream of the stop codon within intron 11 further suggests that Tau mRNA transcripts with IR may escape nonsense-mediated decay (NMD). To study the effects caused by IR, Flag-tagged full-length Tau441 or truncated Tau11i was expressed in 293T cells. As predicted, truncated Tau11i ( Figure 2 B, right well) migrated slower than Tau441 ( Figure 2 B, left well) at a size of ~55 kD and could not be detected by the PHF1 or Tau46 antibodies that recognize the C-terminal domain of Tau. Importantly, it could be specifically detected by the Tau11i-A antibody generated against the amino acid sequence encoded by the retained intron 11 ( Figure 2 B). Interestingly, the Tau11i protein also migrated as a higher molecular weight (HMW) species on denaturing gels and was readily detected by the pT181 antibody, indicating that it underwent oligomerization in 293T cells ( Figure 2B and 5B). This observation is also consistent with the inhibition of Tau monomer polymerization by the C-terminal domain (Martins et al., PloS one, 7(4), e35461 (2012); Abraha et al., J. Cell Sci., 113(21), 3737 - 3745 (2000); Gu et al., J Biol Chem, 295(40), 13812 - 13828 (2020); Wang et al. Proc. Natl. Acad. Sci. USA, 104(24), 10252 - 10257 (2007)) and HMW-Tau undergoes phosphorylation at Thr181 (Zhou et al., Front. Aging Neurosci., 10, 27 (2018)). To eliminate the effects of transient expression, lentiviral infection was used to stably express Flag-Tau441 or Flag-Tau11i in human neural stem cells (NSCs). Similar to 293T cells, the Flag-Tau11i protein was specifically recognized by two different Tau11i antibodies (Tau11i-A and Tau11i-B), but not by Tau46 ( Figure 2 C). Tau11i also continuously oligomerizes to form HMW species in NSCs, although at lower levels, as detected by Tau4R and pT181 antibodies ( Figure 2 C and 5B). Since the formation of HMW species is one of the hallmarks of pathological Tau in AD, other biochemical properties of Tau11i were further examined in these cells. Extraction with the ionic detergent N-lauroylsarcosine (sarkosyl), which can effectively solubilize native-folded proteins, has been widely used to isolate detergent-insoluble protein aggregates from brain tissues of patients with neurodegenerative diseases such as AD (Diner et al., J. Vis. Exp., (128), e55835. (2017)). Compared with Flag-Tau441, more ectopically expressed Flag-Tau11i was present in the sarkosyl-insoluble fractions from 293T cells ( Figure 2 D top blot, right lane), neural progenitor cells (NPCs, Figure 2 D middle blot) and neurons ( Figure 2 D bottom blot). Taken together, the results indicate that ectopically expressed Tau11i undergoes multiple phosphorylations, oligomerizes to form HMW species, and is enriched in the sarkosyl-insoluble fractions.

[0207] Example 4: Detection of Higher Levels of Tau11i Species in AD Brain Regions

[0208] To test whether Tau mRNA transcripts with IR are degraded by NMD or translated in vivo, the hippocampus, amygdala, parietal, frontal, and temporal lobes of 9 healthy controls and 7 AD patients were probed with Tau11i and AT8 antibodies. Consistent with the increased retention of intron 11 in AD samples ( Figure 1 ), two different Tau11i antibodies detected significantly higher levels of a ~50 kD band in AD samples, corresponding to the AT8 band (phosphorylated Tau at S202 / T205) ( Figure 3 A, white arrow. p < 0.003, paired two-tailed t-test, n = 7), when compared to controls. Although Tau11i antibodies failed to detect the HMW form in NSCs ( Figure 2 C), weak high-mobility bands of different sizes corresponding to the AT8 immunoblot were observed in AD frontal lysates ( Figure 3 A, black arrow). These results suggest that Tau mRNA retaining intron 11 escapes NMD and is translated into protein. The presence of premature termination codons and downstream canonical polyadenylation signals in intron 11 may contribute to NMD escape and subsequent translation of novel truncated Tau11i proteins in vivo. In the AD tissues examined, high levels of Tau11i protein correlated with the increased IR observed by transcriptome analysis and qPCR validation. Similar migration patterns on denaturing gels additionally suggest that Tau11i protein may be phosphorylated at serine 202 and threonine 205 (AT8) and oligomerized in AD tissues.

