Modeling of tdp-43 proteinopathies

Abstract

It was discovered herein that neither the nuclear localization signal (NLS) nor the prion-like domain (PLD) of TDP-43 are required for in vitro embryonic stem cell culture and differentiation into motor neurons. ES cells expressing these TDP-43 mutants and differentiated into motor neurons that exhibit an ALS-like phenotype, from which the TDP-43 mutants redistribute to and accumulate in the cytoplasm, and the inability to regulate cryptic exon splicing, such that these cells can serve as a model for TDP-43 proteinopathies for testing candidate therapeutics that can dissipate such proteinopathies. In addition, these ES cells can be used to successfully generate non-human animals, e.g., mice, that also exhibit hallmark symptoms of ALS and can be used to test candidate agents useful for treating TDP-43 proteinopathies.

Description

[0001] Divisional Application Instructions This application is a divisional application of the invention patent application filed on June 26, 2020, with application number 202080045369.8 and invention title "Modeling of TDP-43 protein diseases". Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 867,785, filed June 27, 2019, pursuant to 35 USC § 119(3), the disclosure of which is hereby incorporated by reference in its entirety.

[0003] References to sequence lists submitted as text files via EFS Web The sequence list written in file 10312CN02_ST26..xml is 55.3 kilobytes long and was created on October 24, 2025 (the actual sequence content was created on June 25, 2020), and is hereby incorporated by reference. Technical Field

[0004] This article describes methods for evaluating the biological functions of TDP-43 and its domains, non-human animals and non-human animal cells, and nucleic acids. It also provides TDP-43 proteopathic models comprising such non-human animals, non-human animal cells, or nucleic acids, and methods for their use. Background Technology

[0005] Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that affects motor neurons, leading to limb paralysis and ultimately death from diaphragmatic failure. In autopsies of ALS patients, a nearly universal pathological finding is the accumulation of TDP-43 (trans-activating response DNA-binding protein 43 kDa) in cytoplasmic inclusions.

[0006] TDP-43 is characterized by a nuclear localization signal (NLS) domain, two RNA recognition motifs (RRM1 and RRM2), a putative nuclear export signal (NES) domain, and a glycine-rich prion-like domain (PLD). Similar to members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family, TDP-43 is a major nuclear RNA-binding protein required for cell viability and normal animal development in all mammals. The redistribution of TDP-43 from the nucleus to the cytoplasm and its accumulation in insoluble aggregates are two key diagnostic markers of ALS.

[0007] Although cytoplasmic accumulation of TDP-43 is associated with ALS, the relationship between each domain of TDP-43 and its biological function remains unclear. Summary of the Invention

[0008] This article provides embryonic stem (ES) cells, tissues cultured from them (e.g., primitive ectoderm, embryoid body, motor neurons), and non-human animals derived from them, which express a mutant TDP-43 peptide lacking a functional domain and may exhibit an ALS-like phenotype. Compositions and methods of their preparation and use are also provided. Mutants encoding a mutant TDP-43 peptide lacking a functional domain are also provided. TARDBP The gene and the mutant TDP-43 polypeptide lacking a functional domain are also provided. Exemplary therapeutic oligonucleotides, such as antisense oligonucleotides, are also provided that can restore… TARDBP Self-regulation of expression.

[0009] This article describes non-human animals (e.g., rodents, such as rats or mice) and non-human animal cells (e.g., embryonic stem (ES) cells, embryoid bodies, embryonic stem cell-derived motor neurons (ESMNs), etc.) containing mutations encoding the mutant TDP-43 peptide. TARDBP Genes, for example, in which mutations TARDBP Genes include wild type TARDBP The nucleotide sequence of the gene contains mutations that cause the mutant TDP-43 to contain the amino acid sequence corresponding to the wild-type TDP-43 polypeptide, but with mutations (e.g., one or more of point mutations, substitutions, replacements, insertions, deletions, etc.). In some embodiments, the wild-type... TARDBP The gene contains the sequence described in SEQ ID NO:2 (including its degenerate variant), SEQ ID NO:4 (including its degenerate variant), or SEQ ID NO:6 (including its degenerate variant), which encodes a wild-type TDP-43 polypeptide containing the amino acid sequence described in SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, respectively.

[0010] In some implementations, the mutation encoding the mutant TDP-43 peptide... TARDBP Gene replacement of endogenous genes in non-human animals or non-human animal cells TARDBP Endogenous at loci TARDBP Gene. In some embodiments, mutations in non-human animal cells or non-human animals encoding the mutant TDP-43 polypeptide. TARDBP The gene is heterozygous. For example, in some implementations, in addition to the mutations described herein... TARDBP In addition to genes, non-human animals or non-human animal cells also contain (a) wild-type TARDBP The gene or (b) contains a knockout mutation (e.g., a conditional knockout mutation). TARDBPGenes. In some implementations, conditional knockout mutations contain site-specific recombination recognition sequences, such as loxp Sequences, optionally including site-specific recombination recognition sequences (e.g. loxp The sequence is located flanking the encoding exon (e.g., exon 3). In some implementations, it includes a knockout mutation. TARDBP Genes contain loxp The sequence, which is located TARDBP The gene is deleted from the flanking region of exon 3. In some implementations, the knockout mutation involves the deletion of the entire coding sequence of the TDP-43 peptide.

[0011] In some implementations, non-human animal or non-human animal cells contain (i) endogenous TARDBP At the locus, a mutation encoding the mutant TDP-43 polypeptide was used. TARDBP Endogenous gene replacement TARDBP Genes, and (ii) another endogenous gene on homologous chromosomes. TARDBP The locus contains knockout mutations TARDBP Genetic or wild type TARDBP Gene.

[0012] In some implementations, non-human animal or non-human animal cells are included in endogenous... TARDBP The locus contains conditional knockout mutations. TARDBP Genes, and another endogenous gene on homologous chromosomes. TARDBP The gene locus contains the entire TARDBP missing coding sequence TARDBP Gene.

[0013] In some implementations, mutations in non-human animal cells or non-human animals encoding the mutant TDP-43 polypeptide... TARDBP The genes are homozygous.

[0014] In some implementations, non-human animals or non-human animal cells do not express the wild-type TDP-43 peptide.

[0015] In some implementations, non-human animals or non-human animal cells express wild-type TDP-43 peptide.

[0016] In some embodiments, the non-human animal or non-human animal cells described in any of the preceding claims comprise wild-type cells as described in the control cells. TARDBP Mutations that are equivalent to the mRNA transcription level of a gene TARDBPThe levels of the gene's mRNA transcription, the increased levels of mutant TDP-43 peptide compared to wild-type TDP-43 peptide in control cells, higher concentrations of mutant TDP-43 peptide in the cytoplasm (e.g., in motor neurons) compared to the nucleus, mutant TDP-43 peptide with increased insolubility compared to wild-type TDP-43 peptide, cytoplasmic aggregates containing mutant TDP-43 peptide, increased splicing of hidden exons, and / or decreased levels of alternatively spliced ​​TDP-43 forms. In some embodiments, non-human animals exhibit denervation of muscle tissue composed primarily of fast-twitch muscles (such as the tibialis anterior) and / or normal innervation of muscle tissue composed primarily of slow-twitch muscles (such as the intercostal muscles).

[0017] In some implementations, the non-human animal cells, as described herein, are cultured in vitro. Non-human animal tissues comprising the non-human animal cells described herein are also described herein.

[0018] In some embodiments, non-human animal tissues and / or non-human animal cells are included in the composition.

[0019] In some embodiments, the mutant TDP-43 polypeptide lacks a functional domain compared to the wild-type TDP-43 polypeptide, and the mutant TDP-43 polypeptide is expressed in a non-human animal or non-human animal cell, optionally wherein the wild-type TDP-43 polypeptide comprises a sequence as stated in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5.

[0020] In some embodiments, the mutant TDP-43 peptide lacks a functional domain selected from the group consisting of: nuclear localization signal (NLS), RNA recognition motif 1 (RRM1), RNA recognition motif 2 (RRM2), putative nuclear export signal (E), prion-like domain (PLD), or a combination thereof. In some embodiments, the mutant... TARDBP Genes are non-human animals that contain mutations (e.g., point mutations, substitutions, insertions, deletions, or combinations thereof). TARDBP Genes. In some implementations, non-human animals... TARDBP Genes as described in SEQ ID NO:2 or SEQ ID NO:4. In some implementations, mutations... TARDBP Genes are human TARDBP Genes contain mutations, such as point mutations, substitutions, insertions, deletions, or combinations thereof. In some implementations, mutations... TARDBP In some implementation schemes, people TARDBP The gene is as stated in SEQ ID NO:5.

[0021] In some embodiments, the mutant TDP-43 peptide lacks a functional domain due to one or more of the following: (a) a point mutation in an amino acid in the NLS, (b) a point mutation in an amino acid in the RRM1, (c) a point mutation in an amino acid in the RRM2, (d) a deletion of at least a portion of the nuclear output signal, and (e) a deletion of at least a portion of the prion-like domain. For example, in some embodiments, the mutant TDP-43 peptide comprises the sequence as stated in SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, said sequence further comprising (a) a point mutation in an amino acid in the NLS, (b) a point mutation in an amino acid in the RRM1, (c) a point mutation in an amino acid in the RRM2, (d) a deletion of at least a portion of the nuclear output signal, and (e) a deletion of at least a portion of the prion-like domain. In some embodiments, (a) point mutations in amino acids in the NLS include K82A, K83A, R84A, K95A, K97A, K98A, or combinations thereof; (b) point mutations in the RRM1 include F147L and / or F149L; (c) point mutations in the RRM2 include F194L and / or F229L; (d) deletion of at least a portion of the nuclear output signal includes deletion of amino acids at and between positions 239 and 250 of the wild-type TDP-43 polypeptide; and (e) deletion of at least a portion of the prion-like domain includes deletion of amino acids at and between positions 274 and 414 of the wild-type TDP-43 polypeptide. In some embodiments, the mutant TDP-43 polypeptide comprises K82A, K83A, R84A, K95A, K97A, and / or K98A compared to the wild-type TDP-43 polypeptide, optionally wherein the wild-type TDP-43 polypeptide comprises the sequence as stated in SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5. In some embodiments, the mutant TDP-43 polypeptide lacks a prion-like domain located between amino acids 274 and 414 of the wild-type TDP-43 polypeptide and including the amino acid at said position, optionally wherein the wild-type TDP-43 polypeptide comprises the sequence as stated in SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5. In some embodiments, the mutant TDP-43 polypeptide comprises F147L and F149L compared to the wild-type TDP-43 polypeptide, optionally wherein the wild-type TDP-43 polypeptide comprises the sequence as stated in SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5.In some embodiments, the mutant TDP-43 polypeptide comprises F194L and F229L compared to the wild-type TDP-43 polypeptide, optionally wherein the wild-type TDP-43 polypeptide comprises the sequence as stated in SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5. In some embodiments, the mutant TDP-43 polypeptide lacks a nuclear output signal between and including the amino acids at positions 239 and 250 compared to the wild-type TDP-43 polypeptide, optionally wherein the wild-type TDP-43 polypeptide comprises the sequence as stated in SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5.

[0022] Also provided are the mutant TDP 43 polypeptide described herein and the nucleic acid molecule encoding the mutant TDP 43 polypeptide. In some embodiments, the nucleic acid molecule encoding the mutant TDP-43 polypeptide as described herein further comprises from 5' to 3': a 5' homologous arm, a nucleic acid sequence encoding the mutant TDP-43 polypeptide, and a 3' homologous arm, wherein the nucleic acid undergoes homologous recombination in rodent cells. In some embodiments, the 5' and 3' homologous arms are homologous to rat sequences, such that the nucleic acid is homologous in endogenous rats. TARDBP Homologous recombination occurred at the locus, and the nucleic acid sequence encoding the mutant TDP-43 polypeptide replaced the endogenous one. TARDBP The coding sequence. In some embodiments, the 5' and 3' homologous arms are homologous to mouse sequences, such that the nucleic acid is effective in endogenous mice. TARDBP Homologous recombination occurred at the locus, and the nucleic acid sequence encoding the mutant TDP-43 polypeptide replaced the endogenous one. TARDBP Encoded sequence.

[0023] This document also describes methods for preparing the non-human animal and non-human animal cells described herein. In some embodiments, the methods include modifying the genome of the non-human animal or non-human animal cells to include a mutation encoding the mutant TDP 43 polypeptide. TARDBP The gene, wherein the mutant TDP-43 polypeptide lacks a functional domain compared to wild-type TDP-43, optionally wherein the wild-type TDP-43 polypeptide comprises a sequence as stated in SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5. In some embodiments, the modification includes using a mutation encoding the mutant TDP-43 polypeptide as described herein. TARDBP Endogenous gene replacement TARDBP Gene. In some embodiments, the modification also includes using a gene containing a knockout mutation (e.g., a conditional knockout mutation). TARDBP Endogenous gene replacement TARDBP Genes. In some embodiments, the method further includes eliminating genes containing knockout mutations. TARDBP Cells are cultured under conditions of gene expression.

[0024] This document also describes methods using non-human animals, non-human animal cells, non-human animal tissues, and compositions. In some embodiments, non-human animals, non-human animal cells, non-human animal tissues, and compositions are used in methods, such as methods for identifying therapeutic candidates for treating diseases and / or evaluating the biological function of the TDP-43 domain. In some embodiments for identifying therapeutic candidates, the methods include (a) contacting a non-human animal, non-human animal cell, non-human animal tissue, or composition containing non-human animal cells or tissues (e.g., in vitro cultures) as described herein with a candidate agent, (b) evaluating the phenotype and / or TDP-43 biological activity of the non-human animal, non-human cell, or tissue, and (c) identifying a candidate agent that restores the non-human animal, non-human cell, or tissue to a phenotype and / or TDP-43 biological activity equivalent to that of control cells or tissues expressing wild-type TDP-43 peptides.

[0025] In some embodiments for evaluating the biological function of TDP-4, the method includes (a) modifying embryonic stem (ES) cells to contain a mutation encoding a mutant TDP-43 polypeptide. TARDBP The gene, wherein the polypeptide lacks a functional domain selected from the group consisting of: nuclear localization signal (NLS), first RNA recognition motif (RRM1), first RNA recognition motif (RRM2), putative nuclear output signal (E), prion-like domain (PLD), and combinations thereof; (b) optionally differentiating modified ES cells in vitro and / or obtaining genetically modified non-human animals from modified ES cells; and (c) evaluating the phenotype and / or TDP-43 bioactivity of the genetically modified ES cells, primitive ectoderm derived therefrom, motor neurons derived therefrom, or non-human animals derived therefrom. In some embodiments, as described in claim 39 or claim 40, wherein the phenotype is evaluated by cell culture, fluorescence in situ hybridization, Western blotting analysis, or a combination thereof. In some embodiments, evaluating the phenotype includes measuring the viability of the genetically modified ES cells, primitive ectoderm derived therefrom, motor neurons derived therefrom, or non-human animals derived therefrom. In some embodiments, evaluating the phenotype includes determining the cellular localization of the mutant TDP-43 polypeptide. In some embodiments, evaluating the bioactivity of the mutant TDP-43 peptide includes measuring the splicing products of genes containing hidden exons regulated by TDP-43. In some embodiments, genes containing hidden exons regulated by TDP-43 include Crem, Fyxd2, and Clf1. In some embodiments, assessing the bioactivity of the mutant TDP-43 peptide includes measuring the level of alternatively spliced ​​TDP-43.

[0026] This article also describes oligonucleotides (e.g., antisense oligonucleotides, siRNA, CRISPR / Cas systems, etc.) that can be used as candidates for treating TDP-43 proteinopathy. In some embodiments, the antisense oligonucleotide comprises a gapmer motif targeting a TDP-43 mRNA sequence between a variable 5' and 3' splice site. In some embodiments, the antisense oligonucleotide comprises a gapmer motif targeting a TDP-43 mRNA sequence between a variable 5' and 3' splice site, wherein the variable 5' splice site is associated with a TARDBP genomic location selected from the group consisting of: (a) chromosome 4: 148,618,647; (b) chromosome 4: 148,618,665; and (c) chromosome 4: 148,618,674, and wherein the variable 3' splice site is associated with a TARDBP genomic location on chromosome 4: 148,617,705. In some siRNA implementations, the siRNA comprises a sequence targeting a TDP-43 mRNA sequence between a variable 5' and 3' splice site. In some implementations, the siRNA containing the sequence targets a TDP-43 mRNA sequence between a variable 5' and 3' splice site, wherein the variable 5' splice site is associated with a TARDBP genomic location selected from the group consisting of: (a) chromosome 4: 148,618,647; (b) chromosome 4: 148,618,665; and (c) chromosome 4: 148,618,674, and wherein the variable 3' splice site is associated with a TARDBP genomic location on chromosome 4: 148,617,705. In some CRISPR / Cas system implementations, the system comprises a Cas9 protein and at least one gRNA, wherein the gRNA recognizes sequences located at or near the 5' alternative splice site and / or located at or near the 3' alternative splice site of the TDP-43 mRNA. In some implementations, the CRISPR / Cas system comprises a Cas9 protein and at least one gRNA, wherein the gRNA recognizes a protein located at or near a Cas9 protein. TARDBP The sequence at the genomic location, which is selected from the following groups: (a) chromosome 4: 148,618,647; (b) chromosome 4: 148,618,665; (c) chromosome 4: 148,618,674; (d) chromosome 4: 148,617,705 and combinations thereof. Attached Figure Description

[0027] The patent or application document contains at least one color drawing. Upon request and payment of the necessary fees, our firm will provide a published copy of the patent or application with one or more color drawings.

[0028] Figure 1A description of TDP-43 (not to scale) is provided, along with the relative positions of the nuclear localization signal (NLS; amino acids 82-98), the two RNA recognition motifs (RRM1; amino acids 106-176, and RRM2; amino acids 191-262), the putative relative position of the nuclear export signal (E; amino acids 239-248), the relative position of the prion-like domain (PLD; amino acids 274-414), ALS-related amino acid substitution mutations, and ALS-related C-terminal fragments. Asterisks highlight mutations associated with FTD symptoms in or without ALS. Mutations A90V, S92L, N267S, G287S, G294V, G368S, S375G, A382T, I383V, N390S, and N390D have also been observed in healthy individuals.

[0029] Figure 2A Provided mice TARDBP A diagram of the genome structure (not to scale), depicting exons 1-6 (rectangles), the untranslated region (unfilled rectangles), and the translated region (filled rectangles) starting from the ATG start codon. Figure 2B Amino acid sequence alignments of mouse (m) TDP-43 and human (h) TDP-43 peptides are provided, along with the amino acid positions of the peptides and the common sequences below the mTDP-43 and hTDP-43 sequences. Generally, the boxed regions in the alignments show the nuclear localization signal (NLS: amino acids 82-98), RNA recognition motif 1 (RRM1: amino acids 106-176), RNA recognition motif 2 (RRM2: amino acids 191-262), the putative nuclear export signal (E: amino acids 239-248), and the glycine-rich prion-like domain (PLD: amino acids 274-414). Amino acid mismatches between mouse TDP-43 and human TDP-43 are also boxed and depicted with dashes in the common sequences. Exon junctions are also depicted as vertical lines, representing exons (EX) that are joined at the indicated junctions. The vertical line between amino acids 286 and 287 provides a variable 5' splice site (see [link]). Figure 11A ).

[0030] Figure 3A Two examples are provided TARDBP Explanation of invalid alleles (not proportional): (1) After removal of exon 3 following cre-mediated recombination, alleles containing the lox The conditional knockout allele of exon 3 flanked by the P-site specific recombination recognition site (triangle), hereinafter referred to as "-", and (2) containing the entire TARDBP missing coding sequence TARDBPInvalid alleles, hereinafter referred to as "ΔCDS". The relative positions of exons 1-6 (rectangles), untranslated regions (unfilled rectangles), translated regions (filled rectangles), and the start ATG and stop TGA codons are depicted. Figure 3B Provides various forms of mutation TARDBP Illustrative depiction (not to scale) of the gene-encoded non-restricted mutant TDP-43 polypeptide. Specifically, in these examples and related figures: "WT" refers to wild type. TARDBP Gene, "loxP-Ex3loxP" refers to a mutation in exon 3 containing a fluxed loxP. TARDBP Gene, "-" indicates the absence of wild-type loxP after cre-mediated loxP-Ex3loxP recombination. TARDBP Mutations in the nucleotide sequence of exon 3 of a gene TARDBP Gene, "ΔCDS" refers to the lack of TARDBP Mutation of the entire coding sequence TARDBP Gene, "ΔNLS" refers to a mutation encoding the mutant TDP-43 polypeptide. TARDBP The gene, the polypeptide, contains the following point mutations: K82A, K83A, R84A, K95A, K97A, and K98A. "ΔRRM1" refers to a mutation encoding the mutant TDP-43 polypeptide. TARDBP The gene, the polypeptide, contains the following point mutations: F147L and F149L. "ΔRRM2" refers to a mutation encoding the mutant TDP-43 polypeptide. TARDBP The gene, the polypeptide, contains the following point mutations: F194L and F229L. “ΔE” refers to the mutation encoding the mutant TDP-43 polypeptide. TARDBP The gene, the polypeptide lacks amino acid 239 to 250 of the wild-type TDP-43 polypeptide, and "ΔPLD" refers to a mutation encoding the mutant TDP-43 polypeptide. TARDBP The gene, the polypeptide lacks amino acid 274 to 414 of the wild-type TDP-43 polypeptide.

[0031] For the ΔE and ΔPLD mutant TDP-43 peptides, the diagonal lines represent the missing regions.

[0032] Figure 4 A protocol for differentiating embryonic stem (ES) cells into motor neurons is described. Mutations including those shown in the figure are also illustrated. TARDBPThe ability of ES cells of a gene to remain viable at the ES cell stage after Cre-mediated exon 3 deletion (-), to reach the primitive ectoderm (PE) stage and / or to reach the motor neuron (MN) stage.

[0033] Figure 5 A protocol for evaluating the viability of embryonic stem cell-derived motor neurons (ESMNs) is described. The use of specified mutations following conditional knockout (-) allele activation is also illustrated. TARDBP The result of the activity of ESMN in the gene.

[0034] Figure 6A The TDP-43 region, which is not depicted to scale, is provided by an anti-TDP-43 antibody that recognizes the N-terminus of TDP-43 (α-TDP-43 N-terminus) or the C-terminus of TDP-43 (α-TDP-43 C-terminus). Figure 6B Provided for use such as Figure 6A The depicted protein blots show antibody staining of the cytoplasmic and nuclear fractions of cells that recognize the N-terminus (α-TPD-43 N-terminus) or C-terminus (α-TPD-43 C-terminus) of TDP-43. Cre-mediated exon 3 deletion (-) occurs in the ES cell stage and according to... Figure 4 The protocol described herein involves culturing cells in ES medium, ADFNK medium, ADFNK medium containing retinoic acid and sound hedgehog factor, and ESMN medium to generate embryonic stem cell-derived motor neurons (ESMN). Cytoplasmic and nuclear fractions were isolated from TDP-43 WT / - modified ESMN, ΔNLS / - modified ESMN, ΔE / - modified ESMN, ΔPLD / - modified ESMN, or dying ΔRRM1 / - modified cells. A graph showing the ratio of cytoplasmic to nuclear TDP-43 in control TDP-43 WT / - ESMN (●), ΔNLS / - modified ESMN (▲), ΔRRM1 / - modified cells (▼), or ΔPLD / - modified ESMN (■) is also provided.

[0035] Figure 7 Provides the specified mutations TARDBP Image of gene-modified embryonic stem cell-derived motor neurons (ESMNs) at 40x magnification using fluorescence in situ hybridization. The image shows mutation removal at the ES cell stage. TARDBP Exon 3 (-) of the gene is captured, and according to Figure 4The protocol described herein involves culturing cells in ES medium, ADFNK medium, ADFNK medium containing retinoic acid and sound hedgehog factor, and ESMN medium to generate embryonic stem cell-derived motor neurons (ESMN). Cells are stained with an antibody that recognizes the C-terminus of TDP-43 (α TDP-43 C-terminus; top image) or with anti-MAP2 antibody and DAPI (bottom image).

[0036] Figure 8 Provides the specified mutations TARDBP Image of gene-modified embryonic stem cell-derived motor neurons (ESMNs) at 40x magnification using fluorescence in situ hybridization. The image shows mutation removal at the ES cell stage. TARDBP Exon 3 (-) of the gene is captured, and according to Figure 4 The protocol described herein involves culturing cells in ES medium, ADFNK medium, ADFNK medium containing retinoic acid and sound hedgehog factor, and ESMN medium to generate embryonic stem cell-derived motor neurons (ESMN). Cells are stained with an antibody that recognizes the N-terminus of TDP-43 (α TDP-43 N-terminus; top image) or with anti-MAP2 antibody and DAPI (bottom image).

