Modeling TDP-43 proteinopathy

Abstract

To provide modeling of TDP-43 proteinopathy.SOLUTION: Disclosed herein is the discovery that neither nuclear localization signal (NLS) nor prion-like domain (PLD) of TDP-43 is necessary for embryotic stem cell culture and differentiation into motor neurons in vitro. The capability of ES cells to express TDP-43 variants and differentiate into neurons exhibiting an ALS-like phenotype (the TDP-43 variants redistribute to and aggregate in the cytoplasm, and fail to regulate cryptic exon splicing) allows these cells to act as a model of TDP-43 proteinopathy for the testing of candidate therapeutic agents that may resolve such proteinopathy. Further, these ES cells may be used for successfully generating a non-human animal such as a mouse exhibiting characteristic symptoms of ALS, for use in the search of candidate agents useful for the treatment of TDP-43 proteinopathy.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(3) of U.S. Provisional Application No. 62 / 867,785 (filed June 27, 2019), the disclosure of which is incorporated herein by reference in its entirety.

[0002] Reference to sequence listing submitted as a text file via EFS Web The sequence listing set forth in file 10312WO01_ST25.txt is 35 kilobytes, was created on June 25, 2020, and is incorporated herein by reference.

[0003] Technical Field Described herein are methods for evaluating the biological role(s) of TDP-43 and its domains, non-human animals and non-human animal cells therefor, and nucleic acids therefor. Models of TDP-43 proteinopathy comprising such non-human animals, non-human animal cells, or nucleic acids, and methods of using them are also provided. [Background technology]

[0004] Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that affects motor neurons and causes limb paralysis and eventual death as a result of diaphragm muscle impairment. An almost universal pathological finding in postmortem examination of ALS patient tissue is the accumulation of TDP-43 (transactive response DNA-binding protein 43 kDa) in cytoplasmic inclusions.

[0005] TDP-43 is characterized by having 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 primarily nuclear RNA-binding protein required for the survival of all mammalian cells and normal development of animals. The redistribution of TDP-43 from the nucleus to the cytoplasm and its accumulation in insoluble aggregates are two key diagnostic features of ALS disease.

[0006] Although cytoplasmic accumulation of TDP-43 is associated with ALS, the relationship between each structural domain of TDP-43 and the biological function(s) of TDP-43 is unclear. Summary of the Invention [Means for solving the problem]

[0007] Provided herein are embryonic stem (ES) cells, tissues (e.g., primitive ectoderm, embryoid bodies, motor neurons) cultured therefrom, and non-human animals derived therefrom that express mutant TDP-43 polypeptides lacking functional structural domains and may exhibit ALS-like phenotypes. Also provided are compositions and methods for making and using the same. Also provided are mutated TARDBP genes encoding mutant TDP-43 polypeptides lacking functional structural domains and mutant TDP-43 polypeptides lacking functional structural domains. Also provided are exemplary therapeutic oligonucleotides, e.g., antisense oligonucleotides, that may restore autoregulation of TARDBP expression.

[0008] Described herein are non-human animals (e.g., rodents (e.g., rats or mice)) and non-human animal cells (e.g., embryonic stem (ES) cells, embryoid bodies, embryonic stem cell derived motor neurons (ESMN), etc.) comprising a mutated TARDBP gene encoding a mutant TDP-43 polypeptide, wherein the mutated TARDBP gene comprises the nucleotide sequence of a wild-type TARDBP gene comprising a mutation such that the mutant TDP-43 comprises the amino acid sequence of the corresponding wild-type TDP-43 polypeptide but for the mutation (e.g., one or more of a point mutation, substitution, replacement, insertion, deletion, etc.). In some embodiments, the wild-type TARDBP gene comprises the sequence set forth in SEQ ID NO:2 (including degenerate variants thereof), SEQ ID NO:4 (including degenerate variants thereof), or SEQ ID NO:6 (including degenerate variants thereof), which encode a wild-type TDP-43 polypeptide comprising the amino acid sequence set forth as SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, respectively.