[0209] Next, the solubility of endogenous Tau11i protein was investigated by Sarkosyl extraction. Higher levels of Tau11i protein were found to be enriched in the Sarkosyl-insoluble fraction in the AD hippocampus compared to the AD temporal lobe ( Figure 3 B, white arrow). This suggests that IR-induced C-terminal truncation may reduce the solubility of monomeric Tau11i protein in AD brain tissues. To better understand the cellular localization pattern of Tau11i in brain tissues, we stained control and AD temporal lobe sections with Tau11i and Tau4R (recognizing amino acids 275 - 291) ( Figure 3 C) or α-tubulin ( Figure 6 ). Consistent with the immunoblot results, higher levels of Tau11i staining were observed in AD compared to control temporal lobes ( Figure 3 C and Figure 6 ). Interestingly, Tau11i appears to form granular aggregates that weakly co-localize with the Tau4R fibrillar structures in the AD temporal lobe, suggesting that Tau11i may contribute to pre-tangle assembly rather than causing the pathological neurofibrillary tangle burden ( Figure 3 C).

[0210] Example 5: Reduced Binding of Tau11i to Microtubules and Increased Protein Stability

[0211] The binding of Tau to microtubules can be disrupted by extensive phosphorylation and mutations. Therefore, it was investigated whether IR-induced protein truncation would impair its binding to microtubules in neurons and alter its protein stability in cells. NSCs expressing Flag-Tau441 and Tau11i were differentiated into cortical neurons (day 14) and cultured for an additional week (day 21, n = 2) or one month (day 44, n = 5). Immunostaining with anti-Flag and anti-α-tubulin antibodies showed that the co-localization signal of Tau11i with the microtubule network in neurons was significantly reduced compared to Tau441 ( Figure 4 A, Figure 7 and Figure 8 A), indicating a lower binding efficiency of Tau11i to microtubules. Next, the stability of Tau441 and Tau11i proteins was investigated by time-course monitoring and cycloheximide assays of transiently transfected Tau proteins. The level of Tau11i protein at day 7 was approximately ~5% of that at day 3, while the Tau441 level decreased to approximately 0.4% ( Figure 4 B and Figure 8 B). Consistent with the time-course experiment, Tau11i was also more stable than Tau441 after 8 h of cycloheximide treatment and showed less protein degradation ( Figure 4 C).

[0212] Characterization of Tau11i in different cell types revealed several biochemical properties similar to pathological Tau (Gotz et al., Annu Rev Pathol, 14, 239-261 (2019); Iqbal et al., Nat.Rev.Neurol., 12(1), 15-27. (2016)). Compared to full-length Tau441, truncated Tau11i oligomerizes to form HMW species and is enriched in the Triton X-100-insoluble fraction (Example 3), binds less to the microtubule network in neurons, and exhibits a lower protein turnover rate (Example 5).

[0213] Analysis of different brain regions from different donors by qPCR, immunoblotting, and immunofluorescence showed that the Tau11i protein was significantly elevated in AD tissues compared to healthy controls. Endogenous Tau11i was also observed in the Triton X-100-insoluble fraction in the AD hippocampus, consistent with its binding to protein aggregates. The examples in this disclosure indicate that Tau11i is a novel biomarker for AD. Therefore, the antibody and nucleic acid detection methods presented herein are suitable for diagnostic purposes or for evaluating individuals at risk of developing AD.