[0037] Figure 9A Western blots stained with anti-TDP-43 antibody provided sarkosyl-soluble and sarkosyl-insoluble fractions of the cells. Cre-mediated exon 3 deletion (-) occurs in the ES cell stage and according to... Figure 4 The protocol described herein involves culturing cells in ES medium, ADFNK medium, ADFNK medium containing retinoic acid and sound hedgehog factor, and ESMN medium to generate embryonic stem cell-derived motor neurons (ESMNs). Creatyl-soluble and creatine-insoluble fractions were isolated from cells with TDP-43 WT / - modified ESMN, ΔNLS / - modified ESMN, ΔE / - modified ESMN, ΔPLD / - modified ESMN, or ΔRRM1 / - modified ESMN. A graph showing the ratio of insoluble to soluble TDP-43 expressed by these ESMNs is also provided. Figure 9B Graphs showing TDP-43 mRNA (left; y-axis) or protein (right; y-axis) expression levels are provided. Cre-mediated exon 3 deletion (-) occurs in the ES cell stage and according to... Figure 4The protocol described herein involves culturing cells in ES medium, ADFNK medium, ADFNK medium containing retinoic acid and sound hedgehog factor, and ESMN medium to generate embryonic stem cell-derived motor neurons (ESMN). The mRNA levels of ΔNLS / - modified ESMN, ΔE / - modified ESMN, ΔPLD / - modified ESMN, or dying ΔRRM1 / - modified cells were compared with the control (TDP-43 WT / - modified ESMN (WT / -)). Figure 9C Western blots stained with anti-TDP-43 or anti-GAPDH antibodies against cell lysates are provided. Cre-mediated exon 3 deletion (-) occurs in the ES cell phase and according to Figure 4 The protocol described herein involves culturing cells in ES medium, ADFNK medium, ADFNK medium containing retinoic acid and sound hedgehog factor, and ESMN medium to generate embryonic stem cell-derived motor neurons (ESMN). Cell lysates were isolated from TDP-43 WT / - modified ESMN, ΔNLS / - modified ESMN, ΔE / - modified ESMN, ΔPLD / - modified ESMN, or dying ΔRRM1 / - modified cells after treatment with actinomycin (CHX+) for up to 16 hours. A graph (y-axis) showing the % TDP-43 protein expressed by control TDP-43 WT / - modified ESMN (●), ΔNLS / - modified ESMN (■), ΔRRM1 / - modified cells (▲), or ΔPLD / - modified ESMN (▼) after actinomycin treatment (x-axis; h) is also provided.

[0038] Figure 10 A description of normal and hidden exon splicing occurring in three genes believed to be regulated by TDP-43 is provided (not to scale): Crem , Fyxd2 and Clf1 The graph shows the levels of normal splicing products (filled bars) and abnormal splicing products (patterned bars and unfilled bars). Cre-mediated exon 3 deletion (-) occurs at the ES cell stage and according to... Figure 4 The protocol described herein involves culturing cells in ES medium, ADFNK medium, ADFNK medium containing retinoic acid and sound hedgehog factor, and ESMN medium to generate embryonic stem cell-derived motor neurons (ESMNs). Cells modified with ΔNLS / -, ΔE / -, ΔPLD / -, or ΔRRM1 / - of ESMN and a control (TDP-43 WT / -) are shown. Crem , Fyxd2 and Clf1 The level of hidden exon splicing produced Figure 11A Descriptions of normal and alternative splicing events occurring in the TDP-43 gene are provided (not to scale). Figure 11B A graph showing the levels of TDP-43 mRNA with alternative splicing is provided. Cre-mediated exon 3 deletion (-) occurs in the ES cell phase and according to Figure 4 The protocol described herein involves culturing cells in ADFNK medium, ADFNK medium containing retinoic acid and sound hedgehog factor, and ESMN medium to generate embryonic stem cell-derived motor neurons (ESMNs). The levels of alternatively spliced ​​TDP-43 mRNA produced by unmodified ES cells (WT / WT), ΔNLS / - modified ESMN, ΔE / - modified ESMN, ΔPLD / - modified ESMN, or dying ΔRRM1 / - modified cells are shown.

[0039] Figure 12 Provided an illustration of TDP-43 injection - / - ES cells, TDP-43 ΔNLS / - Modified ES cells, TDP-43 ΔPLD / - Modified ES cells, TDP-43 ΔNLS / WT Modified ES cells, TDP-43 ΔPLD / WT Modified ES cells, TDP-43 WT / - Modified ES cells, TDP-43 loxP-Ex3-loxP / WT Modified ES cells, or wild-type TDP-43 WT / WT A graph showing the survival time of 8-cell embryos with ES cells after fertilization. E3.5 (day 3.5 of embryonic development), E10.5 (day 10.5 of embryonic development), E15.5 (day 15.5 of embryonic development), P0 (day 0 after birth).

[0040] Figure 13A , Figure 13B and Figure 13C Protein imprints of motor neurons isolated from spinal cord tissue of 16-week-old mice (n = 2) were provided. The examined mice expressed proteins from (i) endogenous sources. TARDBP Locus: Mutation in exon 3 containing loxP sidelink TARDBP Gene (loxP-Ex3-loxP), containing a knockout mutation in NLS TARDBP Genes (ΔNLS), or mutations containing prion-like domain deletions. TARDBP Gene (ΔPLD), and (ii) another gene on a homologous chromosome. TARDBP Wild-type (WT) at the gene locus TARDBP Gene. Figure 13AThe cytoplasmic and nuclear fractions of motor neurons stained with the corresponding α-TDP-43 N-terminal antibody or α-TDP-43 C-terminal antibody that recognizes the N-terminus or C-terminus of TDP-43 are shown (see, for example, Figure 6A It also provides a graph showing the ratio of cytoplasmic to nuclear TDP-43 in spinal cord tissues isolated from loxP-Ex3-loxP / WT mice (●), ΔNLS / WT mice (▲), or ΔPLD / WT mice (▼). Figure 13B We provide Western blots of cytoplasmic and nuclear fractions of spinal cord tissue isolated from 16-week-old mice and stained with an antibody that recognizes phosphorylated TDP-43. Figure 13C Provided corresponding α-TDP-43 N-terminal antibodies that recognize the N-terminus or C-terminus of TDP-43 (see, for example) Figure 6A ) or α-TDP-43 C-terminal antibody (see, for example) Figure 6A Western blots of saturated creatine soluble and creatine soluble fractions in stained cells.

[0041] Figure 14 Fluorescence in situ hybridization images at 40x magnification are provided for motor neurons isolated from spinal cord tissue of 16-week-old mice. The examined mice expressed (i) endogenous... TARDBP Locus: Mutation in exon 3 containing loxP sidelink TARDBP Gene (loxP-Ex3-loxP), containing a knockout mutation in NLS TARDBP Genes (ΔNLS), or mutations containing prion-like domain deletions. TARDBP Gene (ΔPLD), and (ii) another gene on a homologous chromosome. TARDBP Wild-type (WT) at the gene locus TARDBP Genes. Cells were stained with an antibody that recognizes the N-terminus of TDP-43 (α TDP-43 M-terminus; top) or with anti-chAT and anti-NeuN antibodies (bottom). A graph also shows the percentage of motor neurons exhibiting cytoplasmic aggregates in animals expressing only wild-type TDP-43 (●), mutant ΔNLS TDP-43 peptide and wild-type TDP-43 peptide (■), mutant ΔNLS TDP-43 peptide and wild-type TDP-43 peptide (■), or mutant ΔPLD TDP-43 peptide and wild-type TDP-43 peptide (▲).

[0042] Figure 15AFluorescence in situ hybridization images of tibialis anterior muscle tissue or intercostal muscle tissue isolated from 16-week-old mice at 10x or 40x magnification are provided. The tissues were stained with antibodies recognizing synaptophysin, krait venom, and / or DAPI. Arrows indicate denervated muscle junctions, and asterisks indicate partially innervated neuromuscular junctions. Figure 15B It is a graph showing the percentage of neuromuscular junctions (NMJ; y-axis) innervated in the tibialis anterior (TA) muscle tissue or intercostal muscles isolated from loxP-Ex3-loxP / WT mice (●), ΔNLS / WT mice (▲), or ΔPLD / WT mice (▼). Detailed Implementation

[0043] Overview TDP-43 is a major nuclear RNA / DNA binding protein that plays a role in RNA processing and metabolism, including RNA transcription, splicing, transport, and stabilization. The RNA-binding property of TDP-43, mediated by binding to the 3'UTR sequence in its own mRNA, appears to be crucial to its autoregulatory activity. (Ayala et al., 2011) EMBO J. 30:277-88. Following cellular stress, TDP-43 localizes to cytoplasmic stress granules and can play a role in stress granule formation. TDP-43 mislocalizes from its normal location in the nucleus to the cytoplasm, where it accumulates. Accumulated TDP-43 is ubiquitinated, hyperphosphorylated, and truncated. Furthermore, cytoplasmic TDP-43 accumulation is a component of almost all ALS cases. Becker et al. (2017) Nature 544:367-371. Ninety-seven percent of ALS cases show post-mortem pathology of cytoplasmic TDP-43 aggregates. The same pathology is seen in approximately 45% of sporadic frontotemporal degeneration (FTLDU). TDP-43, initially identified as the major pathological protein of ubiquitin-positive, tau-negative inclusions in FTLDU, FTLD with motor neuron disease (FTDMND), and ALS / MND (ALS10), is now considered to represent different clinical manifestations of TDP-43 proteasome disorders. Gitcho et al. (2009) Acta Neuropath 118:633-645. Occurs in approximately 3% of familial ALS patients and approximately 1.5% of patients with sporadic ALS. TARDBPB Mutation. Lattante et al. (2013) Hum. Mutat. 34:812-26. In less than 1% of cases, TARDBP Various mutations in genes are associated with ALS. See Figure 1 . like Figure 1 As shown, the associations related to ALS TARDBP Most mutations in the gene can be found in the prion-like domain (PLD). Therefore, understanding all the functions of TDP-43 may illuminate its role in neuropathologies such as ALS, FLTDU, and FLTD.

[0044] It is clear that TDP-43 is essential for cellular and organismal life. TDP-43 depletion leads to embryonic death. Therefore, initial models rely on overexpression of TDP-43 or its mutant forms, or TDP-43 depletion. Various models have been created to evaluate the role of TDP-43 in ALS pathology. (In Tsao et al. (2012)...) Brain Res A review was conducted in 1462:26-39.

[0045] For example, transgenic mice overexpressing the TDP-43 A315T mutant exhibited progressive abnormalities at approximately 3 to 4 months of age and died at approximately 5 months of age. (Wegorzewska et al., 2009) Proc Natl Acad Sci USA 106:18809-814. Although the abnormalities were associated with the presence of TDP-43 C-terminal fragments in the brain and spinal cord of these mutant mice, cytoplasmic TDP-43 aggregates were not detected. These observations led Wegorzewska et al. to propose that neuronal susceptibility to TDP-43-related neurodegeneration is associated with alterations in the function of DNA / RNA-binding proteins, rather than with toxic aggregation. Wegorzewska et al. (2009), Same as above. In contrast, in two independent studies involving TDP-43 overexpression, transgenic mice exhibited neurodegenerative properties, including progressive motor dysfunction associated with cytoplasmic aggregation. (Tsai et al., 2010) J. Exp. Med. 207:1661-1673 and Wils et al. (2010) Proc Natl Acad Sci USA 107:3858-63).

[0046] In loss-of-function studies, pervasive deletion of TDP-43 using conditional knockout mutations resulted in metabolic phenotypes and premature death in mice. (Chiang et al., 2010) Proc Natl Acad Sci USA 107:16320-324. TDP-43 depletion in mouse embryonic stem cells leads to the splicing of hidden exons of certain genes into mRNA, disrupting mRNA translation and promoting nonsense-mediated mRNA decay. (Ling et al., 2015) Science349:650-655. Since postmortem brain tissue from ALS / FTD patients showed impaired repression of hidden exon splicing, this study suggests that TDP-43 normally plays a role in repressing hidden exon splicing and maintaining intron integrity, and that TDP-43 splicing defects can lead to TDP-43-protein disorders in certain neurodegenerative diseases. Ling et al. (2015), Same as above Point mutations in the N-terminus (e.g., NLS) of TDP-43 lead to instability in TDP-43 oligomerization in the nucleus and loss of hidden splicing regulation. Therefore, it is hypothesized that N-terminal-driven head-to-tail oligomerization of TDP-43 plays a role in separating easily aggregated C-terminal domains (e.g., PLD), and thus preventing the formation of pathological aggregates. (Afroz et al., 2017) Nature Communications 8:45.

[0047] One of the initial pathological features in ALS is the retraction of axons from the neuromuscular junction, leading to muscle denervation. This denervation continues, resulting in the loss of motor neuron cell bodies and muscle atrophy. Denervation can be observed through the loss of presynaptic markers of axonal innervation: VAChT, synaptic vesicle protein 2 (SV2), synaptophysin, and neurofilaments. Motor endplates remain but eventually break down and disappear. Recently, knock-in mutations containing disease-related mutations have been observed... TARDBP Dose-dependent denervation was observed in homozygous mice of the gene. (Ebstein (2019)) Cell Reports 26:364-373.

[0048] Although TDP-43 depletion leads to embryonic death, embryonic stem (ES) cells expressing TDP-43 mutants lacking functional domains are shown here to remain viable and differentiate into motor neurons (ESMNs). See , Figures 4 to 5 。 The unique aspect of these observations is the expression of the ES or ESMN mutant TDP-43 peptide, as described herein: (1) Lack of functional structural domains, for example, lack of functional NLS, lack of functional RRM1, lack of functional RRM2, lack of functional E, or lack of functional PLD, and (2) It is expressed at normal levels by endogenous transcription promoters and pre-mRNA splicing signals. See, for example, Figure 2 and Figure 3. 9 .

[0049] Using the ES and ESMN described in this paper, it is shown that RRM1 is required for the vitality of ES cells and motor neurons derived from them. See , Figures 4 to 5 Moreover, (1) the absence of functional NLS or functional PLD and (2) the expression of mutant TDP-43 peptide at normal levels from endogenous loci reproduce two hallmarks of ALS disease in ESMN: (i) The redistribution of TDP-43 from the nucleus to the cytoplasm, and (ii) Accumulation in cytoplasmic contents. See , Figure 6 to Figure 8 .

[0050] Surprisingly, the ΔPLD mutant, a TDP-43 peptide containing functional NLS but lacking PLD, accumulates in the cytoplasm. See, for example Afroz et al. (2017), Same as above Notably, the dotted inclusions formed by the ΔPLD mutant appear to be less abundant and different in nature than those formed by the ΔNLS mutant (i.e., a TDP-43 polypeptide lacking functional NLS but containing PLD). Furthermore, the ALS-like phenotype of ESMN expressing ΔPLD or ΔNLS is associated with reduced repression of hidden exon splicing in these genes, splicing events typically regulated by wild-type TDP-43. Figure 9. The ΔPLD or ΔNLS mutations in ESMN are also shown. TARDBP The correlation between gene expression and a reduction in alternative splicing events involving the 3' untranslated region introns, which result in the alternatively spliced ​​TDP-43 mRNA lacking a sequence or a portion thereof encoding the PLD domain and a stop codon. Figure 10 See also Avendano-Vazquez et al. (2012) Genes& Dev. 26:1679-84; Ayala YM, et al. (2011) EMBO J 30: 277–288. The latter observation suggests that depletion of only wild-type or ALS-related sequences resulting from normal splicing events may be a potential therapeutic for ALS associated with PLD mutations.

[0051] Expression of wild-type from endogenous loci TARDBP Genes and ΔPLD or ΔNLS mutations TARDBP Mice expressing the gene also exhibited hallmarks of TDP-43 proteinopathy. Compared to animals expressing only the wild-type protein, increased mislocalization of TDP-43 from the nucleus to the cytoplasm, phosphorylation of cytoplasmic TDP-43, and cytoplasmic aggregation of TDP-43 were observed in spinal motor neurons of animals expressing the mutant ΔPLD or ΔNLS TDP-43 peptides. Figures 13A to 13B and Figure 14The TDP-43 mutant lacking functional NLS, rather than the TDP-43 mutant lacking PLD, is insoluble. Figure 13C Furthermore, in these mice expressing mutant ΔPLD or ΔNLS TDP-43 proteins, denervation of muscles primarily composed of fast-twitch fibers was observed, rather than denervation of muscles primarily composed of slow-twitch fibers. Figures 15A to 15B ).

[0052] The findings presented in this paper provide not only a method for evaluating TDP-43 mutations in viable embryonic stem (ES) cells and tissues, as well as in non-human animals derived from them (e.g., primitive ectoderm, and motor neurons (ESMNs) derived from it), but also ES cells, ESMN cells, and non-human animals expressing mutant TDP-43 peptides lacking functional domains. ES cells, ESMN cells, and non-human animals (e.g., rodents, such as rats and mice) expressing mutant TDP-43 peptides lacking functional domains can also be used, respectively, as in vitro or in vivo models of TDP-43 proteases, for example, in methods for identifying therapeutic candidates.

[0053] TARDBP gene and TDP-43 polypeptide TARDBP The gene encodes the TDP-43 polypeptide, also known as TAR DNA-binding protein, TARDBP, 43-KD, and TDP43, as well as TDP-43. Wild-type TDP-43 exists in different species. TARDBP The gene and the nucleic acid sequence of the wild-type TDP-43 polypeptide it encodes are well known in the art. For example, wild-type TARDBP The corresponding nucleic acid and amino acid sequences of the gene and wild-type TDP-43 polypeptide can be found in the National Center for Biotechnology Information (NCBI) gene database of the National Library of Medicine (NIH). See example like The website is www.ncbi.nlm.nih.gove / gene / ?term=TARDBP. In some implementations, wild-type mice... TARDBP The gene contains a nucleotide sequence encoding a wild-type mouse TDP-43 polypeptide, said polypeptide comprising the amino acid sequence as stated in GenBank accession number NP_663531 (SEQ ID NO:1), or a variant thereof that differs from it due to conserved amino acid substitutions. In some embodiments, wild-type mice TARDBP The gene contains the nucleic acid sequence as stated in GenBank accession number NM_145556.4 (SEQ ID NO:2), or a variant thereof that differs from it due to the degeneracy and / or conserved codon substitutions of the genetic code. In some embodiments, wild-type rats TARDBPThe gene contains a nucleotide sequence encoding a wild-type rat TDP-43 polypeptide, said polypeptide comprising the amino acid sequence as stated in GenBank accession number NP_001011979 (SEQ ID NO:3), or variants thereof that differ from it due to conserved amino acid substitutions. In some embodiments, wild-type rats TARDBP The gene contains the nucleic acid sequence described in GenBank accession number NM_001011979.2 (SEQ ID NO:4), or a variant thereof that differs from it due to degeneracy and / or conserved codon substitutions in the genetic code. In some embodiments, wild-type humans... TARDBP The gene encodes the TDP-43 polypeptide, which contains the amino acids as stated in GenBank accession number NP_031401.1 (SEQ ID NO:5), or variants thereof that differ from them due to conserved amino acid substitutions. In some embodiments, wild-type human... TARDBP The gene contains the nucleic acid sequence as stated in GenBank accession number NM_007375.3 (SEQ ID NO:6), or a variant thereof that differs from it due to the degeneracy of the genetic code and / or conserved codon substitutions.

[0054] This article describes mutations. TARDBP Genes. Mutations TARDBP Genes can contain knockout mutations. Mutations TARDBP The gene can encode a mutant TDP-43 polypeptide, in which the mutant TDP-43 polypeptide lacks a functional domain. For example, the mutant... TARDBP The gene may contain a nucleotide sequence encoding a TDP-43 domain, said domain comprising point mutations, insertions in part or all of the domain, and / or deletions in part or all of the domain, wherein said point mutations, insertions, and / or deletions result in loss of function of the domain, and wherein the mutations... TARDBP The gene still encodes the TDP-43 polypeptide, although the mutant TDP-43 polypeptide lacks a functional domain due to the mutation. The polypeptide can be referred to as the mutant TDP-43 polypeptide, wherein it contains at least one wild-type TDP-43 domain or a variant thereof and / or wherein it is specifically bound by an anti-TDP-43 antibody or its antigen-binding moiety. Similarly, mutations... TARDBP Genes can be classified in this way, among which mutations TARDBP A gene-encoding mutant TDP-43 polypeptide, for example, a polypeptide containing at least one wild-type TDP-43 domain or a variant thereof and / or a polypeptide that can be specifically bound by an anti-TDP-43 antibody or its antigen-binding moiety.

[0055] The domains of TDP-43 have been identified as nuclear localization signal (NLS), two RNA recognition motifs (RRM1 and RRM2), putative nuclear export signal (E), and glycine-rich prion-like domain (PLD). See Figure 1 And Figure 2. Wild-type TDP-43 peptides include TDP-43 NLS at amino acid 82-99, TDP-43 RRM1 at amino acid 106-176, TDP-43 RRM2 at amino acid 191-262, TDP-43 E at amino acid 239-248, and TDP-43 PLD at amino acid 274-414.

[0056] The classic NLS sequence contains a basic amino acid segment, primarily lysine (K) and arginine (R) residues, and the bipartite NLS contains two clusters of these basic amino acids separated by a linker region containing approximately 10–13 amino acids. Amino acid substitutions and / or deletions in the basic amino acid sequence of the classic NLS can eliminate its function. McLane and Corbett (2009) IUBMB Life 61:697-706. The TDP-43 NLS contains lysine and arginine residues at positions 82, 83, 84, 95, 97, and 98. Wild-type TDP-43 peptides modified to contain amino acid substitutions and / or deletions at positions 82, 83, 84, 95, 97, and / or 98 may lack functional NLS. Mutant TDP-43 peptides lacking functional NLS may contain the amino acid sequence stated in SEQ ID NO:1, which is modified to contain amino acid substitutions and / or deletions at positions 82, 83, 84, 95, 97, and / or 98. Mutant TDP-43 peptides lacking functional NLS may contain the amino acid sequence stated in SEQ ID NO:3, which is modified to contain amino acid substitutions and / or deletions at positions 82, 83, 84, 95, 97, and / or 98. The mutant TDP-43 polypeptide lacking functional NLS may comprise the amino acid sequence stated in SEQ ID NO:5, said amino acid sequence being modified to include amino acid substitutions and / or deletions at positions 82, 83, 84, 95, 97 and / or 98. Therefore, mutations encoding the mutant TDP-43 protein lacking functional TDP-43 NLS... TARDBPThe gene may contain a sequence encoding a TDP-43 polypeptide comprising a sequence as stated in SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, said sequence being modified by substitutions of amino acids at positions consisting of (i) positions selected from 82, 83, 84, 95, 97, and / or 98 and combinations thereof, and / or (ii) deletions of any amino acids at positions 82 and 98 and between them. Mutations encoding mutant TDP-43 proteins lacking functional TDP-43 NLS. TARDBP The gene may comprise a nucleotide sequence encoding an amino acid sequence as stated in SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, said amino acid sequence being modified with amino acid substitutions comprising the group consisting of K82A, K83A, R84A, K95A, K97A, K98A, or combinations thereof. Mutations encoding a mutant TDP-43 protein lacking functional TDP-43 NLS. TARDBP The gene may contain a nucleotide sequence encoding an amino acid sequence as stated in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5, said amino acid sequence being modified to include the following amino acids as substitutes: K82A, K83A, R84A, K95A, K97A and K98A.

[0057] RNA binding generated by a typical RRM is usually achieved through contact between the four-stranded antiparallel β-sheet surface of the typical RRM and the single-stranded RNA. (Melamed et al., 2013) RNA 19:1537-1551. Two highly conserved motifs in the two central β chains, RNP1 (containing K / RGF / YG / AF / YV / I / LXF / Y, where X is any amino acid) and RNP2 (containing I / V / LF / YI / V / LXNL, where X is any amino acid), are the main mediators of RNA binding. Melamed et al. (2013), Same as above .

[0058] The TDP-43 RRM1 located at amino acid positions 106-176 of the wild-type TDP-43 polypeptide contains a shared RNP2 sequence at amino acid positions 106-111 (LIVLGL; SEQ ID NO:7) and a shared RNP1 sequence at amino acid positions 145-152 (KGFGFVRF; SEQ ID NO:8). Previously, W113, T115, F147, F149, D169, R171, and N179 were identified as key residues for nucleic acid binding. Wild-type TDP-43 peptides modified with amino acid substitutions at positions consisting of (i) the group consisting of (i) amino acid substitutions at positions 113, 115, 147, 149, 169, 171, 179 and any combination thereof, (ii) deletions or substitutions of any amino acid at and between positions 106-176, (iii) deletions or substitutions of any amino acid at and between positions 106-111, (iv) deletions or substitutions of any amino acid at and between positions 145-152, or (v) any combination of (i)-(iv) may lack functional RRM1. The mutant TDP-43 polypeptide lacking functional RRM1 may comprise the sequence as stated in SEQ ID NO:1, said sequence being modified to include (i) amino acid substitutions at positions selected from the group consisting of 113, 115, 147, 149, 169, 171, 179 and any combination thereof, (ii) deletions or substitutions of any amino acid at and between positions 106-176, (iii) deletions or substitutions of any amino acid at and between positions 106-111, (iv) deletions or substitutions of any amino acid at and between positions 145-152, or (v) any combination of (i)-(iv). The mutant TDP-43 polypeptide lacking functional RRM1 may comprise the sequence as stated in SEQ ID NO:3, said sequence being modified to include (i) amino acid substitutions at positions selected from the group consisting of 113, 115, 147, 149, 169, 171, 179 and any combination thereof, (ii) deletions or substitutions of any amino acid at and between positions 106-176, (iii) deletions or substitutions of any amino acid at and between positions 106-111, (iv) deletions or substitutions of any amino acid at and between positions 145-152, or (v) any combination of (i)-(iv).The mutant TDP-43 polypeptide lacking functional RRM1 may comprise the sequence as stated in SEQ ID NO:5, said sequence being modified with amino acid substitutions at positions selected from the group consisting of 113, 115, 147, 149, 169, 171, 179 and any combination thereof, (ii) deletions or substitutions of any amino acid at and between positions 106-176, (iii) deletions or substitutions of any amino acid at and between positions 106-111, (iv) deletions or substitutions of any amino acid at and between positions 145-152, or (v) any combination of (i)-(iv). Thus, a mutation encoding the mutant TDP-43 polypeptide lacking functional RRM1 is formed. TARDBP The gene may contain a nucleotide sequence encoding a TDP-43 polypeptide comprising an amino acid sequence as stated in SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, said amino acid sequence being modified to include (i) amino acid substitutions at positions selected from the group consisting of 113, 115, 147, 149, 169, 171, 179, and any combination thereof, (ii) deletions or substitutions of any amino acid at positions 106-176 and between, (iii) deletions or substitutions of any amino acid at positions 106-111 and between, (iv) deletions or substitutions of any amino acid at positions 145-152 and between, or (v) any combination of (i)-(iv). Mutations encoding a mutant TDP-43 polypeptide lacking functional RRM1. TARDBP The gene may contain a nucleotide sequence encoding a TDP-43 polypeptide comprising an amino acid sequence as stated in SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, wherein the amino acid sequence is modified to include the F147L and / or F149L mutations. Mutations encoding a mutant TDP-43 polypeptide lacking functional RRM1. TARDBP The gene may contain a nucleotide sequence encoding a TDP-43 polypeptide comprising an amino acid sequence as stated in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5, wherein the amino acids are modified to include the following amino acids: F147L and F149L.

[0059] The TDP-43 RRM2 located at amino acid positions 191-262 of the wild-type TDP-43 polypeptide contains a shared RNP2 sequence at amino acid positions 193-198 (VFVGRC; SEQ ID NO: 9) and a shared RNP1 sequence at amino acid positions 227-233 (RAFAFVT; SEQ ID NO: 10). F194 and F229 can be considered key residues for nucleic acid binding. Wild-type TDP-43 polypeptides modified with amino acid substitutions at positions selected from the group consisting of (i) substituted amino acids of (i) 194 and / or 229, (ii) deletions or substitutions of any amino acid at and between positions 193-198, (iii) deletions or substitutions of any amino acid at and between positions 227-233, (iv) deletions or substitutions of any amino acid at and between positions 191-262, or (v) any combination of (i)-(iv) may lack functional RRM2. The mutant TDP-43 polypeptide lacking functional RRM2 may comprise the sequence as stated in SEQ ID NO:1, said sequence being modified to include (i) amino acid substitutions at positions selected from the group consisting of 194 and / or 229, (ii) deletions or substitutions of any amino acid at and between positions 193-198, (iii) deletions or substitutions of any amino acid at and between positions 227-233, (iv) deletions or substitutions of any amino acid at and between positions 191-262, or (v) any combination of (i)-(iv). The mutant TDP-43 polypeptide lacking functional RRM2 may comprise the sequence as stated in SEQ ID NO:3, said sequence being modified to include (i) amino acid substitutions at positions selected from the group consisting of 194 and / or 229, (ii) deletions or substitutions of any amino acid at and between positions 193-198, (iii) deletions or substitutions of any amino acid at and between positions 227-233, (iv) deletions or substitutions of any amino acid at and between positions 191-262, or (v) any combination of (i)-(iv). The mutant TDP-43 polypeptide lacking functional RRM2 may comprise the sequence as stated in SEQ ID NO:5, said sequence being modified to include (i) amino acid substitutions at positions selected from the group consisting of 194 and / or 229, (ii) deletions or substitutions of any amino acid at and between positions 193-198, (iii) deletions or substitutions of any amino acid at and between positions 227-233, (iv) deletions or substitutions of any amino acid at and between positions 191-262, or (v) any combination of (i)-(iv). Thus, mutations encoding the mutant TDP-43 polypeptide lacking functional RRM2... TARDBPThe gene may contain a nucleotide sequence encoding a TDP-43 polypeptide comprising an amino acid sequence as stated in SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, wherein the amino acid sequence is modified to include (i) amino acid substitutions at positions 194 and / or 229 of the wild-type TDP-43 polypeptide, (ii) deletions or substitutions of any amino acid at positions 191-262 and between, or (iii) both (i) and (ii). Mutations encoding a mutant TDP-43 polypeptide lacking functional RRM2. TARDBP The gene may contain a nucleotide sequence encoding a TDP-43 polypeptide comprising an amino acid sequence as stated in SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, wherein the amino acid sequence is modified to include the F194L and / or F229L mutations. Mutations encoding a mutant TDP-43 polypeptide lacking functional RRM2 are also possible. TARDBP The gene may contain a nucleotide sequence encoding a TDP-43 polypeptide comprising an amino acid sequence as stated in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5, wherein the amino acid sequence is modified to include the F194L and F229L mutations.

[0060] The nuclear export signal of the wild-type TDP-43 polypeptide may be located at amino acids 239-248. A mutant TDP-43 polypeptide lacking a functional nuclear export signal may comprise the amino acid sequence as stated in SEQ ID NO:1, said amino acid sequence being modified to include the deletion of any amino acid in and between positions 236-251. A mutant TDP-43 polypeptide lacking a nuclear export signal may comprise the amino acid sequence as stated in SEQ ID NO:1, said amino acid sequence being modified to include the deletion of at least amino acids 239-250. A mutant TDP-43 polypeptide lacking a nuclear export signal may comprise the amino acid sequence as stated in SEQ ID NO:3, said amino acid sequence being modified to include the deletion of any amino acid in and between positions 236-251. A mutant TDP-43 polypeptide lacking a nuclear export signal may comprise the amino acid sequence as stated in SEQ ID NO:3, said amino acid sequence being modified to include the deletion of at least amino acids 239-250. The mutant TDP-43 polypeptide lacking a nuclear output signal may comprise the amino acid sequence as stated in SEQ ID NO:5, said amino acid sequence being modified to include the deletion of any amino acid in and between positions 236-251. The mutant TDP-43 polypeptide lacking a nuclear output signal may comprise the amino acid sequence as stated in SEQ ID NO:5, said amino acid sequence being modified to include the deletion of at least amino acids 239-250. Therefore, mutations encoding the mutant TDP-43 polypeptide lacking a functional nuclear output signal... TARDBP The gene may contain a nucleotide sequence encoding a TDP-43 polypeptide comprising an amino acid sequence as stated in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5, wherein the amino acid sequence is modified to include the deletion of amino acids at and between positions 236-251, for example, the deletion of amino acids at and between positions 239-250.

[0061] The prion-like domain (PLD) of the wild-type TDP-43 polypeptide may be located at amino acids 274-414. A mutant TDP-43 polypeptide lacking a functional PLD may comprise the amino acid sequence as stated in SEQ ID NO:1, said amino acid sequence being modified to include the deletion of at least one or all amino acids at and between positions 274-414. A mutant TDP-43 polypeptide lacking a functional PLD may comprise the amino acid sequence as stated in SEQ ID NO:3, said amino acid sequence being modified to include the deletion of at least one or all amino acids at and between positions 274-414. A mutant TDP-43 polypeptide lacking a functional PLD may comprise the amino acid sequence as stated in SEQ ID NO:5, said amino acid sequence being modified to include the deletion of at least one or all amino acids at and between positions 274-414. Therefore, mutations encoding the mutant TDP-43 polypeptide... TARDBP The gene may contain a nucleotide sequence encoding a TDP-43 polypeptide comprising an amino acid sequence as stated in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5, wherein the amino acid sequence is modified to include the deletion of at least one or all of the amino acids at and between positions 274-414.

[0062] mutation TARDBP Genes can contain Figure 3A The structure described in the text. Mutation. TARDBP Genes can encode Figure 3A The mutant TDP-43 polypeptide is described in the text.

[0063] Methods for preparing cells and non-human animals containing and expressing the mutant TARDBP gene As described above, the methods and compositions provided herein allow for the... TARDBP Locus for targeted genetic modification, for example, to produce gene sequences containing mutations. TARDBP The genes are used in the cells and / or to evaluate the biological function of the TDP-43 domain. It is also recognized that additional targeted genetic modifications can be performed. Such systems that allow for these targeted genetic modifications can employ a variety of components, and for ease of reference, the term "targeted genome integration system" in this document generally includes all components required for the integration event (i.e., various nucleases, recognition sites, inserted DNA polynucleotides, targeting vectors, target genome loci, etc.).

[0064] Methods for preparing non-human animal cells expressing mutant TDP-43 peptides and / or for evaluating the biological function of TDP-43 domains may include modifying the cell genome to include the mutant. TARDBP Genes. Mutations TARDBP The gene can encode a mutant TDP-43 polypeptide, wherein the mutant TDP-43 polypeptide lacks a functional domain.

[0065] Methods for preparing non-human animal cells expressing mutant TDP-43 peptides and / or for evaluating the biological function of TDP-43 domains may include modifying the cell genome to include the mutant. TARDBP Genes, wherein the mutations TARDBP Genes contain knockout mutations.

[0066] The methods described herein involve introducing one or more polynucleotide or polypeptide constructs containing various components of a targeted genome integration system into cells. "Introduction" means presenting the sequence (polynucleotide or polypeptide) to the cell in a manner that the sequence enters the cell. The methods described herein do not rely on a specific method for introducing any component of a targeted genome integration system into the cell, but only on the entry of the polynucleotide into at least one cell. Methods for introducing polynucleotides into various cell types are known in the art and include, but are not limited to, stable transfection methods, transient transfection methods, and virus-mediated methods.

[0067] In some embodiments, the cells used in the methods and compositions have DNA constructs that are stably incorporated into their genome. "Stable incorporation" or "stable introduction" means introducing a polynucleotide into a cell such that the nucleotide sequence is integrated into the cell's genome and can be inherited by its offspring. Any approach can be used for various components of a stable incorporation DNA construct or a targeted genome integration system.

[0068] Transfection protocols, and the protocols used to introduce peptide or polynucleotide sequences into cells, can vary. Non-restrictive transfection methods include chemical-based methods, including those using liposomes; nanoparticles; calcium phosphate (Graham). et al. (1973). Virology 52 (2): 456–67, Bacchetti et al. (1977) Proc Natl Acad Sci USA 74(4): 1590–4 and Kriegler, M (1991). Transfer and Expression: A Laboratory Manual (New York: WH Freeman and Company. pp. 96–97); dendritic polymers; or cationic polymers, such as DEAE-dextran or polyethyleneimine. Non-chemical methods include electroporation, ultrasonic perforation, and optical transfection. Particle-based transfection includes the use of gene guns, magnet-assisted transfection (Bertram, J. (2006)). Current Pharmaceutical Biotechnology 7, 277–28). Viral methods can also be used for transfection.

[0069] Various methods disclosed herein can be used to generate mutations. TARDBPCellular modification of genes may include replacing the endogenous TARDBP gene with a mutant TARDBP gene encoding a mutant TDP-43 polypeptide. TARDBP The modification may involve replacing the endogenous TARDBP gene with a TARDBP gene containing a knockout mutation (such as a conditional knockout mutation). The modification may include eliminating the TARDBP gene containing the knockout mutation. TARDBP Cells are cultured under conditions that promote gene expression. This can eliminate... TARDBP Conditions for gene expression may include the expression of recombinase proteins, such as cre-recombinase.

[0070] Such modification methods may include (1) using the methods disclosed herein on the target of interest in pluripotent cells of non-human animals. TARDBP Integration mutations at genomic loci TARDBP Genes are produced in targeted TARDBP Mutations are contained in genomic loci TARDBP Pluripotent cells with genetically modified genes; and (2) selection on the target TARDBP Mutations at genomic loci TARDBP Genetically modified pluripotent cells. Animals can be further produced by (3) introducing genetically modified pluripotent cells into a host embryo of a non-human animal, for example, at the premorula stage; and (4) implanting the host embryo containing the genetically modified pluripotent cells into a surrogate mother to produce an F0 generation derived from the genetically modified pluripotent cells. The non-human animal can be a non-human mammal, rodent, mouse, rat, hamster, monkey, agricultural mammal or domesticated mammal, or fish or bird.

[0071] Pluripotent cells can be human ES cells, non-human ES cells, rodent ES cells, mouse ES cells, rat ES cells, hamster ES cells, monkey ES cells, agricultural mammal ES cells, or domesticated mammal ES cells. In other embodiments, pluripotent cells are non-human cells, mammalian cells, human cells, non-human mammalian cells, human pluripotent cells, human ES cells, human adult stem cells, developmentally restricted human progenitor cells, human iPS cells, rodent cells, rat cells, mouse cells, or hamster cells. In one embodiment, targeted genetic modification leads to mutations. TARDBP Gene.

[0072] Mouse pluripotent cells, totipotent cells, or host embryos can be derived from any mouse strain, including, for example, inbred, hybrid, and outbred strains. Examples of mouse strains include the 129 strain, the C57BL strain (e.g., the C57BL / 6 strain), a mixture of 129 and C57BL / 6 (e.g., 50% 129 and 50% C57BL / 6), the BALB / c strain, and the Swiss Webster strain. Examples of the 129 strain include 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 12951 / SV, 12951 / SvIm), 129S2, 129S4, 129S5, 12959 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, and 129T2 (…). See example like Festival et al. (1999) Revised nomenclature for strain 129 mice, Mammalian Genome 10:836). Examples of C57BL strains include C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr, and C57BL / 01a. Mice can be a mixture of the above-mentioned 129 strains (e.g., the 129S6 (129 / SvEvTac) strain) and the above-mentioned C57BL / 6 strain, a mixture of one or more of the above-mentioned 129 strains, or a mixture of one or more of the above-mentioned C57BL strains. Mice can also be derived from strains other than 129.

[0073] Rat pluripotent cells, totipotent cells, or host embryos can be derived from any rat strain, including, for example, inbred, hybrid, and outbred strains. Examples of rat strains include the ACI rat strain, the Dark Agouti (DA) rat strain, the Wistar rat strain, the LEA rat strain, the Sprague Dawley (SD) rat strain, or the Fisher rat strain, such as Fisher F344 or Fisher F6. Rat pluripotent cells, totipotent cells, or host embryos can also be obtained from strains derived from a mixture of two or more of the above strains. For example, rat pluripotent cells, totipotent cells, or host embryos can be derived from a strain selected from both the DA and ACI strains. The ACI rat strain is characterized by its black-gray coloration and white abdomen and paws. RT1 av1Haplotype. This type of strain can be obtained from various sources, including Harlan Laboratories. An example of a rat ES cell line derived from ACI rats is the ACI.G1 rat ES cell. The Dark Agouti (DA) rat strain is characterized by its wild gray fur and... RT1 av1 Haplotype. These rats are available from a variety of sources, including Charles River and Harlan Laboratories. Examples of rat ES cell lines derived from DA rats are the DA.2B or DA.2C rat ES cell lines. Other examples of rat strains are provided, for example, in US 2014 / 0235933, US 2014 / 0310828, and US 2014 / 0309487, each of which is incorporated herein by reference in its entirety for all purposes.

[0074] For example, germline-transferable rat ES cells can be obtained by culturing isolated rat ES cells on a feeder cell layer in a culture medium containing N2 supplementation, B27 supplementation, about 50 U / mL to about 150 U / mL leukemia suppressor factor (LIF), and an inhibitor combination consisting of a MEK inhibitor and a GSK3 inhibitor, wherein the feeder cell layer is not modified to express LIF, and wherein the rat ES cells: (i) have been modified to include a targeted genetic modification comprising the insertion of at least one heterologous polynucleotide containing a selection marker into the genome of the rat ES cells and are germline-transferable; (ii) have a normal karyotype; (iii) lack c-Myc expression; and (iv) form spherical, free-floating colonies in culture (see, for example, US 2014-0235933 A1 and US 2014-0310828A1, each of which is incorporated herein by reference in its entirety). For example, other examples of rat embryonic stem cell derivation and targeted modification are provided in Yamamoto et al. (“Derivation of rat embryonic stem cells and generation of protease-activated receptor-2 knockout rats,” Transgenic Res. 21:743-755, 2012) and Kwamata and Ochiya (“Generation of genetically modified rats from embryonic stem cells,” Proc.Natl. Acad. Sci. USA 107(32):14223-14228, 2010).

[0075] Nuclear transfer technology can also be used to generate non-human animals. In short, methods for nuclear transfer include the following steps: (1) enucleating an oocyte; (2) separating a donor cell or nucleus to be combined with the enucleated oocyte; (3) inserting the cell or nucleus into the enucleated oocyte to form a reconstructed cell; (4) implanting the reconstructed cell into the uterus of an animal to form an embryo; and (5) allowing the embryo to develop. In such methods, the oocytes are generally removed from a dead animal, although they can also be separated from the oviduct and / or ovary of a living animal. The oocytes can be matured in a variety of culture media known to those skilled in the art prior to enucleation. Enucleation of the oocyte can be performed in a variety of ways well known to those skilled in the art. Insertion of a donor cell or nucleus into the enucleated oocyte to form a reconstructed cell is typically performed prior to fusion by microinjecting the donor cell into the zona pellucida. Fusion can be induced by applying a DC electrical pulse (electrofusion) to the contact / fusion plane, by exposing the cell to a fusion-promoting chemical substance (such as polyethylene glycol), or by inactivating a virus (such as Sendai virus). Reconstructed cells are typically activated by electrical and / or non-electrical means before, during, and / or after the fusion of donor and recipient oocytes. Activation methods include electrical pulses, chemically induced shock, sperm penetration, increasing the level of divalent cations in the oocyte, and decreasing the phosphorylation of cellular proteins in the oocyte (e.g., by kinase inhibitors). Activated reconstructed cells or embryos are typically cultured in media well known to those skilled in the art and then transferred to the uterus of an animal. See, for example, US20080092249, WO / 1999 / 005266A2, US20040177390, WO / 2008 / 017234A1, and U.S. Patent No. 7,612,250, each of which is incorporated herein by reference.

[0076] Other methods are provided for preparing nonhuman animals whose germline contains one or more genetic modifications as described herein, comprising: (a) modifying the target genome of the nonhuman animal in prokaryotic cells using the various methods described herein. TARDBP (a) selecting a genetically modified prokaryotic cell containing the genetic modification at the target genomic locus; (b) isolating the genetically modified targeting vector from the genome of the modified prokaryotic cell; and (d) introducing the genetically modified targeting vector into a pluripotent cell of a non-human animal to produce a target locus. TARDBPThe method involves: (e) selecting genetically modified pluripotent cells containing inserted nucleic acids at genomic loci; (f) introducing the genetically modified pluripotent cells into a non-human animal host embryo at the premorula stage; and (g) implanting the host embryo containing the genetically modified pluripotent cells into a surrogate mother to produce an F0 generation derived from the genetically modified pluripotent cells. In such methods, the targeting vector may include a large targeting vector. The non-human animal may be a non-human mammal, rodent, mouse, rat, hamster, monkey, agricultural mammal, or domesticated mammal. The pluripotent cell may be a human ES cell, a non-human ES cell, a rodent ES cell, a mouse ES cell, a rat ES cell, a hamster ES cell, a monkey ES cell, an agricultural mammal ES cell, or a domesticated mammal ES cell. In other embodiments, the pluripotent cell is a non-human cell, a mammalian cell, a human cell, a non-human mammalian cell, a human pluripotent cell, a human ES cell, a human adult stem cell, a developmentally restricted human progenitor cell, a human iPS cell, a human cell, a rodent cell, a rat cell, a mouse cell, or a hamster cell. In one implementation, targeted genetic modification leads to mutations. TARDBP Mutations in genes, such as those encoding the mutant TDP-43 polypeptide lacking a functional domain. TARDBP Genes and / or mutations containing knockout mutations TARDBP Gene.

[0077] In a further method, the isolation step (c) further includes (c1) linearizing the genetically modified targeting vector (i.e., the genetically modified LTVEC). In a further embodiment, the introduction step (d) further includes (d1) introducing a nuclease agent into pluripotent cells to promote homologous recombination. In one embodiment, the selection steps (b) and / or (e) are performed by applying a selection agent as described herein to prokaryotic or pluripotent cells. In one embodiment, the selection steps (b) and / or (e) are performed via an allele modification (MOA) assay as described herein.

[0078] In some implementations, various genetic modifications of the target genomic loci described herein can be performed using VELOCIGENE® genetic engineering technology, employing LTVECs derived from bacterial artificial chromosome (BAC) DNA in bacterial cells via a series of homologous recombination reactions (BHR) (see [link to implementation details]). For example U.S. Patent No. 6,586,251 and Valenzuela, DM et al. (2003), Nature Biotechnology 21(6): 652-659, the patents and documents mentioned are incorporated herein by reference in their entirety.

[0079] In some implementations, targeted pluripotent and / or totipotent cells containing the various genetic modifications described herein are used as insert donor cells and introduced from the corresponding organism via the VELOCIMOUSE® method. For example In the premorula stage of 8-cell mouse embryos (see...) For example US 7,576,259, US 7,659,442, US 7,294,754, and US2008-0078000 A1, all of which are incorporated herein by reference in their entirety. Non-human animal embryos containing genetically modified pluripotent and / or totipotent cells are incubated to the blastocyst stage and then implanted into a surrogate mother to produce F0 generation. In some embodiments, targeted mammalian ES cells containing the various genetic modifications described herein are introduced into the blastocyst stage embryo. The genetically modified genomic loci (i.e., alleles modified with alleles) can be identified via allele modification (MOA) assays as described herein. TARDBP Non-human animals (genotype loci). The resulting F0 generation non-human animals, derived from genetically modified pluripotent and / or totipotent cells, are crossed with wild-type non-human animals to obtain F1 generation offspring. After genotyping using specific primers and / or probes, F1 non-human animals heterozygous for the genetically modified genomic loci are crossed with each other to produce F2 generation non-human animal offspring homozygous for the genetically modified genomic loci.

[0080] In one embodiment, a method for preparing a mixture containing mutations is provided. TARDBP A cellular approach to gene generation. This method includes: (a) contacting a pluripotent cell with a target construct, the target construct containing a mutation. TARDBP A gene or a mutated portion thereof flanked by 5' and 3' homologous arms; wherein the targeted construct is related to the cellular genome. TARDBP The locus undergoes homologous recombination to form modified pluripotent cells. Methods for preparing non-human animals further include (b) introducing the modified pluripotent cells into a host embryo; and (c) gestating the host embryo in a surrogate mother, wherein the surrogate mother produces cells containing the modified pluripotent cells. TARDBP The offspring of the locus, wherein the genetic modification results in a mutant TDP-43 polypeptide lacking a functional domain.

[0081] In some implementations, mutations are included. TARDBP Genes in cells can be modified to include mutations in ES cells. TARDB Genes are generated and ES cells are cultured in vitro in a differentiation medium. In some embodiments, in vitro culture of ES cells includes differentiating ES cells into primitive ectodermal cells or embryonic stem cell-derived motor neurons (ESMNs).

[0082] Cells and animals The cells disclosed herein (which may be contained in non-human animal tissues or within non-human animals) may contain mutations as disclosed herein. TARDBP Any type of cell containing genes. Cells can contain mutations from non-human animals. TARDBP Genes (e.g., mutations in non-human animals) TARDBP People with genes or mutations TARDBP Gene.