[0214] Example 6: Retention of Tau Intron 11 and Tau11i Protein Can Be Detected in Human Plasma

[0215] To test whether retention of Tau intron 11 can be detected in human plasma, 0.5 ml of control human plasma was lysed with 1.25 ml of RNAzol RT (Sigma). Total RNA was extracted and purified according to the manufacturer's instructions. A cDNA library was generated from 1 μg of total RNA pretreated with DNase I (Thermo Scientific) using a high-capacity cDNA reverse transcription kit (Applied Biosystems). PCR was performed using Phusion DNA polymerase (NEB) with primer pairs as Figure 9 shown in C. Figure 9 Lane 3 of B shows a positive band representing an amplicon spanning the junction of exon 11 and intron 11, indicating that retention of Tau intron 11 is detectable in human plasma.

[0216] Similarly, Tau11i could also be successfully detected in human plasma by applying the Tau11i-specific antibody disclosed herein ( Figure 10 A - B). After Co-IP and SDS-PAGE electrophoresis, the fraction pulled down by the Tau11i-specific antibody Tau11i-B was then digested with trypsin in the gel and subsequently subjected to LC-MS / MS. The mass spectrometry data confirmed that the pulled-down fraction corresponded to Tau species. It should be emphasized that the reference database only includes known Tau variants. For example, Tau isoform F is Tau441 (441 amino acids) which contains 2N4R; isoform D has 383 amino acids which contains 0N4R. Thus, any newly identified form (such as the forms discussed in this disclosure) is not covered in the records of the database. Given that Tau11i-B is specific for Tau11i and does not bind to Tau441 ( Figure 2 C), the above results indicate that Tau11i was detected in human plasma.

[0217] The presence of intron 11-retained Tau and Tau11i proteins circulating in the bloodstream provides a unique opportunity for early and rapid detection of such Tau abnormalities. The accessibility of human plasma as a sample source greatly accelerates the diagnostic process, eliminating the need for more invasive procedures that often pose risks and discomfort to patients. In addition, identifying and quantifying intron 11-retained Tau or Tau11i proteins in human plasma can establish a direct link between their levels and the progression of AD pathology, enabling precise and timely intervention.

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Claims

1. An isolated or recombinant Tau11i-derived polypeptide, comprising the following amino acid sequence: 1) The amino acid sequence HKPGSPVEGEGWDGRVQGV, as shown in SEQ ID NO:14 or an antigenic fragment thereof, or 2) The amino acid sequence SPVEGEGWDGRVQG, as shown in SEQ ID NO:15, or 3) An amino acid sequence resulting from substitution, deletion, addition or insertion of one or two or more amino acids in the amino acid sequence shown in SEQ ID NO:14 or SEQ ID NO:

15.

2. The isolated or recombinant Tau11i-derived polypeptide according to claim 1, wherein, the polypeptide consists of the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:

2.

3. An isolated antibody or an antigen-binding fragment thereof, which specifically binds to a polypeptide comprising: the amino acid sequence HKPGSPVEGEGWDGRVQGV (SEQ ID NO:14) or an 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 shown in SEQ ID NO:14 or SEQ ID NO:

15.

4. The isolated antibody or an antigen-binding fragment thereof according to claim 3, wherein, the polypeptide consists of the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:

2.

5. The isolated antibody or an antigen-binding fragment thereof according to claim 3 or 4, wherein, the antibody or an antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region, the heavy chain variable region comprises the following complementarity-determining regions: HCDR1 having the amino acid sequence shown in SEQ ID NO:31, HCDR2 having the amino acid sequence shown in SEQ ID NO:32 and HCDR3 having the amino acid sequence shown in SEQ ID NO:33, and the light chain variable region comprises the following complementarity-determining regions: LCDR1 having the amino acid sequence shown in SEQ ID NO:36, LCDR2 having the amino acid sequence shown in SEQ ID NO:37 and LCDR3 having the amino acid sequence shown in SEQ ID NO:

38.