[0083] The cell may contain a mutated TARDBP gene encoding a mutant TDP-43 polypeptide lacking a functional domain, and the cell expresses the mutant TDP-43 polypeptide. For example, the cell may contain a mutated TARDBP gene encoding a mutant TDP-43 polypeptide lacking a functional domain. TARDBP The gene, wherein the functional domains include nuclear localization signal (NLS), RNA recognition motif 1 (RRM1), RNA recognition motif 2 (RRM2), putative nuclear export signal (E), prion-like domain (PLD), or combinations thereof. Cells may contain mutations. TARDBP A gene encoding a mutant TDP-43 polypeptide lacking a functional domain due to one or more of the following: (a) a point mutation in amino acids in the NLS (e.g., K82A, K83A, R84A, K95A, K97A, K98A, or a combination thereof), (b) a point mutation in amino acids in the RRM1 (e.g., F147L and / or F149L), (c) a point mutation in amino acids in the RRM2 (F194L and / or F229L), (d) a deletion of at least a portion of the nuclear output signal (e.g., a deletion of amino acids at and between positions 239 and 250 in the wild-type TDP-43 protein), and (e) a deletion of at least a portion of the prion-like domain (e.g., a deletion of amino acids at and between positions 274 and 414 in the wild-type TDP-43 polypeptide). Cells may contain mutations encoding the mutant TDP-43 polypeptide. TARDBP The mutant TDP-43 polypeptide contains the following mutations: K82A, K83A, R84A, K95A, K97A, and K98A, wherein the mutant TDP-43 polypeptide lacks functional NLS. Cells may contain mutations encoding the mutant TDP-43 polypeptide. TARDBP The mutant TDP-43 polypeptide comprises amino acids between positions 274 and 414 of the wild-type TDP-43 polypeptide and includes the deletion of amino acids at said positions, wherein the mutant TDP-43 polypeptide lacks a functional PLD. Cells may contain mutations encoding the mutant TDP-43 polypeptide. TARDBPThe mutant TDP-43 polypeptide contains point mutations F147L and F149L, wherein the mutant TDP-43 polypeptide lacks functional RRM1. Cells may contain mutations encoding the mutant TDP-43 polypeptide. TARDBP The TDP-43 mutant polypeptide contains point mutations F194L and F229L, wherein the mutant polypeptide lacks functional RRM2. Cells may contain mutations encoding the mutant TDP-43 polypeptide. TARDBP The mutant TDP-43 polypeptide contains the deletion of the nuclear output signal between amino acids at positions 239 and 250 of the wild-type TDP-43 polypeptide and includes the amino acids at said positions, wherein the mutant TDP-43 polypeptide lacks functional E.

[0084] Cells can contain mutations TARDBP Genes, which include knockout mutations, such as conditional knockout mutations, TARDBP Deletion of the entire coding sequence of a gene, etc. Cells can contain mutations. TARDBP Genes, which contain conditional knockout mutations, for example, mutations TARDBP Genes may contain site-specific recombination recognition sequences, such as loxp Sequence. Cells can contain mutations. TARDBP Genes that contain exons (e.g., exon 3) flanked by the TDP-43 coding sequence. loxp Sequence. Cells can contain mutations. TARDBP Genes, which contain loxp The sequence is missing, and the coding sequence for TDP-43, such as exon 3, is also lacking. Cells may contain mutations. TARDBP Genes that lack the entire coding sequence of TDP-43, such as mutations that contain the entire coding sequence of the TDP-43 polypeptide deletion. TARDBP Gene.

[0085] In some implementations, the cell may contain mutations. TARDBP Genes, which are inserted into endogenous TARDBP At the gene locus, for example, in its germline genome. In some implementations, the cell contains mutations. TARDBP Genes, such as those containing knockout mutations TARDBP Mutations in genes and / or encoding the mutant TDP-43 polypeptide TARDBP Genes, in their endogenous form TARDBP Replacement of endogenous genes at loci TARDBP Genes. In some implementations, mutations TARDBP Genes and endogenous TARDBP The promoter and / or control element are operatively connected.

[0086] For mutations TARDBPGenes and cells can be heterozygous or homozygous. Diploid organisms have two alleles, one at each locus on a pair of homologous chromosomes. Each pair of alleles represents the genotype at a particular locus. If two identical alleles are present at a particular locus, the genotype is described as homozygous, and if the two alleles are different, the genotype is described as heterozygous.

[0087] Cells may contain (i) endogenous TARDBP Endogenous at locus TARDBP The gene is encoded by a mutation of the mutant TDP-43 polypeptide. TARDBP Gene substitution, and (ii) other endogenous genes on homologous chromosomes TARDPP Mutations containing knockout mutations at gene loci TARDBP Gene.

[0088] Includes mutations TARDBP Cells can express the mutant TDP-43 polypeptide encoded by the gene. (Contains mutations.) TARDBP Cells that express the mutant TDP-43 polypeptide encoded by the gene may or may not express the wild-type TDB-43 polypeptide.

[0089] Includes mutations TARDBP The cell can express the mutant TDP-43 polypeptide encoded by the gene, and is characterized by one or more of the following: (i) mutation TARDBP The mRNA transcript levels of the gene were compared with those of wild-type control cells. TARDBP The levels of the gene's mRNA transcripts were comparable, (ii) the level of mutant TDP-43 peptide was increased compared to the level of wild-type TDP-43 peptide in control cells, (iii) the concentration of mutant TDP-43 peptide in the cytoplasm of cells was found to be higher than that in the nucleus, (iv) mutant TDP-43 peptide showed increased insolubility compared to wild-type TDP-43 peptide, (v) cytoplasmic aggregates containing mutant TDP-43 peptide, (vi) increased splicing of hidden exons of the gene compared to cells expressing wild-type TDP-43, and (vii) the level of TDP-43 mRNA lacking alternative splicing of the sequence encoding TDP-43 PLD was reduced.

[0090] Cells can be cultured in vitro and examined either in vitro or in vivo. For example, cells can be cultured in animals.

[0091] Cells can be eukaryotic cells, including, for example, fungal cells (e.g., yeast), plant cells, animal cells, mammalian cells, non-human mammalian cells, and human cells. The term "animal" includes any member of the animal kingdom, including, for example, mammals, fish, reptiles, amphibians, birds, and worms. Mammalian cells can be, for example, non-human mammalian cells, rodent cells, rat cells, mouse cells, or hamster cells. Other non-human mammals include, for example, non-human primates, monkeys, apes, orangutans, cats, dogs, rabbits, horses, bulls, deer, bison, livestock (e.g., cattle species such as cows, steers, etc.; sheep species such as sheep, goats, etc.; and pig species such as pigs and boars). Birds include, for example, chickens, turkeys, ostriches, geese, ducks, etc. Domesticated animals and agricultural animals are also included. The term "non-human" does not include humans. In some embodiments: animals can be human or non-human animals, including but not limited to mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including but not limited to monkeys and chimpanzees. In some implementations, the non-human animal cells are rodent cells, such as rat cells or mouse cells.

[0092] Non-human animals can be from any genetic background. For example, suitable mice can be from the 129 strain, the C57BL / 6 strain, a mixture of 129 and C57BL / 6, the BALB / c strain, or the Swiss Webster strain. Examples of the 129 strain include 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / Svlm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, and 129T2. See, for example Festing et al. (1999) Mammalian Genome 10:836, the referenced document is incorporated herein by reference in its entirety for all purposes. Examples of the C57BL strain include C57BL / A, C57BL / An, C57BL / GrFa, C57BL / Kal_wN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr, and C57BL / Ola. Suitable mice may also be derived from a mixture of the above-mentioned 129 strain and the above-mentioned C57BL / 6 strain (e.g., 50% 129 and 50% C57BL / 6). Similarly, suitable mice may be derived from a mixture of the above-mentioned 129 strain or a mixture of the above-mentioned BL / 6 strain (e.g., the 129S6 (129 / SvEvTac) strain).

[0093] Similarly, rats can be derived from any rat strain, including, for example, the ACI rat strain, the Dark Agouti (DA) rat strain, the Wistar rat strain, the LEA rat strain, the Sprague Dawley (SD) rat strain, or the Fischer rat strain, such as Fisher F344 or Fisher F6. Rats can also be obtained from hybrid strains derived from two or more of the above strains. For example, suitable rats could be derived from the DA or ACI strains. The ACI rat strain is characterized by its black-gray coloration and white abdomen and feet. RT1 av1 Haplotype. This strain can be obtained from various sources, including Harlan Laboratories. The Dark Agouti (DA) rat strain is characterized by its wild gray fur and... RT1 av1 Haplotype. These rats can be obtained from a variety of sources, including Charles River and Harlan Laboratories. Some suitable rats may be derived from inbred rat strains. See, for example US 2014 / 0235933, the patent described herein is incorporated herein by reference in its entirety for all purposes.

[0094] Cells can also be in any type of undifferentiated or differentiated state. For example, cells can be totipotent, pluripotent (e.g., human pluripotent cells or non-human pluripotent cells, such as mouse embryonic stem (ES) cells or rat ES cells), or non-pluripotent cells. Totipotent cells include undifferentiated cells capable of producing any cell type, and pluripotent cells include undifferentiated cells with the ability to develop into more than one differentiated cell type. Such pluripotent and / or totipotent cells can be, for example, ES cells or ES-like cells, such as induced pluripotent stem (iPS) cells. ES cells include embryo-derived totipotent or pluripotent cells that are capable of developing into any tissue of the embryo after being introduced into the embryo. ES cells may be derived from the inner cell mass of the blastocyst and are capable of differentiating into cells of any of the three vertebrate germ layers (endoderm, ectoderm, and mesoderm).

[0095] Cells can also be derived from ES cells. For example, cells can be neuronal cells (e.g., ES cell-derived motor neurons (ESMN)), primitive ectoderm-like cells, embryoid cells, etc.

[0096] The cells described herein can also be germ cells (e.g., sperm or oocytes). Cells can be mitotically competent or mitotically inactive, meiotically competent or meiotically inactive. Similarly, cells can be primary somatic cells or cells that are not primary somatic cells. Somatic cells include any cell that is not a gamete, germ cell, gametophyte, or undifferentiated stem cell.

[0097] Suitable cells as described in this article also include primary cells. Primary cells include cells or cell cultures directly isolated from an organism, organ, or tissue. Primary cells include cells that are neither transformed nor immortalized. They include any cells obtained from an organism, organ, or tissue that have not previously been passaged in tissue cultures or have previously been passaged in tissue cultures but cannot be passaged in tissue cultures indefinitely.

[0098] Other suitable cell types mentioned in this article include immortalized cells. Immortalized cells comprise cells from multicellular organisms that do not normally proliferate indefinitely but have evaded normal cellular senescence due to mutations or alterations, and can instead continue to divide. Such mutations or alterations can occur naturally or be intentionally induced. Several types of immortalized cells are well known. Immortalized or primary cells include cells commonly used for culture or for expressing recombinant genes or proteins.

[0099] The cells described in this article also include single-cell stage embryos (i.e., fertilized oocytes or zygotes). Such single-cell stage embryos can be derived from any genetic background (e.g., for mice BALB / c, C57BL / 6, 129, or combinations thereof), can be fresh or frozen, and can be derived from natural breeding or in vitro fertilization.

[0100] A systematic approach using the expression of the mutant TDP-43 peptide Includes mutations TARDBP Cells and non-human animals (as well as tissues or animals containing such cells) that express mutant TDP-43 peptides lacking functional domains, as described herein, provide models for studying TDP-43 domain function and / or TDP-43 protein disorders. For example, cells containing mutant TDP-43 peptides... TARDBP Cells or non-human animals that express the mutant TDP-43 polypeptide lacking a functional domain encoded by the gene can exhibit phenotypic features of TDP-43 proteinopathy. In some embodiments, such as (a) embryonic stem cell-derived motor neurons (ESMNs) containing mutant TDP-43 proteins... TARDBP The gene expresses a mutant TDP-43 polypeptide lacking a functional domain encoded by it and / or (b) isolates from non-human animals that have endogenous expression of the gene. TARDBP The locus contains mutations TARDBP Endogenous gene replacement TARDBP Cells that express the TDP-43 mutant peptide from the gene may be characterized by one or more of the following: (i) mutation TARDBP The mRNA transcript levels of the gene were compared with those of wild-type control cells. TARDBPThe levels of the gene's mRNA transcripts were comparable, (ii) the level of mutant TDP-43 peptide was increased compared with the level of wild-type TDP-43 peptide in control cells, (iii) the concentration of mutant TDP-43 peptide in the cytoplasm of cells was found to be higher than that in the nucleus, (iv) mutant TDP-43 peptide showed increased insolubility compared with wild-type TDP-43 peptide, (v) cytoplasmic aggregates containing mutant TDP-43 peptide, (vi) the splicing of hidden exons of the gene was increased compared with cells expressing wild-type TDP-43, and (vii) the level of TDP-43 mRNA with alternative splicing lacking the sequence encoding TDP-43 PLD was reduced.

[0101] Therefore, it contains mutations TARDBP Cells (and tissues or animals containing such cells) that express the mutant TDP-43 polypeptide lacking a functional domain, as described herein, also provide therapeutic candidates for identifying one or more symptoms of TDP-43 proteases (e.g., cytoplasmic accumulation of the mutant TDP-43 polypeptide) and / or restoring the biological function of the wild-type TDP-43 polypeptide (e.g., inhibiting hidden exon splicing and / or increasing the level of alternatively spliced ​​TDP-43 mRNA). In some embodiments, by making the mutant TDP-43 polypeptide contain... TARDBP The effect of a therapeutic agent is determined by contacting cells that express the TDP-43 mutant polypeptide, which lacks a functional domain, with the therapeutic agent. Contact can be conducted in vitro. Contact may include administration of the therapeutic agent to an animal.

[0102] In some embodiments, the assay includes determining the effect on the phenotype and / or genotype of cells or animals exposed to the drug. In some embodiments, the assay includes determining the batch-to-batch variability of the drug (in some embodiments, the assay includes determining the difference between the effect on cells or animals described herein exposed to the administered drug and the effect on control cells or animals (e.g., expressing wild-type TDP-43)).

[0103] Exemplary parameters that can be measured in non-human animals (or in cells isolated from them and / or using cells isolated from them) to assess the pharmacokinetic properties of a drug include, but are not limited to, agglutination, autophagy, cell division, cell death, complement-mediated hemolysis, DNA integrity, drug-specific antibody titers, drug metabolism, gene expression arrays, metabolic activity, mitochondrial activity, oxidative stress, phagocytosis, protein biosynthesis, protein degradation, protein secretion, stress response, target tissue drug concentration, non-target tissue drug concentration, transcriptional activity, etc.

[0104] Oligonucleotides that selectively reduce full-length TDP-43 mRNA Figure 11A The full-length TDP-43 precursor mRNA is shown, along with normal (top) and variable (bottom) splicing events occurring at its 3' end. As illustrated, exon 6 encodes the prion-like domain (PLD) in the full-length TDP-43 protein, formed by normal splicing events, with the coding sequence terminating at the end of the PLD. Two new exons (7 and 8) are formed by variable splicing events from one of at least three variable 5' splicing sites within exon 6 to a downstream variable 3' splicing site (e.g., adjacent to the new exon 7). There is evidence of a second variable splicing event from variable exon 7 to variable exon 8.

[0105] In mice, the variable 5' splicing site at or at the beginning of exon 6 described herein was mapped to the following locations: (a) chromosome 4: 148,618,647; (b) chromosome 4: 148,618,665; and (c) chromosome 4: 148,618,674. The variable 3' splicing site in exon 7 was mapped to location 4: 148,617,705. A second variable splicing event from exon 7 to exon 8 occurred from chromosome 4: 148,617,566 to chromosome 4: 148,616,844. Those skilled in the art will be able to identify other... TARDBP Genes (e.g., human genes) TARDBP Similar variable 5' and 3' splicing sites in genes.

[0106] Alternative splicing from the variable 5' splice site in exon 6 to the downstream variable 3' splice site is predicted to produce an mRNA in which most of the PLD-coding sequence is replaced by a sequence encoding a PLD-deficient TDP-43 polypeptide. For example, from (a) chromosome 4: 148,618,647; (b) chromosome 4: 148,618,665; and (c) chromosome 4: 148,618,674 to chromosome 4: 148,617,705 (and human...) TARDBP Alternative splicing at any corresponding location in the gene can produce an mRNA in which most of the PLD coding sequence is replaced by a variable mRNA, which is predicted to encode a truncated form of TDP-43 lacking the PLD, wherein the PLD is replaced by 18 amino acids. This second alternative splicing event does not produce any new form of the TDP-43 protein because the open reading frame stops at exon 7, upstream of the 5'-splicing site of exon 7.

[0107] TDP-43 lacking PLD was observed to support viability, particularly in motor neurons, and in neurons expressing ΔPLD or ΔNLS mutations. TARDBPThe reduced levels of this alternatively spliced ​​TDP-43 mRNA in cells, along with its ALS-like phenotype, indicate that this alternatively spliced ​​TDP-43 mRNA and its truncated translational products do not cause TDP-43 proteases and may even provide protection against them. The application of siRNAs, antisense oligonucleotides, and / or CRISPR / Cas9 systems designed to eliminate or inactivate TDP-43 mRNA isoforms encoding PLD-containing protein forms can deplete TDP-43 variants prone to pathological aggregation while preserving the alternatively spliced ​​mRNA that produces truncated, PLD-free TDP-43 protein. The truncated form of TDP-43 is resistant to pathological aggregation while still supporting cellular life, particularly the activity of motor neurons.

[0108] Therefore, the treatment strategy will consist of searching for active antisense oligonucleotides (ASOs) or siRNAs that target only those TDP-43 mRNA sequences containing sequences encoding PLD (e.g., those mRNAs containing sequences encoded by a genomic sequence following an alternative splice site within exon 6). As a non-limiting example, ASOs or siRNAs could target those containing sequences following a codon encoding an alternative 5' splice site that results in the removal of the PLD domain. TARDBP The sequence of the transcribed gene is the mRNA. An ASO or siRNA designed to target this region of TDP-43 mRNA will recognize only the full-length TDP-43 mRNA encoding the TDP-43 polypeptide containing the PLD, while preserving alternatively spliced ​​TDP-43 mRNA encoding a truncated and potentially protected TDP-43 polypeptide lacking the PLD. In other words, such an ASO or siRNA should not be able to recognize or enhance the degradation of alternatively spliced ​​TDP-43 mRNA. The ASO or siRNA may target the TDP-43 mRNA sequence encoding amino acids 287-414 of the TDP-43 polypeptide, or any 3' untranslated region upstream of the 3' alternative splice site of exon 7. The ASO may promote the degradation of said mRNA via RNase H-mediated cleavage (e.g., via a -5-10-5 spacer). The siRNA may promote mRNA degradation and / or protein synthesis through RNA interference.

[0109] Another treatment strategy would be to use the CRISPR / Cas system to selectively target and delete cross-links. TARDBP The genomic sequence of the variable 5' splice site within exon 6 and the downstream 3' splice site (e.g., at exon 7). In this way, only the mRNA encoding the truncated, PLD-deficient TDP-43 polypeptide can be transcribed.

[0110] A. Antisense oligonucleotides and siRNA Antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) targeting sequences within precursor mRNA can enhance the degradation of unwanted isoforms. As designed herein, ASOs or siRNAs can be used to disrupt TDP-43 mRNA encoding PLD while preserving alternatively spliced ​​TDP-43 mRNA. To reduce the level of full-length TDP-43 mRNA only, ASOs or siRNAs can target TDP-43 mRNA containing the sequence from the alternative 5' splice site within exon 6 to (ii) the downstream alternative 3' splice site, for example, TDP-43 mRNA containing the sequence of amino acids 287-414 encoding the TDP-43 polypeptide and / or any 3' untranslated region upstream of the alternative splice site. See also, Figure 11A In some implementations, the variable 5' splice site within exon 6 is selected from the group consisting of the following: TARDBP Genomic location correlations: (a) mouse chromosome 4: 148,618,647; (b) mouse chromosome 4: 148,618,665; (c) mouse chromosome 4: 148,618,674; and (d) human chromosome 4. TARDBP Any corresponding location in the gene. In some implementations, the downstream variable 3' splice site corresponds to mouse chromosome 4:148,617,705 or human chromosome 4. TARDBP Related to the corresponding location in the gene.

[0111] The antisense oligonucleotide or siRNA targeting the TDP-43 mRNA sequence encoding PLD may have chemically modified subunits arranged in a pattern or motif to confer properties such as enhanced inhibitory activity, increased binding affinity to the target nucleic acid, or resistance to degradation by nucleases in vivo.

[0112] Antisense oligonucleotides typically contain at least one region modified to confer increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity to target nucleic acids, and / or increased inhibitory activity. The second region of the antisense oligonucleotide may optionally serve as a substrate for the cellular endonuclease RNase H, which cleaves the RNA strand of the RNA:DNA duplex.

[0113] In some embodiments, the antisense oligonucleotide is a homogeneous sugar-modified oligonucleotide. The antisense oligonucleotide may include a spacer motif. In the spacer, an inner region having multiple nucleotides supporting RNase H cleavage is located between an outer region having multiple nucleotides chemically different from the nucleosides of the inner region. In the case of an antisense oligonucleotide with a spacer motif, the spacer segment generally acts as a substrate for endonuclease cleavage, while the wing segments contain modified nucleosides. In some embodiments, the regions of the spacer are distinguished by the type of sugar moiety comprising each distinct region. In some embodiments, the types of sugar moiety used to distinguish the regions of the spacer may include β-D-ribonucleosides, β-D-deoxyribonucleosides, 2'-modified nucleosides (such 2'-modified nucleosides may include 2'-MOE and 2'-O-CH3, etc.), and bicyclic sugar-modified nucleosides. In some embodiments, the wings may include several modified sugar moieties, including, for example, 2'-MOE. In some embodiments, the wings may include several modified and unmodified sugar moieties. In some embodiments, the wing may include various combinations of 2'-MOE nucleoside and 2'-deoxyribonucleoside.

[0114] Each distinct region may contain a homogeneous sugar motif, a variant, or an alternating sugar motif. Wing-spacer-wing motifs are often described as “XYZ”, where “X” represents the length of the 5’ wing, “Y” represents the length of the spacer, and “Z” represents the length of the 3’ wing. “X” and “Z” may contain homogeneous, variant, or alternating sugar motifs. In some embodiments, “X” and “Y” may comprise one or more 2’-deoxyribonucleosides. “Y” may comprise a 2’-deoxyribonucleoside. As used herein, spacer bodies described as “XYZ” have a configuration such that the spacer is positioned immediately adjacent to each of the 5’ and 3’ wings. Therefore, there are no intercalated nucleotides between the 5’ wing and the spacer, or between the spacer and the 3’ wing. Any of the antisense compounds described herein may have a spacer body motif. In some embodiments, “X” and “Z” are the same; in other embodiments, they are different. In some embodiments, Y is between 8 and 15 nucleotides. X, Y, or Z can be any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or more nucleotides. Therefore, the spacers described herein include, but are not limited to, for example, 5-10-5, 5-10-4, 4-10-4, 4-10-3, 3-10-3, 2-10-2, 5-9-5, 5-9-4, 4-9-5, 5-8-5, 5-8-4, 4-8-5, 5-7-5, 4-7-5, 5-7-4, or 4-7-4.

[0115] Antisense oligonucleotides targeting the TDP-43 mRNA sequence encoding PLD can have a 5-10-5 spacer motif.

[0116] The antisense oligonucleotide targeting the TDP-43 mRNA sequence encoding PLD may contain a narrowed-space motif. The narrowed-space antisense oligonucleotide targeting TDP-43 mRNA may have 9, 8, 7, or 6 2'-deoxynucleotide spacer segments positioned adjacent to and between 5, 4, 3, 2, or 1 chemically modified nucleosides. The chemically modified nucleosides may contain bicyclic sugars. The bicyclic sugars may contain 4' to 2' bridges selected from: 4'-(CH2)nO-2' bridges, where n is 1 or 2; and 4'-CH2-O--CH2-2'. The bicyclic sugars may contain 4'-CH(CH3)-O-2' bridges. The chemical modification may contain a non-bicyclic 2'-modified sugar moiety, such as 2'-O-methylethyl or 2'-O-methyl. In some embodiments, the antisense oligonucleotide comprises a spacer motif targeting the TDP-43 mRNA sequence between a variable 5' and 3' splice site, wherein the variable 5' splice site is located within exon 6, for example, wherein the variable 5' splice site is selected from the group consisting of... TARDBP Genomic location correlations: (a) mouse chromosome 4: 148,618,647; (b) mouse chromosome 4: 148,618,665; (c) mouse chromosome 4: 148,618,674; and (d) human chromosome 4. TARDBP Any corresponding location in the gene, and wherein the variable 3' splice site is associated with the TARDBP genomic location on chromosome 4: 148,617,705. In some embodiments, the siRNA comprises a sequence targeting the TDP-43 mRNA sequence between the variable 5' and 3' splice sites, wherein the variable 5' splice site is located within exon 6, for example, wherein the variable 5' splice site is selected from the group consisting of TARDBP Genomic location correlations: (a) mouse chromosome 4: 148,618,647; (b) mouse chromosome 4: 148,618,665; (c) mouse chromosome 4: 148,618,674; and (d) human chromosome 4. TARDBP Any corresponding location in the gene, and wherein the variable 3' splice site is associated with the TARDBP genomic location on chromosome 4: 148,617,705.

[0117] The antisense oligonucleotide or siRNA can be uniformly modified to target the TDP-43 mRNA sequence encoding PLD. In some embodiments, each nucleoside is chemically modified. In some embodiments, the chemical modification comprises a non-bicyclic 2'-modified sugar moiety. In some embodiments, the 2'-modified sugar moiety comprises 2'-O-methoxyethyl. In some embodiments, the 2'-modified sugar moiety comprises 2'-O-methyl.

[0118] ASO or siRNA can also be covalently linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the resulting ASO or siRNA. Typical conjugate groups include cholesterol and lipid moieties. Other conjugate groups include carbohydrates, phospholipids, biotin, phenazine, folic acid, phenidine, anthraquinones, acridine, fluorescein, rhodamine, coumarin, and dyes.

[0119] ASO or siRNA can also be modified to have one or more stabilizing groups, typically linked to one or both ends. These stabilizing groups include cap structures. These end modifications protect ASO or siRNA with terminal nucleic acids from exonuclease degradation and can facilitate intracellular delivery and / or localization. The cap may be present at the 5' end (5' cap) or the 3' end (3' cap), or at both ends. Cap structures are well-known and include, for example, inverse deoxygenated baseless caps.

[0120] ASO or siRNA can be of any length suitable for binding to a target nucleic acid (e.g., TDP-43 precursor mRNA) and having the desired effect. For example, the length of an ASO can be about 12 to about 30, about 12 to about 24, about 13 to about 23, about 14 to about 22, about 15 to about 21, about 16 to about 20, about 17 to about 19, or about 18 nucleotides. As another example, an ASO can be about 8 to about 80, about 12 to about 50, about 15 to about 30, about 18 to about 24, about 19 to about 22, or about 20 linked nucleotides. Optionally, the length of the ASO can be approximately 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, or 43. Approximately 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or approximately 80 linked nucleosides. For example, an ASO may consist of approximately 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or approximately 25 linked nucleosides. In one specific instance, an ASO may be approximately 15 to approximately 25 linked nucleosides.

[0121] ASO or siRNA can be complementary and / or specifically hybridize with target nucleic acids (e.g., TDP-43 precursor mRNA, such as the mRNA sequence encoding PLD). When a sufficient number of nucleotides in the ASO are hydrogen-bonded to the corresponding nucleotides in the target nucleic acid, the ASO and the target nucleic acid are complementary, thus producing the desired effect. Specific hybridization means that the ASO has sufficient complementarity with the target nucleic acid to induce the desired effect, while exhibiting minimal or no effect on non-target nucleic acids under the conditions of desired specific binding (e.g., physiological conditions).

[0122] Some ASOs or siRNAs are at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% complementary to the isolength portion of the TDP-43 precursor mRNA. Optionally, the ASO may be approximately 100% complementary to the isolength portion of the TDP-43 precursor mRNA. The percentage of complementarity between the ASO and the target nucleic acid can be determined using conventional methods. For example, an ASO in which 18 out of 20 nucleotides are complementary to the target region and therefore specifically hybridizes will represent 90% complementarity. The percentage of complementarity between the ASO and the target nucleic acid region can be routinely determined using the well-known BLAST program (Basic Local Alignment Search Tool) and the PowerBLAST program. See, for example Altschul et al. (1990) J. Mol. Biol 215:403 410 and Zhang and Madden (1997) Genome Res .7:649-656). The percentage of homology, sequence identity, or complementarity can be determined, for example, by the Gap program (Wisconsin sequence analysis package, version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.) using default settings, which uses the Smith and Waterman algorithm ( Adv. Appl. Math ., 1981, 2, 482489).

[0123] The non-complementary nucleotides between the ASO or siRNA and the TDP-43 precursor mRNA can be inclusive, provided that the ASO or siRNA can still specifically hybridize with the target nucleic acid. Furthermore, the ASO or siRNA can hybridize on one or more segments of the TDP-43 precursor mRNA such that intercalated or adjacent segments do not participate in hybridization events (e.g., loop structures, mismatches, or hairpin structures). The non-complementary nucleotides may be located at the 5' or 3' end of the ASO or siRNA. Optionally, the one or more non-complementary nucleotides may be located internally within the ASO or siRNA. When two or more non-complementary nucleotides are present, they can be continuous (i.e., connected) or discontinuous.

[0124] B. Deletion of the genomic sequence encoding TDP-43 PLD As demonstrated in this paper, cells remained viable despite expressing only the mutant TDP-43 peptide lacking a functional PLD. This paper also describes a clustered regularly spaced short palindromic repeat (CRISPR) / CRISPR-associated (Cas) system, or one or more components of a CRISPR / Cas system, which can be used to extract endogenous proteins from cells (e.g., embryonic stem cells) lacking the endogenous proteins described herein. TARDBP A protein-like domain (or a portion thereof) at a gene locus. The CRISPR / Cas system can remove genomic sequences from cells (e.g., embryonic stem cells) that are located at or near a short 5' splice site in exon 6 and a 3' splice site in or near exon 7. Such components include, for example, Cas proteins and / or guide RNA (gRNA), which may comprise two separate RNA molecules; for example, a targeting RNA (e.g., CRISPR RNA (crRNA) and an activating RNA (e.g., tracrRNA); or a single guide RNA (e.g., a single-molecule gRNA (sgRNA)). In some embodiments, the CRISPR / Cas system comprises a Cas9 protein and at least one gRNA, wherein the gRNA recognizes a gene locus located at or near a Cas9 protein. TARDBP The sequence at the genomic location, which is selected from the following groups: (a) chromosome 4: 148,618,647; (b) chromosome 4: 148,618,665; (c) chromosome 4: 148,618,674; (d) chromosome 4: 148,617,705 and combinations thereof.

[0125] The CRISPR / Cas system includes transcripts and other elements involved in Cas gene expression or guiding its activity. The CRISPR / Cas system can be, for example, a type I, type II, or type III system. Optionally, the CRISPR / Cas system can be a type V system (e.g., subtype VA or subtype VB). In endogenous... TARDBP The sequence encoding the TDP-43 prion-like domain (or a portion thereof) at the locus, or the sequence between the 5' alternative splice site (e.g., the sequence encoding amino acid 288) and the 3' alternative splice site (e.g., adjacent to the alternative exon 7), can be deleted by site-specific cleavage of nucleic acids using a CRISPR complex (containing a guide RNA (gRNA) complexed with the Cas protein).

[0126] The CRISPR / Cas system described herein may include Cas proteins (e.g., Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), Cse4). (CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cu1966 and their homologs or modified forms) and / or one or more guide RNAs (gRNAs) targeting gRNA recognition sequences. The CRISPR / Cas system described herein may also include at least one expression construct comprising nucleic acid encoding a Cas protein (e.g., capable of operatively linking to a promoter) and / or DNA encoding gRNA.

[0127] Cas protein TARDBP Site-specific binding and cleavage of a gene can occur at a location determined by both: (i) base pairing complementarity between the gRNA and the target DNA; and (ii) a short motif in the target DNA called a pre-intercalation sequence adjacent motif (PAM). The PAM can be lateralized to a guide RNA recognition sequence. Optionally, the guide RNA recognition sequence can be lateralized to the 3' end of the PAM. Optionally, the guide RNA recognition sequence can be lateralized to the 5' end of the PAM. For example, the cleavage site of a Cas protein can be approximately 1 to approximately 10 base pairs or approximately 2 to approximately 5 base pairs (e.g., 3 base pairs) upstream or downstream of the PAM sequence. In some cases (e.g., when using Streptococcus pyogenes), (S. pyogenesWhen the target DNA is Cas9 or closely related to Cas9, the PAM sequence of the non-complementary strand can be 5'-N1GG-3', where N1 is any DNA nucleotide and is immediately adjacent to the 3' of the guide RNA recognition sequence of the non-complementary strand of the target DNA. Therefore, the PAM sequence of the complementary strand will be 5'-CCN2-3', where N2 is any DNA nucleotide and is immediately adjacent to the 5' of the guide RNA recognition sequence of the complementary strand of the target DNA. In some such cases, N1 and N2 can be complementary, and the N1-N2 base pair can be any base pair (e.g., N1=C and N2=G; N1=G and N2=C; N1=A and N2=T; or N1=T and N2=A). In Staphylococcus aureus (… S. aureus In the case of Cas9, PAM can be NNGRRT or NNGRR, where N can be A, G, C or T, and R can be G or A.

[0128] As disclosed herein, guide RNA may be provided in any form. In some embodiments, gRNA may be provided in the form of RNA, as two molecules (crRNA and tracrRNA separately) or as a single molecule (sgRNA), and optionally as a complex with a Cas protein. gRNA may also be provided in the form of DNA encoding gRNA. In some embodiments, the DNA encoding gRNA may encode a single RNA molecule (sgRNA) or a single RNA molecule (e.g., crRNA and tracrRNA separately) (wherein the single RNA molecule may be provided as a single DNA molecule, or as separate DNA molecules encoding crRNA and tracrRNA respectively).

[0129] In one embodiment, the CRISPR / Cas system as described herein comprises a Cas9 protein or a protein derived from Cas9 of a type II CRISPR / Cas system and / or at least one gRNA, wherein the at least one gRNA is encoded by DNA encoding crRNA and / or tracrRNA.

[0130] Targeted genetic modifications can be generated by contacting cells with a Cas protein and one or more guide RNAs that hybridize to one or more guide RNA recognition sequences within a target genomic locus. At least one of the one or more guide RNAs can form a complex with at least one of the one or more guide RNA recognition sequences and direct the Cas protein to at least one of the one or more guide RNA recognition sequences, and the Cas protein can cleave the target genomic locus within at least one of the one or more guide RNA recognition sequences. The cleavage caused by the Cas protein can produce double-strand breaks or single-strand breaks (e.g., if the Cas protein is a nicking enzyme). The terminal sequences resulting from the double-strand breaks or single-strand breaks can then be recombined.

[0131] C. Methods for introducing oligonucleotides This article provides a variety of methods and compositions to allow the introduction of oligonucleotides into cells. Methods for introducing oligonucleotides into various cell types are known and include, for example, stable transfection methods, transient transfection methods, and virus-mediated methods.

[0132] Transfection protocols, and protocols used to introduce oligonucleotides into cells, can vary. Non-restrictive transfection methods include chemical-based methods that use liposomes; nanoparticles; calcium phosphate (Graham et al. (1973)). Virology 52 (2): 456–467, Bacchetti et al. (1977) Proc. Natl. Acad. Sci. USA 74(4): 1590–1594, and Kriegler, M (1991). Transfer and Expression: A Laboratory Manual. New York: WH Freeman and Company. pp. 96–97); dendritic polymers; or cationic polymers, such as DEAE-dextran or polyethyleneimine. Non-chemical methods include electroporation, ultrasonic perforation, and optical transfection. Particle-based transfection includes transfection using gene guns or magnet-assisted transfection (Bertram (2006)). Current Pharmaceutical Biotechnology 7, 277–28). Viral methods can also be used for transfection.

[0133] Oligonucleotides can also be introduced into cells via electroporation, intracytoplasmic injection, viral transfection, adenovirus, adeno-associated virus, lentivirus, retrovirus, transfection, lipid-mediated transfection, or nuclear transfection. Nuclear transfection is an improved electroporation technique that allows nucleic acid substrates to be delivered not only to the cytoplasm but also across the nuclear membrane and into the nucleus. Furthermore, the methods disclosed herein typically require far fewer cells than conventional electroporation (e.g., approximately 2 million cells compared to 7 million cells required for conventional electroporation). In one example, LONZA was used. ® Nuclear transfection was performed using the NUCLEOFECTOR™ system.

[0134] Introducing oligonucleotides into cells (e.g., fertilized eggs) can also be accomplished via microinjection. In fertilized eggs (i.e., single-cell stage embryos), microinjection can be performed into the maternal and / or paternal pronuclei or into the cytoplasm. If microinjection is performed into only one pronucleus, the paternal pronucleus is preferred due to its larger size. Methods for performing microinjection are well known. See, for example , Nagy et al (Nagy A, Gertsenstein M, Vintersten K, Behringer R., 2003, Manipulating the Mouse Embryo, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); See also Meyer et al. (2010) Proc. Natl. Acad. Sci. USA 107:15022-15026 and Meyer et al. (2012) ) Proc. Natl. Acad. Sci. USA 109:9354-9359.

[0135] Other methods for introducing oligonucleotides into cells may include, for example, carrier delivery, particle-mediated delivery, exosome-mediated delivery, lipid nanoparticle-mediated delivery, cell-penetrating peptide-mediated delivery, or implantable device-mediated delivery. As specific examples, oligonucleotides may be introduced into cells or non-human animals in carriers such as poly(lactic acid) (PLA) microspheres, poly(D,L-lactic acid-coglycolic acid) (PLGA) microspheres, liposomes, micelles, reverse micelles, lipid helices, and lipid microtubules.

[0136] The introduction of oligonucleotides can also be accomplished via virus-mediated delivery, such as AAV-mediated delivery or lentivirus-mediated delivery. Other exemplary viruses / viral vectors include retroviruses, adenoviruses, vaccinia viruses, poxviruses, and herpes simplex viruses. Viruses can infect dividing cells, non-dividing cells, or both. Viruses can integrate into the host genome or optionally not integrate into the host genome. Such viruses can also be engineered to reduce immunity. Viruses can be replicative or can be replication-defective (e.g., defective in one or more genes necessary for additional rounds of viral particle replication and / or packaging). Viruses can induce transient expression, long-term expression (e.g., at least 1 week, 2 weeks, 1 month, 2 months, or 3 months), or permanent expression. Exemplary viral titers (e.g., AAV titers) include 10-1. 12 10 13 10 14 10 15 and 10 16 One vector genome / mL.

[0137] The ssDNA AAV genome consists of two open reading frames, Rep and Cap, which are side-joined by two inverted terminal repeat sequences to allow the synthesis of complementary DNA strands. When constructing the AAV transfer plasmid, the transgene is placed between the two ITRs, and Rep and Cap can be provided in trans form. In addition to Rep and Cap, AAV also requires a helper plasmid containing genes from adenovirus. These genes (E4, E2a, and VA) mediate AAV replication. For example, the transfer plasmid, Rep / Cap, and helper plasmid can be transfected into HEK293 cells containing the adenovirus gene E1+ to produce infectious AAV particles. Alternatively, Rep, Cap, and the adenovirus helper genome can be synthesized into a single plasmid. Similar packaging cells and methods can be used for other viruses, such as retroviruses.

[0138] Several AAV serotypes have been identified. These serotypes differ in the cell types they infect (i.e., their tropism), thus allowing preferential transduction of specific cell types. Serotypes in CNS tissues include AAV1, AAV2, AAV4, AAV5, AAV8, and AAV9. Serotypes in heart tissues include AAV1, AAV8, and AAV9. Serotypes in kidney tissues include AAV2. Serotypes in lung tissues include AAV4, AAV5, AAV6, and AAV9. Serotypes in pancreatic tissues include AAV8. Serotypes in photoreceptor cells include AAV2, AAV5, and AAV8. Serotypes in retinal pigment epithelium tissues include AAV1, AAV2, AAV4, AAV5, and AAV8. Serotypes in skeletal muscle tissues include AAV1, AAV6, AAV7, AAV8, and AAV9. Serotypes in liver tissues include AAV7, AAV8, and AAV9, especially AAV8.

[0139] Taste tropism can be further refined through pseudotyped packaging, which is a mixture of capsids and genomes from different viral serotypes. For example, AAV2 / 5 represents a virus containing the genome of serotype 2 packaged in a capsid of serotype 5. Using pseudotyped viruses can improve transduction efficiency and alter taste tropism. Mixed capsids derived from different serotypes can also be used to alter viral taste tropism. For example, AAV-DJ contains a mixed capsid from eight serotypes and exhibits high infectivity across a wide range of cell types in vivo. AAV-DJ8 is another example, exhibiting the characteristics of AAV-DJ but with enhanced brain uptake. AAV serotypes can also be modified through mutations. Examples of AAV2 mutations include Y444F, Y500F, Y730F, and S662V. Examples of AAV3 mutations include Y705F, Y731F, and T492V. Examples of AAV6 mutations include S663V and T492V. Other pseudo-packaged / modified AAV variants include AAV2 / 1, AAV2 / 6, AAV2 / 7, AAV2 / 8, AAV2 / 9, AAV2.5, AAV8.2, and AAV / SASTG.

[0140] To accelerate transgene expression, complementary AAV (scAAV) variants can be used. Because AAV relies on the cell's DNA replication mechanism to synthesize the complementary strand of the AAV single-stranded DNA genome, transgene expression may be delayed. To address this delay, scAAV containing a complementary sequence that spontaneously anneals upon infection can be used, thereby eliminating the need for host cell DNA synthesis. However, single-stranded AAV (ssAAV) vectors can also be used.

[0141] The introduction of oligonucleotides can also be accomplished via lipid nanoparticle (LNP)-mediated delivery. Lipid formulations protect biomolecules from degradation while improving their cellular uptake. Lipid nanoparticles are particles comprising multiple lipid molecules physically associated with each other through intermolecular forces. These lipid nanoparticles include microspheres (including monolayer and multilayer vesicles, such as liposomes), dispersed phases in emulsions, micelles, or internal phases in suspensions. Such lipid nanoparticles can be used to encapsulate one or more oligonucleotides for delivery. Formulations containing cationic lipids can be used to deliver polyanionic compounds, such as nucleic acids. Other lipids that may be included are neutral lipids (i.e., uncharged or zwitterionic lipids), anionic lipids, auxiliary lipids that enhance transfection, and occult lipids that increase the duration of nanoparticle presence in vivo. Examples of suitable cationic lipids, neutral lipids, anionic lipids, auxiliary lipids, and occult lipids can be found in WO 2016 / 010840 A1, which is incorporated herein by reference in its entirety for all purposes.

[0142] Internal administration can be performed via any suitable route, including, for example, parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, local, intranasal, or intramuscular. Systemic administration routes include, for example, oral and parenteral routes. Examples of parenteral routes include intravenous, intra-arterial, intraosseous, intramuscular, intradermal, subcutaneous, intranasal, and intraperitoneal routes. A specific example is intravenous infusion. Nasal drops and intravitreal injection are other specific examples. Local administration routes include, for example, intrathecal, intravenous, intraparenchymal (e.g., local intraparenchymal delivery to the striatum (e.g., into the caudate nucleus or putamen), cerebral cortex, precentral gyrus, hippocampus (e.g., into the dentate gyrus or CA3 area), temporal cortex, amygdala, frontal cortex, thalamus, cerebellum, medulla, hypothalamus, tectum, tegmentum, or substantia nigra), intraocular, intraorbital, subconjunctival, intravitreal, subretinal, and transscleral routes. Compared to systemic administration (e.g., intravenous), significantly smaller amounts of the component are required to be effective when administered locally (e.g., intraparenchymal or intravitreal). Local administration can also reduce or eliminate the incidence of potential toxic side effects that would occur with systemic administration of therapeutically effective amounts of the component.

[0143] A common method used in cell culture to facilitate the uptake of reagents (such as antisense oligonucleotides) involves transfecting nucleic acids with cationic lipids. Mixing cationic lipids with negatively charged nucleic acids yields a complex that can cross the cell membrane and release the active nucleic acid into the cell's cytoplasm. Electroporation of reagents (such as antisense oligonucleotides) into cells is also possible. This method is highly effective and useful for cell lines that are not readily transfected with lipids.

[0144] If the cells are in vivo (e.g., in an animal), they can be administered to the animal by any suitable means. For example, administration can include parenteral routes such as intraperitoneal, intravenous, and subcutaneous. Parenteral administration means administration by injection or infusion. Parenteral administration includes subcutaneous, intravenous, intramuscular, intra-arterial, intraperitoneal, or intracranial administration (e.g., intrathecal or intraventricular administration).

[0145] In some methods, administration is achieved by means of introducing the reagent to neurons or the nervous system. This can be accomplished, for example, through peripheral delivery or by direct delivery to the nervous system. See, for example, Evers et al. (2015) Adv. Drug Deliv. Res. References 87:90-103 are incorporated herein by reference in their entirety for all purposes.

[0146] For reagents (such as antisense oligonucleotides) to reach the nervous system, they must first cross the vascular barrier, which consists of the blood-brain barrier or the blood-spinal barrier. One mechanism that can be used to cross the vascular barrier is receptor-mediated endocytosis. Another usable mechanism is a cell-penetrating peptide (CPP)-based delivery system. Different CPPs use different cell translocation pathways, depending on the cell type and the deliverable. For example, systemically delivered antisense oligonucleotides labeled with arginine-rich CPPs can cross the blood-brain barrier. Another usable delivery mechanism is exosomes, which are extracellular vesicles known to mediate cell-to-cell communication through the transfer of proteins and nucleic acids. For example, intravenous injection of exosomes transduced with a short viral peptide derived from rabies virus glycoprotein (RVG) can result in crossing the blood-brain barrier and delivery to the brain.

[0147] Techniques that bypass the vascular barrier by direct infusion into the cerebrospinal fluid (CSF) can also be used. For example, a reagent (e.g., an antisense oligonucleotide) can be infused into the intraventricular ventricle (ICV), after which the reagent (e.g., the antisense oligonucleotide) must pass through the ependymal cell layer lined within the ventricular system to enter the brain parenchyma. Intrathecal (IT) delivery refers to the delivery of a reagent (e.g., an antisense oligonucleotide) into the subarachnoid space of the spinal cord. From here, the reagent (e.g., the antisense oligonucleotide) must pass through the pia mater to enter the brain parenchyma. The reagent (e.g., the antisense oligonucleotide) can be delivered via an outflow catheter connected to an implantable cartridge using either ICT or IT. The drug can be injected into the cartridge and delivered directly to the CSF. Intranasal administration is an alternative delivery route that can be used.

[0148] The scope of this invention is defined by the appended claims and is not limited to the specific embodiments described herein; those skilled in the art will recognize various modifications that are equivalent to the embodiments described herein or otherwise within the scope of the claims. Generally, unless explicitly specified otherwise, terminology is consistent with its meaning as understood in the art. References cited herein, or relevant portions thereof, are incorporated herein by reference in their entirety.

[0149] The use of ordinal terms such as “first,” “second,” and “third” in claims to modify claim elements does not imply any priority, order, or temporal sequence of actions of one claim element relative to another claim element, but is merely a marker to distinguish one claim element with a specific name from another element with the same name (only for the use of ordinal terms).

[0150] Unless explicitly specified to the contrary, the article "a / an" in the specification and claims shall be understood to include a plural reference. Unless explicitly specified to the contrary or otherwise apparent from the context, a claim or description including "or" among one or more members of the group is considered satisfied if one, more than one, or all members of the group are present in, used in, or otherwise associated with a given product or method. This invention includes embodiments in which only one member of the group is present in, used in, or otherwise associated with a given product or method. This invention also includes embodiments in which more than one or all members of the group are present in, used in, or otherwise associated with a given product or method. Furthermore, it should be understood that, unless otherwise specified or unless a contradiction or inconsistency would be apparent to a person skilled in the art, this invention covers all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., derived from one or more of the enumerated claims depend on another claim of the same basic claim (or any other related claim). When elements are presented in list form (e.g., in Markush group or similar form), it should be understood that each subgroup of said elements is also disclosed, and any one or more elements may be removed from said group. It should be understood that, generally, when an embodiment or aspect of the invention is referred to as including a particular element, feature, etc., it consists of or substantially consists of such elements, features, etc. For simplicity, these embodiments are not specifically described herein in so many words in every instance. It should also be understood that any embodiment or aspect of the invention may be expressly excluded by the claims, whether or not a specific exclusion is set forth in the specification.

[0151] "Comparison" includes " Comparison "Controls" are used as a standard for comparison, as understood in the art. Typically, controls are used to enhance the integrity of the experiment by separating variables, thereby obtaining conclusions about those variables. In some embodiments, controls are performed simultaneously with the test reaction or assay to provide a reaction or assay for comparison. Comparison "Also includes " Comparison of movements things". “ control animals "It may have modifications as described herein, modifications different from those described herein, or no modifications (i.e., wild-type animals). In one experiment, "" test (That is, the variable is tested). In the second experiment, "" was not applied. Comparison"(The variable is tested). In some embodiments, the control is a historical control (i.e., a previously performed test or determination, or a previously known quantity or result). In some embodiments, the control is or includes a printed or otherwise preserved record. The control can be a positive control or a negative control."

[0152] "Determine," "measure," "evaluate," "assess," "determine," and "analyze" encompass any form of measurement and include determining the presence of an element. These terms include both quantitative and / or qualitative determinations. Determination can be relative or absolute. "Determining the presence" can be used to determine the existence of something. Existence quantity and / or determination of its existence.

[0153] The terms “nucleic acid” and “polynucleotide”, used interchangeably herein, include polymers of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, or their analogues or modifications. These include single-stranded, double-stranded, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers containing purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural, or derivatized nucleotide bases.

[0154] The terms “protein,” “polypeptide,” and “peptide,” used interchangeably herein, include polymeric forms of amino acids of any length, including both coding and non-coding amino acids and chemically or biochemically modified or derivatized amino acids. The terms also include modified polymers, such as polypeptides having a modified peptide backbone. The term “domain” refers to any portion of a protein or polypeptide that has a specific function or structure. Unless otherwise stated, any domain mentioned herein refers to the TDP-43 domain.

[0155] The term "wildtype" includes entities that have the structure and / or activity found in normal (compared to mutated, diseased, altered, etc.) states or conditions. Wildtype genes and polypeptides often exist in many different forms (e.g., alleles).