6. The isolated antibody or an antigen-binding fragment thereof according to claim 5, wherein, the antibody or an antigen-binding fragment thereof comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:29 and / or a light chain variable region having the amino acid sequence shown in SEQ ID NO:

34.

7. An oligonucleotide for amplifying a polynucleotide sequence spanning the exon 11 and intron 11 junction of the Tau gene.

8. The oligonucleotide according to claim 7, wherein, the oligonucleotide consists of the nucleotide sequence shown in SEQ ID NO:7 or SEQ ID NO:

8.

9. A polynucleic acid molecule encoding the following: i) the polypeptide according to claim 1 or 2, or ii) an antibody or an antigen-binding fragment thereof according to any one of claims 3 to 6.

10. The polynucleic acid molecule according to claim 9, wherein, the polynucleic acid molecule is cDNA.

11. A method for detecting the polypeptide according to claim 1 or 2, the method comprising contacting a sample suspected of comprising the polypeptide with an antibody or an antigen-binding fragment thereof according to any one of claims 3 to 6.

12. A method for diagnosing Alzheimer's disease or identifying a subject at risk of developing Alzheimer's disease, wherein, the method comprises: (a) contacting a sample derived from the subject with an antibody or an antigen-binding fragment thereof according to any one of claims 3 to 6; or (b) performing a nucleic acid detection assay to evaluate the abundance of intron 11-retained mRNA in a sample derived from the subject; or (c) contacting a sample derived from the subject with a Tau11i epitope-specific aptamer that specifically binds to a polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO: 1, 2, 13, 14 or 15.

13. The method according to claim 12, wherein, the nucleic acid detection assay in (b) comprises amplifying a polynucleotide sequence spanning the exon 11 and intron 11 junction of the Tau gene.

14. The method according to claim 13, wherein, the amplification comprises using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 7 and / or SEQ ID NO:

8.

15. The method according to any one of claims 11-14, wherein, the sample is brain tissue, cerebrospinal fluid, plasma, saliva, tear or urine.

16. A method for screening a candidate therapeutic or prophylactic drug for Alzheimer's disease, comprising using any one of the following as an indicator: a) reducing the amount of Tau protein in a subject with Alzheimer's disease or a subject at risk of developing Alzheimer's disease, or b) inhibiting the production of Tau protein in a subject with Alzheimer's disease or a subject at risk of developing Alzheimer's disease, wherein, the Tau protein comprises the amino acid sequence of the polypeptide according to claim 1 or 2.

17. A method for preparing a Tau11i-specific antibody, comprising a) injecting the polypeptide according to claim 1 or 2 into an animal to induce an immune response against the polypeptide; b) isolating an antibody that binds to the polypeptide; c) selecting the antibody of (b) that binds to Tau11i but not to full-length Tau, thereby generating a Tau11i-specific antibody.

18. The method according to claim 17, comprising a) injecting at least one Tau11i-derived polypeptide according to claim 1 or 2 into at least one non-human mammal to form at least one B cell specific for Tau11i; b) selecting at least one B cell specific for the Tau11i; c) Fusing the selected B cells with at least one immortalized cell to produce at least one hybridoma, wherein, the hybridoma is capable of producing at least one antibody or fragment thereof that is specific for the Tau11i; and d) Optionally isolating the antibody from the hybridoma and sequencing the variable heavy chain and variable light chain.

19. A method for screening and / or selecting Tau11i epitope-specific aptamers, the method comprising: Performing systematic evolution of ligands by exponential enrichment (SELEX) technology to identify Tau11i epitope-specific aptamers, wherein the SELEX technology comprises providing a polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO: 1, 2, 13, 14 or 15 as a target ligand.

20. A Tau11i-specific aptamer that is capable of binding to a polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO: 1, 2, 13, 14 or 15.

21. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 3 to 6.

22. The kit according to claim 21, further comprising the isolated polypeptide according to claim 1.

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