[0156] The term "endogenous" refers to the location, nucleic acid, or amino acid sequence found or naturally present within cells or animals. For example, endogenous in non-human animals. TARDBP Sequence refers to the endogenous sequence that naturally exists in non-human animals. TARDBP Wild type at the gene locus TARDBP sequence.

[0157] The term "locus" refers to a specific location on the chromosome of an organism's genome, where a gene (or important sequence), DNA sequence, polypeptide coding sequence, or position is located. For example, " TARDBP "Locus" can refer to TARDBP Gene, TARDBP DNA sequence, TARDBP 2-encoded sequence or TARDBP The location is a specific position on the chromosome of an organism's genome that has been identified as containing such sequences. TARDBP "Locus" can contain TARDBP Regulatory elements of a gene include, for example, enhancers, promoters, 5' and / or 3' untranslated regions (UTRs), or combinations thereof.

[0158] The term "gene" refers to a DNA sequence in a chromosome that encodes a product (e.g., RNA product and / or polypeptide product) and includes coding regions interrupted by non-coding introns and sequences located near the 5' and 3' ends of the coding region, such that the gene corresponds to full-length mRNA (including the 5' and 3' untranslated sequences). Other non-coding sequences of a gene include regulatory sequences (e.g., promoters, enhancers, and transcription factor binding sites), polyadenylation signals, internal ribosome entry sites, silencers, insulating sequences, and matrix attachment regions. These sequences may be located near the coding region of the gene (e.g., within 10 kb) or at distant sites, and they influence the level or rate of transcription and translation of the gene.

[0159] The term "allele" refers to a variant form of a gene. Some genes have multiple different forms, located at the same location or locus on a chromosome. Diploid organisms specifically have two alleles, each located at an endogenous locus on a homologous chromosome. Each pair of alleles represents the genotype at a particular locus. If two identical alleles are present at a particular locus, the genotype is described as homozygous, and if the two alleles are different, the genotype is described as heterozygous.

[0160] "Operationally linked" includes juxtaposition, where the components are in a relationship that allows them to function in the intended manner. "Operationally linked" control sequences to coding sequences are linked in a manner that enables expression of the coding sequence under conditions compatible with the control sequences. "Operationally linked" sequences include both expression control sequences adjacent to the gene of interest and expression control sequences that function trans- or at a distance to control the expression of the gene of interest. The term "expression control sequence" includes polynucleotide sequences necessary to influence the expression and processing of the coding sequence to which it is linked. "Expression control sequences" include: appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences stabilizing cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak concordant sequences); sequences that enhance protein stability; and sequences that enhance protein secretion when desired. The nature of such control sequences varies depending on the host organism. For example, in prokaryotes, such control sequences generally include promoters, ribosome binding sites, and transcription termination sequences, while in eukaryotes, such control sequences typically include promoters and transcription termination sequences. The term "control sequence" is intended to include components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, such as leader sequences and fusion mate sequences.

[0161] A “phenotype” includes a trait, or a class or group of traits exhibited by a cell or organism. In some embodiments, a particular phenotype may be associated with a particular allele or genotype. In some embodiments, a phenotype may be discrete; in some embodiments, a phenotype may be continuous. A phenotype may include cell viability or cell fitness. A phenotype may include the expression level of a protein (e.g., the mutant TDP-43 peptide), cellular localization, and / or solubility / stability characteristics, each of which can be determined using well-known methods such as Western blot analysis, fluorescence in situ hybridization, qualitative RT-PCR, etc.

[0162] A promoter is a regulatory region of DNA that typically contains a TATA box that guides RNA polymerase II to initiate RNA synthesis at the appropriate transcription start site of a specific polynucleotide sequence. Promoters may additionally contain other regions that influence the transcription initiation rate. The promoter sequences disclosed herein regulate the transcription of operable polynucleotides. Promoters may be active in one or more of the cell types disclosed herein (e.g., eukaryotic cells, non-human mammalian cells, human cells, rodent cells, pluripotent cells, single-cell stage embryos, differentiated cells, or combinations thereof). Promoters may be, for example, constitutively active promoters, conditional promoters, inducible promoters, time-restricted promoters (e.g., developmentally regulatory promoters), or spatially restricted promoters (e.g., cell-specific or tissue-specific promoters). Examples of promoters can be found, for example, in WO2013 / 176772, which is incorporated herein by reference in its entirety for all purposes.

[0163] "Reference" includes a standard or control agent, cell, animal, cohort, individual, population, sample, sequence, or value to be compared with the agent, cell, animal, cohort, individual, population, sample, sequence, or value of interest. In some embodiments, the testing and / or determination of the reference agent, cell, animal, cohort, individual, population, sample, sequence, or value is performed substantially simultaneously with the testing or determination of the agent, cell, animal, cohort, individual, population, sample, sequence, or value of interest. In some embodiments, the reference agent, cell, animal, cohort, individual, population, sample, sequence, or value is a historical reference optionally embodied in a tangible medium. In some embodiments, reference may refer to a control. "Reference" also includes "reference cell." A "reference cell" may have modifications as described herein, modifications different from those described herein, or no modification (i.e., wild-type cells). Generally, as those skilled in the art will understand, the reference agent, animal, cohort, individual, population, sample, sequence, or value is determined or characterized under conditions equivalent to those used to determine or characterize the agent, cell, animal (e.g., mammal), cohort, individual, population, sample, sequence, or value of interest.

[0164] The term "variant" refers to a nucleotide sequence that differs from a reference nucleotide sequence (e.g., by one nucleotide) or a protein sequence that differs from a reference amino acid sequence (e.g., by one amino acid), but retains the biological function of the reference sequence. In some embodiments, variants differ from the reference sequence due to the degeneracy and / or conserved codon / amino acid substitutions of the genetic code.

[0165] In the case of two polynucleotide or polypeptide sequences, "sequence identity" or "identity" refers to the same residues in two sequences when compared within a specified comparison window to achieve maximum correspondence. When using a sequence identity percentage for proteins, dissimilar residue positions are often due to conserved amino acid substitutions, where an amino acid residue is replaced by another amino acid residue with similar chemical properties (e.g., charge or hydrophobicity) and therefore does not alter the functional properties of the molecule. When the sequence differences lie in conserved substitutions, the sequence identity percentage can be adjusted upwards to correct for the conservatism of the substitution. Sequences that differ by a class of conserved substitutions are thus considered to have "sequence similarity" or "similarity." The means of making this adjustment are well known. Typically, this involves counting conserved substitutions as partial mismatches rather than complete mismatches, thereby increasing the sequence identity percentage. Thus, for example, where the score for identical amino acids is 1 and the score for non-conservative substitutions is zero, the score for conserved substitutions is between zero and 1. The score for conserved substitutions is calculated, for example, as implemented in the program PC / GENE (Intelligenetics, Mountain View, California).

[0166] The "Sequence Identity Percentage" is a value determined by comparing two optimally aligned sequences (the maximum number of perfectly matching residues) within a comparison window. The polynucleotide sequence portion within the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence (excluding additions or deletions) to achieve optimal alignment between the two sequences. The percentage is calculated by determining the number of positions in both sequences containing the same nucleic acid base or amino acid residue to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the sequence identity percentage. Unless otherwise specified (e.g., the shorter sequence includes linked heterologous sequences), the comparison window is the full length of the shorter of the two sequences being compared.

[0167] Unless otherwise stated, sequence identity / similarity values ​​include those obtained using GAP version 10 with the following parameters: % identity and % similarity of nucleotide sequences using a GAP weight of 50 and a length weight of 3, and an nwsgapdna.cmp score matrix; % identity and % similarity of amino acid sequences using a GAP weight of 8 and a length weight of 2, and a BLOSUM62 score matrix; or any equivalent procedure thereof. An “equivalence procedure” includes any sequence comparison procedure that, for any two sequences considered, produces alignments with the same nucleotide or amino acid residue match and the same percentage of sequence identity when compared to corresponding alignments generated by GAP version 10.

[0168] The term "conservative amino acid substitution" refers to the substitution of a normally present amino acid in a sequence with a different amino acid having similar size, charge, or polarity. Examples of conservative substitution include the substitution of one nonpolar (hydrophobic) residue for another nonpolar residue, such as isoleucine, valine, or leucine. Similarly, examples of conservative substitution include the substitution of one polar (hydrophilic) residue for another, such as between arginine and lysine, glutamine and asparagine, or glycine and serine. Additionally, the substitution of one basic residue for another basic residue, such as lysine, arginine, or histidine, or the substitution of one acidic residue for another acidic residue, such as aspartic acid or glutamic acid, are further examples of conservative substitution. Examples of nonconservative substitution include the substitution of polar (hydrophilic) residues, such as cysteine, glutamine, glutamic acid, or lysine, with nonpolar (hydrophobic) amino acid residues, such as isoleucine, valine, leucine, alanine, or methionine, and / or the substitution of nonpolar residues with polar residues. Table 1 below summarizes typical amino acid classifications.

[0169] Table 1. Amino acid classification

[0170] The term "in vitro" includes artificial environments and processes or reactions that occur within artificial environments (e.g., test tubes). The term "in vivo" includes natural environments (e.g., cells, organisms, or the body) and processes or reactions that occur within those natural environments. The term "ex vivo" includes cells that have been removed from an individual and processes or reactions that occur within such cells.

[0171] Non-limiting exemplary implementations include the following.

[0172] Implementation Scheme 1. A non-human animal cell containing a mutation encoding a mutant TDP-43 polypeptide. TARDBP Gene, The mutant TDP-43 peptide lacks the functional domains found in the wild-type TDP-43 peptide, and The non-human animal or non-human animal cell expresses the mutant TDP-43 polypeptide. Optionally, the wild-type TDP-43 polypeptide comprises a sequence as stated in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5.

[0173] Implementation Scheme 2. The non-human animal cell as described in Implementation Scheme 1, wherein the mutant TDP-43 polypeptide lacks a functional domain including nuclear localization signal (NLS), RNA recognition motif 1 (RRM1), RNA recognition motif 2 (RRM2), putative nuclear export signal (E), prion-like domain (PLD), or a combination thereof.

[0174] Implementation Scheme 3. The non-human animal cell as described in Implementation Scheme 1 or Implementation Scheme 2, wherein the non-human animal cell is an embryonic stem (ES) cell, embryoid body, or embryonic stem cell-derived motor neuron (ESMN).

[0175] Implementation Scheme 4. The non-human animal cell as described in any of the preceding implementation schemes, wherein the mutation TARDBP Genes are mutations in non-human animals TARDBP Gene.

[0176] Implementation Scheme 5. A non-human animal cell as described in any one of Implementation Schemes 1-3, wherein the mutation TARDBP The gene is a mutant human TARDBP Gene.

[0177] Implementation Scheme 6. The non-human animal cell as described in any of the preceding embodiments, wherein the mutant TDP-43 polypeptide lacks a functional domain due to one or more of the following: (a) Point mutations of amino acids in NLS, (b) Point mutations in amino acids in RRM1, (c) Point mutations in amino acids in RRM2, (d) The absence of at least a portion of the nuclear output signal, and (e) At least a portion of the prion-like domain is missing.

[0178] Implementation Scheme 7. Non-human animal cells as described in Implementation Scheme 6, wherein (a) The point mutations of amino acids in the NLS include K82A, K83A, R84A, K95A, K97A, K98A, or combinations thereof. (b) The point mutations in RRM1 include F147L and / or F149L. (c) The point mutations in RRM2 include F194L and / or F229L. (d) The deletion of at least a portion of the nuclear output signal includes the deletion of amino acids at and between positions 239 and 250 of the wild-type TDP-43 polypeptide, and (e) The deletion of at least a portion of the prion-like domain includes the deletion of amino acids at and between positions 274 and 414 of the wild-type TDP-43 polypeptide.

[0179] Implementation Scheme 8. The non-human animal cell as described in any of the preceding implementation schemes, wherein the mutant TDP-43 polypeptide comprises K82A, K83A, R84A, K95A, K97A, and K98A.

[0180] Implementation Scheme 9. The non-human animal cell as described in any of the preceding embodiments, wherein the mutant TDP-43 polypeptide lacks the prion-like domain between amino acids at positions 274 to 414 of the wild-type polypeptide and includes the amino acid at said position.

[0181] Implementation Scheme 10. The non-human animal cell as described in any of the preceding implementation schemes, wherein the mutant TDP-43 polypeptide comprises F147L and F149L.

[0182] Implementation Scheme 11. The non-human animal cell as described in any of the preceding implementation schemes, wherein the mutant TDP-43 polypeptide comprises F194L and F229L.

[0183] Implementation Scheme 12. The non-human animal cell as described in any of the preceding embodiments, wherein the mutant TDP-43 polypeptide lacks nuclear output signals between and including the amino acids at positions 239 and 250.

[0184] Implementation Scheme 13. The non-human animal cell as described in any of the preceding implementation schemes, wherein the mutation encoding the mutant TDP-43 polypeptide is... TARDBP Endogenous gene replacement TARDBP Endogenous at loci TARDBP Gene.

[0185] Implementation Scheme 14. The non-human animal cell as described in Implementation Scheme 13, wherein the non-human animal cell is adapted to the mutation encoding the mutant TDP-43 polypeptide. TARDBP The genes are heterozygous.

[0186] Implementation Scheme 15. The non-human animal cell as described in Implementation Scheme 13, wherein the non-human animal cell is adapted to the mutation encoding the mutant TDP-43 polypeptide. TARDBP The genes are homozygous.

[0187] Implementation Scheme 16. The non-human animal cell as described in any one of Implementation Schemes 1-14, wherein the non-human animal cell further comprises a cell containing a knockout mutation. TARDBP Gene.

[0188] Implementation Scheme 17. Non-human animal cells as described in Implementation Scheme 16, wherein the knockout mutation includes a conditional knockout mutation.

[0189] Implementation Scheme 18. Non-human animal cells as described in Implementation Scheme 16 or Implementation Scheme 17, wherein the knockout mutation comprises a site-specific recombination recognition sequence.

[0190] Implementation Scheme 19. The non-human animal cell as described in any one of Implementation Schemes 16-18, wherein the knockout mutation comprises loxp sequence.

[0191] Implementation Scheme 20. The non-human animal cell as described in Implementation Scheme 19, wherein... loxp Sequence side insertion containing knockout mutations TARDBP Exon 3 of the gene.

[0192] Implementation Scheme 21. The non-human animal cell as described in Implementation Scheme 16, wherein the knockout mutation comprises the deletion of the entire coding sequence of the TDP-43 peptide.

[0193] Implementation Scheme 22. The non-human animal cell as described in any one of Implementation Schemes 16-21, wherein the non-human animal cell is for the modified TARDBP The locus is heterozygous and contains (i) In the endogenous nature of a chromosome TARDBP At the gene locus, endogenous TARDBP The gene is encoded by the mutation of the TDP-43 mutant polypeptide. TARDBP Gene replacement, and (ii) the endogenous nature of another homologous chromosome TARDBP At the locus, containing the knockout mutation TARDBP Genetic or wild type TARDBP Gene.

[0194] Implementation Scheme 23. The non-human animal cell as described in any of the preceding implementation schemes, wherein the non-human animal cell does not express the wild-type TDP-43 polypeptide.

[0195] Implementation Scheme 24. The non-human animal cell as described in any one of Implementation Schemes 1-22, wherein the non-human animal cell expresses the wild-type TDP-43 polypeptide.

[0196] Implementation Scheme 25. The non-human animal cell as described in any of the preceding implementation schemes, comprising: (i) Wild-type cells compared to control cells TARDBP The mutation is equivalent to the level of mRNA transcription of the gene. TARDBP mRNA transcription level of genes (ii) The increased level of the mutant TDP-43 peptide compared to the level of wild-type TDP-43 peptide in control cells. (iii) The mutant TDP-43 peptide is found in higher concentrations in the cytoplasm of, for example, motor neurons than in the nucleus. (iv) Mutant TDP-43 peptide with increased insolubility compared to wild-type TDP-43 peptide. (v) Cytoplasmic aggregates containing the mutant TDP-43 polypeptide. (vi) Splicing of additional hidden exons, and / or (vii) Reduce the level of variable splicing in the TDP-43 form.

[0197] Implementation Scheme 26. A non-human animal cell comprising (i) endogenous [material] on a chromosome. TARDBP At the gene locus, the stated TARDBP Conditional knockout mutations in genes, and (ii) the endogenous genes on another homologous chromosome. TARDBP At the locus, the entire TARDBP Missing coding sequence.

[0198] Implementation Scheme 27. The non-human animal cell as described in any of the preceding implementation schemes, wherein the cell is an embryonic stem (ES) cell, a primitive ectoderm cell, or a motor neuron (ESMN) derived from a motor neuron.

[0199] Implementation Scheme 28. The non-human animal cell as described in any of the preceding implementation schemes, wherein the non-human animal cell is a rodent cell.

[0200] Implementation Scheme 29. The non-human animal cell as described in any of the preceding implementation schemes, wherein the non-human animal cell is a rat cell.

[0201] Implementation Scheme 30. The non-human animal cell as described in any one of Implementation Schemes 1-28, wherein the non-human animal cell is a mouse cell.

[0202] Implementation Scheme 31. The non-human animal cell as described in any of the preceding implementation schemes, wherein the non-human animal cell is cultured in vitro.

[0203] Implementation Scheme 32. A non-human animal tissue comprising non-human animal cells as described in any of the preceding implementation schemes.

[0204] Implementation Scheme 33. A composition comprising non-human animal cells or tissues as described in any of the preceding embodiments.

[0205] Implementation Scheme 34. A method for preparing a non-human animal or non-human animal cell expressing a mutant TDP-43 polypeptide, comprising modifying the genome of the non-human animal or non-human animal cell to include a mutation encoding the mutant TDP-43 polypeptide. TARDBPThe gene, wherein the mutant TDP-43 polypeptide lacks a functional domain compared to the wild-type TDP-43, and optionally the wild-type TDP-43 polypeptide comprises a sequence as stated in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5.

[0206] Implementation Scheme 35. The method of Implementation Scheme 34, wherein the modification comprises using the mutation encoding the mutant TDP-43 polypeptide. TARDBP Endogenous gene replacement TARDBP Gene.

[0207] Implementation Scheme 36. The method as described in Implementation Scheme 34 or Implementation Scheme 35, wherein the modification further includes using a method containing a knockout mutation. TARDBP Endogenous gene replacement TARDBP Gene.

[0208] Implementation Scheme 37. The method of Implementation Scheme 36, wherein the knockout mutation includes a conditional knockout mutation.

[0209] Implementation Scheme 38. The method of implementation scheme 37, further comprising eliminating the knockout mutation. TARDBP The cells were cultured under conditions that facilitated gene expression.

[0210] Implementation Scheme 39. A method for identifying therapeutic candidates for treating a disease, the method comprising: (a) Contacting the candidate agent with the non-human animal cells or tissues of any one of embodiments 1-31 or the composition of embodiment 32. (b) Evaluate the phenotype and / or TDP-43 bioactivity of the said nonhuman cells or tissues, and (c) Identify the candidate agents that restore nonhuman cells or tissues to a phenotype and / or TDP-43 bioactivity comparable to that of control cells or tissues expressing wild-type TDP-43 peptides.

[0211] Implementation Scheme 40. A method for evaluating the biological function of the TDP-43 domain, comprising: (a) Modifying embryonic stem (ES) cells to include a mutant encoding the TDP-43 polypeptide TARDBP The mutant TDP-43 polypeptide lacks functional domains selected from the group consisting of: nuclear localization signal (NLS), first RNA recognition motif (RRM1), first RNA recognition motif (RRM2), putative nuclear export signal (E), prion-like domain (PLD), and combinations thereof. (b) Optionally, differentiating the modified ES cells in vitro and / or obtaining genetically modified non-human animals from the modified ES cells, and (c) Evaluate the phenotype and / or TDP-43 bioactivity of the genetically modified ES cells, primitive ectoderms derived therefrom, motor neurons derived therefrom, or non-human animals derived therefrom.

[0212] Implementation Scheme 41. The method of implementation scheme 39 or implementation scheme 40, wherein the phenotype is evaluated by cell culture, fluorescence in situ hybridization, Western blot analysis, or a combination thereof.

[0213] Implementation Scheme 42. The method of any one of Implementation Schemes 39-41, wherein evaluating the phenotype includes measuring the viability of the genetically modified ES cells, primitive ectoderm derived therefrom, motor neurons derived therefrom, or non-human animals derived therefrom.

[0214] Implementation Scheme 43. The method of any one of Implementation Schemes 39-42, wherein the evaluation phenotype includes determining the cellular localization of the mutant TDP-43 peptide.

[0215] Implementation Scheme 44. The method of any one of Implementation Schemes 39-43, wherein evaluating the bioactivity of the mutant TDP-43 polypeptide comprises measuring the splice product of a gene containing a hidden exon regulated by TDP-43.

[0216] Implementation Scheme 45. The method as described in Implementation Scheme 44, wherein the gene containing a hidden exon regulated by TDP-43 includes Crem, Fyxd2, Clf1 .

[0217] Implementation Scheme 46. The method of any one of Implementation Schemes 39-45, wherein evaluating the bioactivity of the mutant TDP-43 peptide includes measuring the level of alternatively spliced ​​TDP-43.

[0218] Implementation Scheme 47. An antisense oligonucleotide comprising a spacer motif of a TDP-43 mRNA sequence containing a PLD targeting a TDP-43 polypeptide and / or a non-translated sequence downstream of exon 6 and upstream of exon 7. Optionally, the TDP-mRNA comprises the sequence between a variable 5' splice site in exon 6 and a downstream variable 3' splice site. Optionally, the variable 5' splice site is selected from the group consisting of the following. TARDBPGenomic location correlations: (a) mouse chromosome 4: 148,618,647; (b) mouse chromosome 4: 148,618,665; (c) mouse chromosome 4: 148,618,674; and (d) human chromosome 4. TARDBP Any corresponding location in the gene, and / or the variable 3' splice site therein associated with the TARDBP genomic location on chromosome 4: 148,617,705.

[0219] Implementation Scheme 48. An siRNA comprising a sequence of a PLD targeting a TDP-43 polypeptide and / or a TDP-43 mRNA sequence comprising an untranslated sequence downstream of exon 6 and upstream of exon 7. Optionally, the TDP-mRNA sequence is located between the variable 5' splice site and the downstream variable 3' splice site within exon 6. Optionally, the variable 5' splice site is selected from the group consisting of the following. TARDBP Genomic location correlations: (a) mouse chromosome 4: 148,618,647; (b) mouse chromosome 4: 148,618,665; (c) mouse chromosome 4: 148,618,674; and (d) human chromosome 4. TARDBP Any corresponding location in the gene, and / or the variable 3' splice site therein associated with the TARDBP genomic location on chromosome 4: 148,617,705.

[0220] Implementation Scheme 49. A CRISPR / Cas system comprising a Cas9 protein and at least one gRNA, wherein the gRNA recognizes a sequence located at or near a site encoding an alternative splicing site, the sequence producing a variable mRNA encoding a truncated PLD-deficient TDP-43 polypeptide. Optionally, the variable splicing site includes a variable 5' splicing site within exon 6 and a downstream variable 3' splicing site. Optionally, the variable 5' splice site is selected from the group consisting of the following. TARDBP Genomic location correlations: (a) mouse chromosome 4: 148,618,647; (b) mouse chromosome 4: 148,618,665; (c) mouse chromosome 4: 148,618,674; and (d) human chromosome 4. TARDBP Any corresponding location in the gene, and / or the variable 3' splice site therein associated with the TARDBP genomic location on chromosome 4: 148,617,705.

[0221] Implementation Scheme 50. A non-human animal comprising the embryonic stem cells described in Implementation Scheme 2.

[0222] Implementation Scheme 51. A non-human animal containing a mutation encoding a mutant TDP-43 polypeptide. TARDBP Gene, Compared to the wild-type TDP-43 peptide, the mutant TDP-43 peptide lacks a functional domain, and The non-human animals described therein express the mutant TDP-43 polypeptide. Optionally, the wild-type TDP-43 polypeptide comprises a sequence as stated in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5.

[0223] Implementation Scheme 52. The non-human animal as described in Implementation Scheme 51, wherein the mutant TDP-43 polypeptide lacks a functional domain including nuclear localization signal (NLS), RNA recognition motif 1 (RRM1), RNA recognition motif 2 (RRM2), putative nuclear export signal (E), prion-like domain (PLD), or a combination thereof.

[0224] Implementation Scheme 53. A non-human animal as described in Implementation Scheme 51 or Implementation Scheme 52, wherein the mutation TARDBP Genes are mutations in the aforementioned non-human animals. TARDBP Gene.

[0225] Implementation Scheme 54. A non-human animal as described in any one of Implementation Schemes 51-53, wherein the mutation TARDBP The gene is a mutant human TARDBP Gene.

[0226] Implementation Scheme 55. A non-human animal as described in any one of Implementation Schemes 51-54, wherein the mutant TDP-43 polypeptide lacks a functional domain due to one or more of the following: (a) Point mutations of amino acids in NLS, (b) Point mutations in amino acids in RRM1, (c) Point mutations in amino acids in RRM2, (d) The absence of at least a portion of the nuclear output signal, and (e) At least a portion of the prion-like domain is missing.

[0227] Implementation Scheme 56. Non-human animals as described in Implementation Scheme 55, wherein (a) The point mutations of amino acids in the NLS include K82A, K83A, R84A, K95A, K97A, K98A, or combinations thereof. (b) The point mutations in RRM1 include F147L and / or F149L. (c) The point mutations in RRM2 include F194L and / or F229L. (d) The deletion of at least a portion of the nuclear output signal includes the deletion of amino acids at and between positions 239 and 250 of the wild-type TDP-43 polypeptide, and (e) The deletion of at least a portion of the prion-like domain includes the deletion of amino acids at and between positions 274 and 414 of the wild-type TDP-43 polypeptide.

[0228] Implementation Scheme 57. The non-human animal as described in any one of Implementation Schemes 51-56, wherein the mutant TDP-43 polypeptide comprises K82A, K83A, R84A, K95A, K97A, and K98A.

[0229] Implementation Scheme 58. A non-human animal as described in any one of Implementation Schemes 51-57, wherein the mutant TDP-43 polypeptide lacks the prion-like domain between amino acids at positions 274 to 414 of the wild-type polypeptide and includes the amino acid at said position.

[0230] Implementation Scheme 59. The non-human animal as described in any one of Implementation Schemes 51-58, wherein the mutant TDP-43 polypeptide comprises F147L and F149L.

[0231] Implementation Scheme 60. The non-human animal as described in any one of Implementation Schemes 51-59, wherein the mutant TDP-43 polypeptide comprises F194L and F229L.

[0232] Implementation Scheme 61. A non-human animal as described in any one of Implementation Schemes 51-60, wherein the mutant TDP-43 polypeptide lacks nuclear output signals between and including the amino acids at positions 239 and 250.

[0233] Implementation Scheme 62. A non-human animal as described in any one of Implementation Schemes 51-61, wherein the mutation encoding the mutant TDP-43 polypeptide is... TARDBP Endogenous gene replacement TARDBP Endogenous at loci TARDBP Gene.

[0234] Implementation Scheme 63. The non-human animal as described in Implementation Scheme 62, wherein the non-human animal has the mutation encoding the mutant TDP-43 polypeptide. TARDBP The genes are heterozygous.

[0235] Implementation Scheme 64. The non-human animal as described in any one of Implementation Schemes 51-63, wherein the non-human animal further comprises a knockout mutation. TARDBP Gene.

[0236] Implementation Scheme 65. The non-human animal as described in Implementation Scheme 64, wherein the knockout mutation includes a conditional knockout mutation.

[0237] Implementation Scheme 66. The non-human animal as described in Implementation Scheme 64 or Implementation Scheme 65, wherein the knockout mutation comprises a site-specific recombination recognition sequence.

[0238] Implementation Scheme 67. A non-human animal as described in any one of Implementation Schemes 64-66, wherein the knockout mutation comprises loxp sequence.

[0239] Implementation Scheme 68. The non-human animal as described in Implementation Scheme 67, wherein... loxp Sequence side insertion containing knockout mutations TARDBP Exon 3 of the gene.

[0240] Implementation Scheme 69. The non-human animal as described in Implementation Scheme 64, wherein the knockout mutation comprises the deletion of the entire coding sequence of the TDP-43 peptide.

[0241] Implementation Scheme 70. A non-human animal as described in any one of Implementation Schemes 64-69, wherein the non-human animal is heterozygous for the modified TARDBP locus and contains (i) At an endogenous TARDBP locus on a chromosome, the endogenous TARDBP gene is replaced by the mutant TARDBP gene encoding the mutant TDP-43 polypeptide, and (ii) the endogenous nature of another homologous chromosome TARDBP At the locus, containing the knockout mutation TARDBP Genetic or wild type TARDBP Gene.

[0242] Implementation Scheme 71. A non-human animal as described in any one of Implementation Schemes 50-70, wherein the non-human animal expresses a wild-type TDP-43 polypeptide.

[0243] Implementation Scheme 72. The non-human animal as described in any one of Implementation Schemes 50-71, comprising: (i) Wild-type compared to control animals TARDBP The mutation is equivalent to the level of mRNA transcription of the gene. TARDBP mRNA transcription level of genes (ii) The increased level of the mutant TDP-43 peptide compared to the level of the wild-type TDP-43 peptide in control animals. (iii) The mutant TDP-43 peptide is found in higher concentrations in the cytoplasm of, for example, motor neurons than in the nucleus. (iv) Mutant TDP-43 peptide with increased insolubility compared to wild-type TDP-43 peptide. (v) Cytoplasmic aggregates containing the mutant TDP-43 polypeptide. (vi) Splicing of additional hidden exons, (vii) Reduce the level of variable splicing in the TDP-43 form. (viii) Denervation and / or innervation of muscle tissue primarily composed of fast-twitch muscles (such as the tibialis anterior muscle) (ix) Normal nerve innervation of muscle tissue mainly composed of slow-twitch muscles (such as intercostal muscles).

[0244] Implementation Plan 73. A non-human animal containing endogenous... TARDBP The locus contains conditional knockout mutations. TARDBP Genes, and another endogenous gene on homologous chromosomes. TARDBP The gene locus contains the entire TARDBP missing coding sequence TARDBP Gene.

[0245] Implementation Scheme 74. The non-human animal as described in any one of Implementation Schemes 50-73, wherein the non-human animal is a rodent.

[0246] Implementation Scheme 75. A non-human animal as described in any one of Implementation Schemes 50-74, wherein the non-human animal is a rat.

[0247] Implementation Scheme 76. The non-human animal as described in any one of Implementation Schemes 50-74, wherein the non-human animal is a mouse.

[0248] Implementation Scheme 77. A method for identifying therapeutic candidates for treating a disease, the method comprising: (a) Contacting the candidate agent with a non-human animal as described in any one of embodiments 50-76, (b) Evaluate the phenotype and / or TDP-43 bioactivity of the said non-human animals, and (c) Identify the candidate agents that restore the phenotype and / or TDP-43 bioactivity of the non-human.

[0249] Implementation Scheme 78. A mutant TDP-43 polypeptide comprising a sequence as stated in SEQ ID NO:1, 3 or 5, said sequence being modified to include one or more of the following: (a) Point mutations of amino acids in NLS, (b) Point mutations in amino acids in RRM1, (c) Point mutations in amino acids in RRM2, (d) The absence of at least a portion of the nuclear output signal, and (e) At least a portion of the prion-like domain is missing.

[0250] Implementation Scheme 79. The mutant TDP-43 polypeptide as described in Implementation Scheme 78, wherein (a) The point mutations of amino acids in the NLS include K82A, K83A, R84A, K95A, K97A, K98A, or combinations thereof. (b) The point mutations in RRM1 include F147L and / or F149L. (c) The point mutations in RRM2 include F194L and / or F229L. (d) The deletion of at least a portion of the nuclear output signal includes the deletion of amino acids at and between positions 239 and 250 of the wild-type TDP-43 polypeptide, and (e) The deletion of at least a portion of the prion-like domain includes the deletion of amino acids at and between positions 274 and 414 of the wild-type TDP-43 polypeptide.

[0251] Implementation Scheme 80. The mutant TDP-43 polypeptide as described in Implementation Scheme 78 or Implementation Scheme 79, comprising K82A mutation, K83A mutation, R84A mutation, K95A mutation, K97A mutation and / or K98A mutation.

[0252] Implementation Scheme 81. The mutant TDP-43 polypeptide as described in any one of Implementation Schemes 78-80, comprising the deletion of a prion-like domain between amino acids at positions 274 to 414 of the wild-type polypeptide and including the amino acids at said positions.

[0253] Implementation Scheme 82. The mutant TDP-43 polypeptide as described in any one of Implementation Schemes 78-81, wherein the mutant TDP-43 polypeptide comprises the F147L mutation and / or the F149L mutation.

[0254] Implementation Scheme 83. The mutant TDP-43 polypeptide as described in any one of Implementation Schemes 78-82, wherein the mutant TDP-43 polypeptide comprises the F194L mutation and / or the F229L mutation.

[0255] Implementation Scheme 84. The mutant TDP-43 polypeptide as described in any one of Implementation Schemes 78-83, wherein the mutant TDP-43 polypeptide lacks and includes the nuclear output signal between and including the amino acids at positions 239 and 250.

[0256] Implementation Scheme 85. A nucleic acid comprising a nucleic acid sequence encoding a mutant TDP-43 polypeptide as described in any one of Implementation Schemes 78-84.

[0257] Implementation Scheme 86. The nucleic acid as described in Implementation Scheme 85, further comprising from 5' to 3': a 5' homologous arm, the nucleic acid sequence encoding the mutant TDP-43 polypeptide, and a 3' homologous arm, wherein the nucleic acid undergoes homologous recombination in rodent cells.

[0258] Implementation Scheme 87. A nucleic acid as described in Implementation Scheme 86, wherein the 5' and 3' homologous arms are homologous to a rat sequence, such that the nucleic acid is effective in endogenous rats. TARDBP Homologous recombination occurs at the locus, and the nucleic acid sequence encoding the mutant TDP-43 polypeptide replaces the endogenous one. TARDBP Encoded sequence.

[0259] Implementation Scheme 88. A nucleic acid as described in Implementation Scheme 86, wherein the 5' and 3' homologous arms are homologous to a mouse sequence, such that the nucleic acid is effective in endogenous mice. TARDBP Homologous recombination occurs at the locus, and the nucleic acid sequence encoding the mutant TDP-43 polypeptide replaces the endogenous one. TARDBP Encoded sequence.

[0260] Implementation Scheme 89. A method for selectively reducing TDP-43 mRNA encoding a TDP-43 polypeptide containing PLD in cells while retaining a variable TDP-43 mRNA encoding a truncated, PLD-deficient TDP-43, the method comprising introducing into the cells: (i) An antisense oligonucleotide comprising a spacer motif of a PLD that targets and encodes a TDP-43 polypeptide and / or a TDP-43 mRNA sequence comprising untranslated sequences downstream of exon 6 and upstream of exon 7. (ii) A siRNA comprising a PLD targeting and encoding a TDP-43 polypeptide and / or a TDP-43 mRNA sequence comprising an untranslated sequence downstream of exon 6 and upstream of exon 7, and / or (iii) A CRISPR / Cas system comprising a Cas9 protein and at least one gRNA, wherein the gRNA recognizes a sequence located at or near a site encoding an alternative splicing site, the sequence producing an alternative mRNA encoding a truncated PLD-deficient TDP-43 polypeptide.

[0261] Implementation Scheme 90. The method as described in Implementation Scheme 89, wherein: (i) The antisense oligonucleotide is the ASO described in embodiment 47. (ii) The siRNA is the siRNA described in embodiment 48, and / or (iii) The CRISPR / Cas system is the CRISPR / Cas system described in Implementation Scheme 49.

[0262] Implementation Scheme 91. The method as described in Implementation Scheme 89 or Implementation Scheme 90, wherein the cells are in vivo.

[0263] Sequence Description

[0264] Example The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.

[0265] Example 1: Expression Mutation TARDBP The generation of embryonic stem cells Since TDP-43 is essential for viability, embryonic stem (ES) cells containing a conditional knockout on a first endogenous TDP-43 allele and a mutation on another second endogenous TDP-43 allele can be generated, such that wild-type TDP-43 from the first endogenous allele maintains the viability of the ES cells until conditional activation, after which the role of the mutant TDP-43 peptide expressed by the second allele can be determined.

[0266] To evaluate the biological, biochemical, and / or pathogenic effects of various TDP-43 domains, mouse ES cells were modified to include: (i) in endogenous TARDBP (ii) a conditional knockout mutation at the locus, and (ii) another mutation on a homologous chromosome. TARDBP Mutation of the TDP-43 peptide at the gene locus TARDBP In the mutant TDP-43 polypeptide, one of its five domains—nuclear localization signal (NLS), RNA recognition motif 1 (RRM1), RNA recognition motif 2 (RRM2), putative output signal (E), or prion-like domain (PLD)—is either altered or missing in a manner predicted to eliminate its function. See Figure 3. Containing mutations TARDBP The (1) phenotype and (2) bioactivity of the mutant TDP-43 peptide in cells expressing the gene lacking functional NLS, RRM1, RRM2, E, or PLD were analyzed as described in Examples 2 and 3, respectively.

[0267] The conditional alleles were designed based on previously published work that showed that deletion of exon 3 of TDP-43 does not produce a functional protein. (Chiang et al., 2010) Proc Natl Acad Sci USA 107:16320-324. Endogenous mice TARDBP Exon 3 lateralization of genes loxp Site. Following Cre-mediated recombination, the deletion at genomic coordinates chr4:147995844-147996841 was affected. As described in this paper, mutations were used... TARDBP The gene was further modified to include exon 3, which is flanked by loxP. As a control, mouse ES cells were also generated that were modified with a conditional knockout mutation in one allele and a deletion from the start codon to the stop codon in exon 2 (genomic coordinates chr4:147992370-147999471) in another allele.

[0268] Example 2: Expression Mutation TARDBP Phenotypic analysis of genes in cells The phenotypes of the embryonic stem (ES) cells, primitive ectoderms derived therefrom, or motor neurons (ESMNs) derived therefrom generated in Example 1 were analyzed by evaluating the viability of the cells and the localization and stability of the mutant TDP-43 peptide.

[0269] It is noteworthy that ES cells expressing the mutant TDP-43 peptide lacking functional NLS or functional PLD are viable; although cells expressing the mutant TDP-43 peptide lacking functional PLD appear to be less fit. Figure 4 ES cells and ESMN expressing the mutant TDP-43 peptide lacking functional RRM1 or RRM2 showed no activity. Figure 4 and Figure 5 .

[0270] The mutant TDP-43 peptide, lacking functional NLS, redistributes from the nucleus to the cytoplasm in ESMN, and the mutant TDP-43 accumulates in large aggregate-like inclusions in many ALS pathologies. (Figure 6 to...) Figure 8 The lack of functional NLS leads to the massive accumulation of mutant TDP-43 peptide in the cytoplasm, accompanied by the disappearance of nuclear staining. Figures 7 to 8 The mutant TDP-43 peptide lacking functional PLD also redistributed into the cytoplasm of ESMN and accumulated in punctate inclusions, which appeared to be less abundant and different in nature than those produced by the mutant TDP-43 peptide lacking functional NLS. (Figures 6 to 6) Figure 8Although nuclear staining was preserved, the absence of PLD resulted in the mutant TDP-43 peptide being mislocalized to the cytoplasm to the greatest extent. Figures 7 to 8 .

[0271] The mutant TDP-43 peptide lacking functional NLS or PLD exhibits increased solubility of the mutant TDP-44. Figure 9A The solubility of mutant TDP-43 peptides lacking functional E or RRM1 did not change compared to wild-type TDP-43 peptides. Figure 9A Although there was no difference in mRNA expression levels among any mutant TDP-43 peptides, increased protein levels were observed in mutant TDP-43 peptides lacking functional NLS, PLD, or RRM1. Figure 9B Since the mRNA expression levels of these mutant TDP-43 peptides are comparable to those of wild-type TDP-43, the increased protein levels may be due to increased stability of the mutant TDP-43 peptides. Figure 9C .

[0272] The materials and methods used to analyze the phenotype of cells expressing the mutant TDP-43 peptide lacking a functional domain are described below.

[0273] Cell culture The ability of the mutant TDP-43 protein, as the only form of the protein expressed in cells, to support the viability of embryonic stem (ES) cells and the motor neurons (ESMNs) derived from them was tested by differentiation in culture. ES cells were cultured for 2 days in embryonic stem cell medium (ESM; DMEM + 15% fetal bovine serum + penicillin / streptomycin + glutamine + non-essential amino acids + nucleosides + β-mercaptoethanol + sodium pyruvate + LIF), with the medium changed daily during this period. One hour before trypsin digestion, the ES medium was replaced with 7 mL of ADFNK medium (advanced DMEM / F12 + neural basal medium + 10% gene knockout serum + penicillin / streptomycin + glutamine + β-mercaptoethanol). The ADFNK medium was withdrawn, and the ESCs were trypsinized with 0.05% trypsin-EDTA. The pelleted cells were resuspended in 12 mL of ADFNK and grown in suspension for two days. Cells were cultured for 4 days in ADFNK supplemented with retinoic acid (RA), a smoothing agonist, and purmorphamine to obtain limb-like motor neurons (ESMN). The isolated motor neurons were plated and matured in embryonic stem cell-derived motor neuron medium (ESMN; neural basal medium + 2% horse serum + B27 + glutamine + penicillin / streptomycin + β-mercaptoethanol + 10 ng / mL GDNF, BDNF, CNTF). In the ES cell stage ( Figure 4 (Figures 6 to 9) or seven days after slab laying ( Figure 5 The conditional knockout allele was activated using cre recombinase delivered via electroporation.

[0274] Intracellular localization of mutant TDP-43 peptide Intracellular localization of the TDP-43 mutant was analyzed using an antibody recognizing the N-terminus of the TDP-43 peptide (α-TDP-43 N-terminus) and an antibody recognizing the C-terminal prion-like domain of the TDP-43 peptide (α-TDP-43 C-terminus) (Proteintech, Rosemont, IL). Soluble cytoplasmic protein extracts were prepared by incubating ES cell-derived MN on ice for 10 min in ice-cold lysis buffer (10 mM KCl, 10 mM Tris-HCl, pH 7.4, 1 mM MgCl2, 1 mM DTT, 0.01% NP-40) supplemented with protease and phosphatase inhibitors (Roche). Cells were then passaged five times using a 27-gauge syringe. The protein supernatant containing the soluble cytoplasmic extract was collected after centrifugation at 4000 rpm for 5 min at 4°C. Insoluble nuclear protein extracts were prepared by resuspending the precipitate in an equal volume of RBS-100 buffer supplemented with protease and phosphatase (10 mM Tris-HCl pH 7.4, 2.5 mM MgCl2, 100 mM NaCl, 0.1% NP-40). An equal volume of 2X SDS sample buffer was added to each fraction, and the sample was heated to 90 °C. Each fraction was then loaded onto a 14% SDS gel and electrophoresed at 225 V for 50 min. TDP-43 was subsequently blotted using either α-TDP-43-N-terminal antibody or α-TDP-43-C-terminal antibody, the latter of which does not recognize the PLD deletion mutant. Densitometric measurements were performed using ImageJ. Figure 6B The ratio of cytoplasmic to nuclear TDP-43 was plotted and statistically analyzed using GraphPad for Prism. Figure 6B ; Figure 9B The image on the right.

[0275] Fluorescence in situ hybridization (FISH) ES cell-derived MNs were plated on polyornithine / laminin-coated coverslips and cultured for 7 days. The coverslips were fixed by immersion in ice-cold 4% PFA for 15 minutes and washed in 1x PBS. Cells were blocked with 5% normal donkey serum diluted in Tris-buffered saline (pH 7.4) containing 0.2% Triton X-100 (TBS-T) and incubated overnight at 4°C in primary antiserum (TDP-43 C-terminus and MAP2) diluted in TBS-T containing 5% normal donkey serum. After washing with TBS-T, cells were incubated at room temperature for 1 hour with species-specific secondary antibodies conjugated to Alexa 488 and 568 (1:1,000; Life Technologies, Carlsbad, CA, USA). After washing with TBS-T, the stained tissue coverslips were mounted on microscope slides from Flouromount (Southern Biotech, Birmingham, AL, USA) and imaged at 40x magnification using a Leica 710LSM confocal microscope. Figure 7 and Figure 8 .

[0276] Solubility of mutant TDP-43 peptide This proposal is adapted from Jo et al. (2014). Nature Communications5:3496. 500 μL of ice-cold soluble buffer (0.1 M MES (pH 7), 1 mM EDTA, 0.5 mM MgSO4, 1 M sucrose) containing 50 mM N-ethylmaleimide (NEM), 1 mM NaF, 1 mM Na3VO4, 1 mM PMSF, and 10 μg / mL each of aprotinin, leucostin, and pepsin. Cells were passaged 3–5 times using a 21-gauge needle, followed by 3–5 passages using a 23-gauge needle. An equal volume of homogenate was then collected from each sample and centrifuged at 50,000 xg for 20 min at 4 °C. The remaining fraction was stored at -80 °C. Remove the supernatant and resuspend each precipitate in 700 μL of RAB buffer (100 mM MES (pH 6.8), 10% sucrose, 2 mM EGTA, 0.5 mM MgSO4, 500 mM NaCl, 1 mM MgCl2, 10 mM NaH2PO4, 20 mM NaF) containing 1% N-lauroyl sarcosyl sarcosyl and protease inhibitors (1 mM PMSF, 50 mM NEM, and 10 μg / mL each of aprotinin, leucopeptide, and pepsin). Vortex for 1 min at room temperature, then incubate overnight at 4 °C using a rotating inverting motion. Centrifuge the samples at 200,000 xg for 30 min at 12 °C and collect the supernatant as the sarcosyl soluble fraction. The precipitate was resuspended in 700 μL of RAB buffer and passaged 3–5 times using a 26-gauge needle to completely disperse it, thus producing the sarcosyl-insoluble fraction. Equal volumes of the sarcosyl-soluble and insoluble fractions were then aliquoted, and an equal volume of 2X SDS sample buffer was added to each. The samples were heated to 90°C. Equal volumes of each fraction were then loaded onto a 14% SDS gel and electrophoresed at 225 V for 50 min, followed by Western blotting with TDP-43. Densitometric analysis was performed using ImageJ. Figure 9A We used GraphPad for Prism to plot the ratio of soluble to insoluble TDP-43 and performed statistical analysis. Figure 9A .

[0277] Expression level of mutant TDP-43 peptide The expression level of the TDP-43 mutant was analyzed using Western blot analysis as described herein. Messenger RNA levels in this embodiment were determined using quantitative polymerase chain reaction.

[0278] Total RNA was extracted from each sample and reverse transcribed using primers spanning the normal exon 4 and exon 5 junction and a probe detecting the mouse TDP-43 locus region. DROSHA qPCR was performed using probes and primers from an off-the-shelf kit.

[0279] Specifically, as described in Example 1, RNA was isolated from embryonic stem cell-derived motor neurons (ESMN).

[0280] Total RNA was isolated using the Direct-zol RNA Miniprep plus kit according to the manufacturer's protocol (Zymo Research). The total RNA was treated with DNase using the Turbo DNA-free kit according to the manufacturer's protocol (Invitrogen) and diluted to 20 ng / μL. Reverse transcription (RT) and PCR were performed in a one-step reaction using the Quantitect Probe RT-PCR kit (Qiagen). The qRT-PCR reaction contained 2 μL of RNA and 8 μL of a mixture containing RT-PCRMaster mix, ROX dye, RT mix, and 20X gene-specific primer-probe mix, for a final volume of 10 μL.

[0281] Unless otherwise specified, the final primer and probe concentrations were 0.5 μM and 0.25 μM, respectively. qRT-PCR was performed on a ViiA™ 7 Real-Time PCR Detection System (ThermoFisher). PCR reactions were performed in quadruplicate in 45 cycles of RT step at 45°C for 10 min, followed by 95°C for 10 min, and then two cycles of 95°C for 5 s and 60°C for 30 s in optical 384-well plates. The sequences of primers and probes used in the analysis (Pan assay) are provided in Table 2 below.

[0282]

[0283] Stability of mutant TDP-43 peptide Two days later, ES cell colonies were isolated and cultured in ADFNK medium. On day 2, the medium was changed and supplemented with retinoic acid (100 nM to 2 μM) (Sigma) and Sonic hedgehog (Shh-N; 300 nM) (Curis Inc.), and embryoids (EB) were cultured for 4 days. On day 4, the embryoids were treated with actinomycete ketone (100 μg / ml) to inhibit new protein synthesis. The medium was changed every 4 hours, and fresh actinomycete ketone was added. Cell lysates were collected at specified time points and analyzed by immunoblotting using TDP-43 and GAPDH antibodies. Figure 9C .

[0284] Example 3: Analysis of the biological activity of TDP-43 mutant against TDP-43 Hidden exons typically possess GU-rich TDP-43 binding sites, and TDP-43 has been shown to repress the recognition of hidden exons, thereby promoting normal splicing. Loss of TDP-43 leads to the loss of normal mRNA and protein levels in regulated genes. TDP-43 also binds to the 3' end of its own transcript as a negative feedback autoregulatory loop to maintain TDP-43 levels. This study tested bioactive mutant TDP-43 peptides lacking functional domains by evaluating their ability to continue repressing hidden exon splicing and / or participating in their autoregulatory loops.

[0285] Analysis of wild type TARDBP Mutations in the gene or encoding a mutant TDP-43 polypeptide lacking functional RRM1, NLS, or PLD TARDBP The expression products of three genes containing hidden exons in the ESMN heterozygote of the gene are known to be repressed by wild-type TDP-43: Crem , Fyxd2 and Clf1 . Figure 10 Normal splicing was observed in all ESMNs expressing mutant TDP-43 peptides lacking functional RRM1, NLS, or PLD. Crem , Fyxd2 and Clf1 The product was analyzed, and it was found that the amount of normal splicing product was comparable to that of ESMN expressing wild-type TDP-43 peptide. Figure 10 However, compared with ESMN expressing wild-type TDP-43 peptide, ESMN expressing mutant TDP-43 peptide lacking functional RRM1, NLS, or PLD showed increased splicing of hidden exons. Figure 10 This data indicates that mutant TDP-43 peptides lacking functional RRM1, NLS, or PLD cannot inhibit [the immune response]. Crem , Fyxd2 and Clf1 Hidden exon splicing of genes. Figure 10 .

[0286] Analysis of wild type TARDBP Mutations in the gene or encoding a mutant TDP-43 polypeptide lacking functional NLS, RRM1, RRM2, E, or PLD TARDBP The level of TDP-43 mRNA in the ESMN heterozygote of the gene through alternative splicing. Figure 11BCompared to control ESMN expressing wild-type TDP-43 peptide, ESMN expressing mutant TDP-43 peptide lacking functional NLS, RRM1, E, or PLD exhibited reduced levels of alternatively spliced ​​TDP-43 mRNA. Figure 11B ESMN expressing the mutant TDP-43 peptide lacking functional E exhibits a considerable level of alternatively spliced ​​TDP-43 mRNA. Figure 11B This data, combined with the data provided in Example 2, indicates that ESMN expressing TDP-43 mutants lacking functional NLS or PLD exhibits an ALS phenotype. Figure 5 This suggests that a strategy aimed at reducing the level of normally spliced ​​TDP-43 mRNA while preserving alternatively spliced ​​TDP-43 mRNA may have a therapeutic effect on TDP-43-related pathologies.

[0287] The materials and methods used to analyze the phenotype of cells expressing the mutant TDP-43 peptide lacking a functional domain are described below.

[0288] Quantitative Polymerase Chain Reaction Total RNA was extracted from each sample and reverse transcribed using primers for side-joined splice regions and probes to detect regions at interrogation loci (Crem, Fxyd2, Clf1, TDP-43). Detectable regions for interrogating Crem, Fxyd2, and Clf1 genes included those spanning mouse sequence junctions in both normal and hidden exons of each interrogated gene. Detectable regions for interrogating TDP-43 included those spanning alternative splice regions. qPCR for DROSHA was performed using probes and primers from an off-the-shelf kit.

[0289] Specifically, RNA was isolated from embryonic stem cell-derived motor neurons (ESMNs) differentiated as described in Example 2. Total RNA was isolated using the Direct-zol RNA Miniprep plus kit according to the manufacturer's protocol (Zymo Research). The total RNA was treated with DNase using the Turbo DNA-free kit according to the manufacturer's protocol (Invitrogen) and diluted to 20 ng / μL. Reverse transcription (RT) and PCR were performed in a one-step reaction using the Quantitect Probe RT-PCR kit (Qiagen). The qRT-PCR reaction contained 2 μL of RNA and 8 μL of a mixture containing RT-PCR Master Mixture, ROX dye, RT Mixture, and 20X gene-specific primer-probe mixture, for a final volume of 10 μL.

[0290] Unless otherwise specified, the final primer and probe concentrations are 0.5 μM and 0.25 μM, respectively. qRT-PCR was performed on a ViiA™ 7 real-time PCR detection system (ThermoFisher). PCR reactions were performed in quadruplicate in 50 cycles of RT step at 45°C for 10 min, followed by 95°C for 10 min, and then two cycles of 95°C for 5 s and 60°C for 30 s in an optical 384-well plate.

[0291] Using primers containing the nucleotide sequences described in SEQ ID NO:14 and SEQ ID NO:15 and primers containing the nucleotide sequence described in SEQ ID NO:16, a process for evaluating the nucleotide sequence from... Crem Productivity of exon 1 to exon 2 Crem spliced ​​qRT-PCR. Crem The splicing of exon 1 to hidden exons was evaluated using primers containing nucleotide sequences as stated in SEQ ID NO:17 and SEQ ID NO:18 and primers containing nucleotide sequences as stated in SEQ ID NO:19. Crem The splicing of hidden exon to exon 2 was evaluated using primers containing nucleotide sequences as stated in SEQ ID NO:20 and SEQ ID NO:21 and primers containing nucleotide sequences as stated in SEQ ID NO:22.

[0292] Using primers containing the nucleotide sequences described in SEQ ID NO:23 and SEQ ID NO:24 and primers containing the nucleotide sequence described in SEQ ID NO:25, an evaluation was performed for... Fyxd2 Productivity of exon 3 to exon 4 Fyxd2 spliced ​​qRT-PCR. Fyxd2 The splicing of exon 3 to hidden exons was evaluated using primers containing nucleotide sequences as stated in SEQ ID NO:26 and SEQ ID NO:27 and primers containing nucleotide sequences as stated in SEQ ID NO:28. Fyxd2 The splicing of hidden exon to exon 4 was evaluated using primers containing nucleotide sequences as stated in SEQ ID NO:29 and SEQ ID NO:30 and primers containing nucleotide sequences as stated in SEQ ID NO:31.

[0293] Using primers containing nucleotide sequences as stated in SEQ ID NO:32 and SEQ ID NO:33 and primers containing nucleotide sequences as stated in SEQ ID NO:34, the production of... Crlf1 qRT-PCR of splice products. Crlf1 The splicing of exon 1 to hidden exons was evaluated using primers containing nucleotide sequences as stated in SEQ ID NO:35 and SEQ ID NO:36 and primers containing nucleotide sequences as stated in SEQ ID NO:37. Crlf1 The splicing of hidden exon to exon 2 was evaluated using primers containing nucleotide sequences as stated in SEQ ID NO:38 and SEQ ID NO:39 and primers containing nucleotide sequences as stated in SEQ ID NO:40.

[0294] TDP-43 mRNAs lacking sequences encoding the PLD domain were evaluated using primers containing nucleotide sequences as stated in SEQ ID NO:41 and SEQ ID NO:42 and primers containing nucleotide sequences as stated in SEQ ID NO:43.

[0295] The sequences of primers and probes used in each qPCR analysis (normal and hidden splicing) in this embodiment are provided in Table 3 below.

[0296] Table 3

[0297] Example 4: Expression Mutation TDP-43 protein The production of mice Although the absence of TDP-43 leads to embryonic death, it is only caused by endogenous factors. TARDBP Embryonic stem cells expressing the mutant ΔNLS TDP-43 gene or the mutant ΔPLD TDP-43 gene at the locus are viable and can differentiate into motor neurons in vitro. This data suggests the possibility that endogenous... TARDBP Embryonic stem cells expressing a mutant TDP-43 peptide lacking a functional domain at a locus can be viable and can be used to create animal models of TDP-43 protein disorders. For example, such embryonic stem cells can be used to generate non-human animals, such as mice, expressing a mutant TDP-43 protein lacking a functional domain to examine the role of the TDP-43 domain in normal and pathobiological processes.

[0298] To generate embryos or animals expressing mutant TDP-43 protein lacking functional NLS or PLD domains, the VelociMouse® method (Dechiara, TM, (2009), Methods Mol Biol 530:311-324; Poueymirou et al. (2007), Nat. Biotechnol. 25:91-99) was used, which included the following targeting ES cells. (i) In endogenous TARDBP At the locus, there is exon 3 (loxP-Ex3-loxP) with conditional side-attached loxP. TARDBP Gene, null alleles following Cre-mediated deletion of exon 3 of loxP (-), knockout mutation in NLS (ΔNLS), deletion of prion-like domain (ΔPLD), or wild-type TARDBP Gene (WT), See Figure 3A ,as well as (ii) another on a homologous chromosome TARDBP At the gene locus, wild type (WT) TARDBP The gene or the null allele (-) following Cre-mediated lateral loxP exon 3 deletion was injected into uncompacted 8-cell stage Swiss Webster embryos. Embryo viability was examined after fertilization, and the ability to produce live F0 generation mice was assessed.

[0299] Consistent with previous experiments, the functional TDP-43 protein (TDP-43) was lacking. - / - Embryos of this type are not viable and cannot survive beyond E3.5. Figure 12 ) phase. Similarly, only expressing TDP-43 protein (TDP-43) lacking functional NLS. ΔNLS / - ) or only expressing TDP-43 protein lacking functional PLD (TDP-43 ΔPLD / - Embryos that are not viable, although such embryos have a longer lifespan ( Figure 12 ).Depend on TARDPB One allele at a locus expresses wild-type TDP-43 protein, rescuing TDP-43 protein lacking functional NLS from another allele on a homologous chromosome. ΔNLS / - ) or TDP-43 protein lacking functional PLD (TDP-43 ΔPLD / - ) embryo ( Figure 12 ).

[0300] F0 generation mice were successfully produced from 8-cell stage Swiss Webster embryos injected with ES cells, wherein the ES cells contain (i) In endogenous TARDBP At the locus, the wild-type gene (WT) contains exon 3 deletion mediated by cre-mediated lateral loxP. TARDBP Gene (-), exon 3 with loxP lateralization (loxP-Ex3-loxP), knockout mutation in NLS (ΔNLS), deletion of prion-like domain (ΔPLD), See Figure 3A ,as well as (iii) Another homologous chromosome TARDBPAt the gene locus, wild type (WT) TARDBP Gene.

[0301] Example 4: Phenotypic analysis of mice expressing mutant TDP-43 peptide lacking functional domains The phenotypes of the animals produced in Example 3 were analyzed by evaluating the localization, phosphorylation status, and solubility of the TDP-43 peptide in spinal cord tissue or motor neurons isolated from animals. Additionally, the denervation or innervation of the animal muscles was determined.

[0302] The cytoplasmic and nuclear fractions of motor neurons derived from the spinal cord of 16-week-old mice were evaluated by Western blot analysis as follows: (1) antibodies that recognize the N-terminus of wild-type TDP-43 protein and thus bind to wild-type TDP-43, ΔNLS TDP-43 and ΔPLD TDP-43, (2) antibodies that recognize the C-terminus of wild-type TDP-43 protein and thus bind to wild-type TDP-43 and ΔNLS TDP-43, but not ΔPLD TDP-4, or (3) antibodies that recognize its phosphorylated form of TDP-43.

[0303] like Figures 13A to 13C As shown, wild-type and ΔNLS mutant TDP-43 proteins were detected at an expected size of approximately 43 kDa, while the ΔPLD mutant was detected at an expected size of approximately 30 kDa. Similar to ESMN analyzed in Example 2, even in the presence of wild-type TDP-43 protein, mutant TDP-43 peptides lacking functional NLS or PLD redistributed from the nucleus to the cytoplasm in spinal cord tissue. Figure 13A Approximately 43 kDa of phosphorylated TDP-43 peptide was detected in the cytoplasm of motor neurons derived from the spinal cord of mice expressing mutant ΔNLS or ΔPLD peptides, but was not detected in mice expressing only wild-type TDP-43 peptide. Figure 13B In all the samples examined, no phosphorylated TDP-43 in the nuclei of motor neurons remained undetectable. Figure 13B Since the phosphorylation site is at amino acid positions 409 / 410, it is not surprising that phosphorylated TDP-43 peptides lacking functional PLD were not detected. Figure 13B Motor neurons in the spinal cord of 16-week-old mice expressing the ΔNLS mutant TDP-43 protein, which contains a functional mutation in the NLS domain, showed an overall increased level of insoluble TDP-43 protein. Figure 13C The solubility of TDP-43 protein did not appear to increase in mice expressing the ΔPLD mutant TDP-43 protein. The ΔPLD mutant appeared to be soluble, as it was not detected in the insoluble fraction. Figure 13C .

[0304] A subset of mouse motor neurons expressing either the ΔNLS mutant TDP-43 protein containing a functional mutation in the ΔNLS domain or the ΔPLD TDP-43 mutant protein lacking a functional PLD exhibited extensive cytoplasmic TDP-43 aggregation. Figure 14 Compared with mice expressing the mutant TDP-43 peptide lacking functional NLS, mice expressing the ΔPLD mutant protein showed a lower frequency of cytoplasmic aggregation in motor neurons. Figure 14 .

[0305] Since denervation is one of the earliest pathological features in ALS, denervation of muscles primarily containing fast-twitch fibers (tibialis anterior) or slow-twitch fibers (intercostal muscles) was analyzed. Mislocalization of TDP-43 resulted in partially innervated endplates (*) and denervation of muscles primarily containing fast-twitch fibers rather than slow-twitch fibers (arrows). Figures 15A to 15B .

[0306] The data presented in this article suggest that the animals described herein can serve as a valuable disease model for ALS. In typical ALS patients, distal rapid fatigue (FF) motor units are the first to be affected, and neurogenic changes in muscles can be observed prior to motor neuron loss. Similarly, skeletal muscle denervation also occurs before motor neuron loss in the most widely used “ALS” model, the SOD1 G93A mouse, with FF motor units involved early and preferentially. In contrast, proximal muscles, primarily innervated by slow fibers (such as the intercostal muscles and diaphragm), are typically unaffected until the very end—and denervation of these muscles is fatal. Denervation of the intercostal muscles is expected as the disease progresses.

[0307] The materials and methods used to analyze the phenotypes of mice expressing both (a) the mutant TDP-43 peptide lacking functional NLS or PLD and (b) the wild-type TDP-43 peptide are described below.

[0308] Intracellular localization and phosphorylation detection of mutant TDP-43 peptide Intracellular localization of TDP-43 mutants was analyzed using antibodies recognizing the N-terminus of the TDP-43 peptide (α-TDP-43 N-terminus) and antibodies recognizing the C-terminal prion-like domain of the TDP-43 peptide (α-TDP-43 C-terminus) (Proteintech, Rosemont, IL). Soluble cytoplasmic protein extracts were prepared from whole spinal cord tissue by incubating on ice for 10 min in ice-cold lysis buffer (10 mM KCl, 10 mM Tris-HCl, pH 7.4, 1 mM MgCl2, 1 mM DTT, 0.01% NP-40) supplemented with protease and phosphatase inhibitors (Roche). Cells were then passaged five times using a 27-gauge syringe. The protein supernatant containing the soluble cytoplasmic extract was collected after centrifugation at 4000 rpm for 5 min at 4°C. Insoluble nuclear protein extracts were prepared by resuspending the precipitate in an equal volume of RBS-100 buffer supplemented with protease and phosphatase (10 mM Tris-HCl pH 7.4, 2.5 mM MgCl2, 100 mM NaCl, 0.1% NP-40). An equal volume of 2X SDS sample buffer was added to each fraction, and the sample was heated to 90°C. Then, an equal volume of each fraction was loaded onto a 14% SDS gel and electrophoresed at 225 V for 50 min, followed by analysis with α-TDP-43 N-terminal antibody (…). Figure 13A ), α-TDP-43-C-terminal antibody ( Figure 13A ), or detect α-phospho-TDP-43 antibody at amino acid 409 / 410 phosphorylation of TDP-43 ( Figure 13B (Cosmo Bio USA; Catalog No. CAC-TIP-PTD-M01) Western blotted TDP-43. Neither the α-TDP-43 C-terminal antibody nor the α-phosphate TDP-43 antibody recognized the PLD deletion mutant. Densitometric measurements were performed using ImageJ. Figure 13A and 13B The ratio of cytoplasmic to nuclear TDP-43 was plotted and statistically analyzed using GraphPad for Prism. Figure 13A (See the image below).

[0309] Fluorescence in situ hybridization (FISH) The spinal cord was isolated from the spine, fixed overnight in 4% PFA (or immunostained with FUS for 1 hour), and washed in 1xPBS. Spinal cord segments were embedded in 4% low-melting-point agarose (Promega) and processed into free-floating sections (70 μm) using a vibratory microtome (Leica VT1000S). Free-floating spinal cord sections were blocked with 5% normal donkey serum diluted in Tris-buffered saline (pH 7.4) containing 0.2% Triton X-100 (TBS-T) and incubated overnight at room temperature in primary antiserum diluted in TBS-T containing 5% normal donkey serum. The primary antibodies used were: ChAT (1:250) EMD Millipore Cat AB144P; TDP-43 (1:10,000) Proteintech 10782-2-AP; and NeuN (1:500) EMD Millipore MAB377. After washing with TBS-T, tissue sections were incubated at room temperature for 4 hours with species-specific secondary antibodies conjugated to Alexa 488, 555, 647 (1:1,000; Life Technologies, Carlsbad, CA, USA), Cy3, or Cy5 (1:500 dilution; Jackson Immunoresearch Labs, West Grove, PA, USA). After washing with TBS-T, the stained tissue sections were mounted on Flouromount G (Southern Biotech, Birmingham, AL, USA) microscope slides and imaged using an LSM 510 confocal microscope at 40x magnification and 1.5x zoom. Figure 14 ).

[0310] Solubility of mutant TDP-43 peptide This proposal is adapted from Jo et al. (2014). Nature Communications5:3496. 500 μL of ice-cold soluble buffer (0.1 M MES (pH 7), 1 mM EDTA, 0.5 mM MgSO4, 1 M sucrose) containing 50 mM N-ethylmaleimide (NEM), 1 mM NaF, 1 mM Na3VO4, 1 mM PMSF, and 10 μg / mL each of aprotinin, leucopeptide, and pepsin. Cells from the spinal cord tissue of 16-week-old mice were lysed by 3–5 passages using a 21-gauge needle, followed by 3–5 passages using a 23-gauge needle. Equal volumes of homogenate were then collected from each sample and centrifuged at 50,000 xg for 20 min at 4 °C, with the remainder stored at -80 °C. Remove the supernatant and resuspend each precipitate in 700 μL of RAB buffer (100 mM MES (pH 6.8), 10% sucrose, 2 mM EGTA, 0.5 mM MgSO4, 500 mM NaCl, 1 mM MgCl2, 10 mM NaH2PO4, 20 mM NaF) containing 1% N-lauroyl sarcosyl sarcosyl and protease inhibitors (1 mM PMSF, 50 mM NEM, and 10 μg / mL each of aprotinin, leucopeptide, and pepsin). Vortex for 1 min at room temperature, then incubate overnight at 4 °C using a rotating inverting motion. Centrifuge the samples at 200,000 x g for 30 min at 12 °C and collect the supernatant as the sarcosyl soluble fraction. The precipitate was resuspended in 700 μL of RAB buffer and passaged 3–5 times using a 26-gauge needle to completely disperse it, thus producing the sarcosyl-insoluble fraction. Equal volumes of the sarcosyl-soluble and insoluble fractions were then aliquoted, and an equal volume of 2X SDS sample buffer was added to each. The samples were heated to 90°C. Equal volumes of each fraction were then loaded onto a 14% SDS gel and electrophoresed at 225 V for 50 min, followed by Western blotting with TDP-43. Densitometric analysis was performed using ImageJ. Figure 13C The ratio of soluble to insoluble TDP-43 was plotted using GraphPad for Prism, and statistical analysis was performed. Figure 13C ).

[0311] Denervation research For muscle analysis, the tibialis anterior (TA) and intercostal muscles were dissected, fixed for 2 hours by immersion in 4% PFA, and washed in 1x phosphate-buffered saline, pH 7.4 (PBS). The muscles were then equilibrated in a sucrose gradient (0.1 M phosphate-buffered saline containing 10%–20%–30% sucrose, pH 7.4), inlaid with OCT compounds (Sakura, Torrance, CA), and frozen at -20°C. Serial sections (30 μm thick) were cut using a cryostat (Leica CM 3050S). The frozen muscle sections (30 μm) were stained with an anti-synaptophysin (invitrogen) antibody to identify presynaptic terminals and stained with Alexa 488-conjugated α-BTX (Invitrogen) to detect postsynaptic acetylcholine receptors. Images were acquired using a Zeiss Pascal LSM 510 confocal microscope with x10 and x40 objectives. The percentage (%) of NMJ innervation is determined by dividing the total number of overlapping regions between VAChT and α-BTX signals (total number of nerve-innervated endplates) by the number of α-BTX signal regions (total number of endplates).

Claims

1. A non-human animal cell, a non-human animal tissue comprising the non-human animal cell, a composition comprising the non-human animal cell or the non-human animal tissue, or a non-human animal genome, wherein the non-human animal cell or genome comprises a mutation encoding a mutant TDP-43 polypeptide. TARDBP Gene, The mutant TDP-43 peptide lacks the functional domains found in the wild-type TDP-43 peptide, including nuclear localization signal (NLS), RNA recognition motif 1 (RRM1), RNA recognition motif 2 (RRM2), putative nuclear export signal (NES), prion-like domain (PLD), or combinations thereof. The non-human animal cells, non-human animal tissues, or non-human animal cells containing the genome of the non-human animal express the mutant TDP-43 polypeptide. Optionally, the wild-type TDP-43 polypeptide comprises a sequence as shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:

5.

2. A method for preparing a non-human animal or non-human animal cell expressing a mutant TDP-43 polypeptide, comprising modifying the genome of the non-human animal or the non-human animal cell to include a mutation encoding the mutant TDP-43 polypeptide. TARDBP The gene, wherein the mutant TDP-43 polypeptide lacks a functional domain compared to the wild-type TDP-43, and optionally the wild-type TDP-43 polypeptide comprises a sequence as shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:

5.

3. A method for identifying therapeutic candidates for treating a disease, the method comprising: (a) Contacting the non-human animal cell of claim 1, the non-human animal tissue, the composition comprising the non-human animal cell or the non-human animal tissue, or a non-human animal cell comprising the non-human animal genome of claim 1 with the therapeutic candidate. (b) Evaluate the phenotype and / or TDP-43 bioactivity of the said nonhuman cells or tissues, and (c) Identify the therapeutic candidate that restores the nonhuman animal cells or tissues to a phenotype and / or TDP-43 bioactivity equivalent to that of control nonhuman animal cells or tissues expressing wild-type TDP-43 polypeptide.

4. A method for evaluating the biological function of the TDP-43 domain, comprising: (a) Modifying embryonic stem (ES) cells to include a mutant encoding the TDP-43 polypeptide TARDBP The mutant TDP-43 polypeptide lacks functional domains selected from the group consisting of: nuclear localization signal (NLS), first RNA recognition motif (RRM1), second RNA recognition motif (RRM2), putative nuclear export signal (NES), prion-like domain (PLD), and combinations thereof. (b) Optionally, differentiating the modified ES cells in vitro and / or obtaining genetically modified non-human animals from the modified ES cells, and (c) Evaluate the phenotype and / or TDP-43 bioactivity of the genetically modified ES cells, primitive ectoderms derived therefrom, motor neurons derived therefrom, or non-human animals derived therefrom.

5. An antisense oligonucleotide comprising a spacer motif targeting a TDP-43 mRNA sequence, the TDP-43 mRNA sequence encoding a PLD of a TDP-43 polypeptide and / or comprising an untranslated sequence downstream of exon 6 and upstream of exon 7.

6. An siRNA comprising a sequence targeting a TDP-43 mRNA sequence, said TDP-43 mRNA sequence encoding a PLD of the TDP-43 polypeptide and / or comprising an untranslated sequence downstream of exon 6 and upstream of exon 7.

7. A CRISPR / Cas system comprising a Cas9 protein and at least one gRNA, wherein the at least one gRNA recognizes a sequence located at or near a sequence encoding an alternative splicing site, the alternative splicing site producing an alternative mRNA encoding a truncated PLD-deficient TDP-43 polypeptide.

8. A mutant TDP-43 polypeptide comprising a sequence modified to include one or more of the following, as shown in SEQ ID NO:1, 3, or 5: (a) Point mutations of amino acids in NLS, (b) Point mutations in amino acids in RRM1, (c) Point mutations in amino acids in RRM2, (d) At least a portion of the nuclear output signal (NES) is missing, and (e) At least a portion of the prion-like domain (PLD) is missing.

9. A nucleic acid comprising a nucleic acid sequence encoding the mutant TDP-43 polypeptide of claim 8, optionally wherein the nucleic acid further comprises, from 5' to 3': a 5' homologous arm, the nucleic acid sequence encoding the mutant TDP-43 polypeptide, and a 3' homologous arm, wherein the nucleic acid undergoes homologous recombination in rodent cells.

10. A method for selectively reducing TDP-43 mRNA encoding a TDP-43 polypeptide containing PLD in cells while retaining a variable TDP-43 mRNA encoding a truncated TDP-43 polypeptide lacking PLD, the method comprising introducing into the cells: (i) An antisense oligonucleotide comprising a spacer motif targeting a TDP-43 mRNA sequence, the TDP-43 mRNA sequence encoding a PLD of a TDP-43 polypeptide and / or comprising untranslated sequences downstream of exon 6 and upstream of exon 7. (ii) An siRNA comprising a sequence targeting a TDP-43 mRNA sequence, said TDP-43 mRNA sequence encoding a PLD of the TDP-43 polypeptide and / or comprising untranslated sequences downstream of exon 6 and upstream of exon 7, and / or (iii) A CRISPR / Cas system comprising a Cas9 protein and at least one gRNA, wherein the at least one gRNA recognizes a sequence located at or near a sequence encoding an alternative splicing site, the alternative splicing site producing an alternative mRNA encoding a truncated PLD-deficient TDP-43 polypeptide.

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