[0009] In some embodiments, the mutated TARDBP gene encoding the mutant TDP-43 polypeptide replaces the endogenous TARDBP gene at the endogenous TARDBP locus of the non-human animal or non-human animal cell. In some embodiments, the non-human animal cell or non-human animal is heterozygous for the mutated TARDBP gene encoding the mutant TDP-43 polypeptide. For example, in some embodiments, the non-human animal or non-human animal cell further comprises, in addition to the mutated TARDBP gene as described herein, (a) a wild-type TARDBP gene or (b) a TARDBP gene comprising a knockout mutation, e.g., a conditional knockout mutation. In some embodiments, the conditional knockout mutation comprises a site-specific recombination recognition sequence, e.g., a loxp sequence, and optionally, the site-specific recombination recognition sequence (e.g., a loxp sequence) flanks a coding exon, e.g., exon 3. In some embodiments, the TARDBP gene comprising the knockout mutation comprises a loxp sequence flanking the deleted exon 3 of the TARDBP gene. In some embodiments, the knockout mutation comprises a deletion of the entire coding sequence of the TDP-43 peptide.

[0010] In some embodiments, the non-human animal or non-human animal cell (i) comprises a replacement of the endogenous TARDBP gene at the endogenous TARDBP locus with a mutated TARDBP gene encoding a mutant TDP-43 polypeptide, and (ii) comprises either a TARDBP gene comprising a knockout mutation or a wild-type TARDBP gene at the other endogenous TARDBP locus of the homologous chromosome.

[0011] In some embodiments, the non-human animal or non-human animal cell comprises a TARDBP gene that comprises a conditional knockout mutation at the endogenous TRADBP locus and a TARDBP gene that comprises a deletion of the entire TARDBP coding sequence at the other endogenous TARDBP locus of the homologous chromosome.

[0012] In some embodiments, the non-human animal cell or the non-human animal is homozygous for a mutated TARDBP gene encoding a mutant TDP-43 polypeptide.

[0013] In some embodiments, the non-human animal or non-human animal cell does not express a wild-type TDP-43 polypeptide.

[0014] In some embodiments, the non-human animal or non-human animal cell expresses a wild-type TDP-43 polypeptide.

[0015] In some embodiments, the non-human animal or non-human animal cell of any one of the preceding claims comprises an mRNA transcription level of the mutated TARDBP gene comparable to the mRNA transcription level of the wild-type TARDBP gene in a control cell, comprises an increased level of the mutant TDP-43 polypeptide compared to the level of the wild-type TDP-43 polypeptide in a control cell, comprises a higher concentration of the mutant TDP-43 polypeptide found, for example, in the cytoplasm rather than the nucleus of a motor neuron, comprises a mutant TDP-43 polypeptide that is increased insolubility compared to wild-type TDP-43 polypeptide cytoplasmic aggregates that comprise the mutant TDP-43 polypeptide, comprises increased splicing of cryptic exons, and / or a reduced level of alternatively spliced ​​TDP-43 forms. In some embodiments, the non-human animal exhibits 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.

[0016] In some embodiments, the non-human animal cells as described herein are cultured in vitro. Also described herein are non-human animal tissues comprising the non-human animal cells as described herein.

[0017] In some embodiments, non-human animal tissue and / or non-human animal cells are included in the composition.

[0018] In some embodiments, the mutant TDP-43 polypeptide lacks a functional structural domain compared to a wild-type TDP-43 polypeptide, and the non-human animal or non-human animal cell expresses the mutant TDP-43 polypeptide, and optionally, the wild-type TDP-43 polypeptide comprises the sequence set forth as SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5.

[0019] In some embodiments, the mutated TDP-43 polypeptide lacks a functional structural domain selected from the group consisting of a nuclear localization signal (NLS), an RNA recognition motif 1 (RRM1), an RNA recognition motif 2 (RRM2), a putative nuclear export signal (E), a prion-like domain (PLD), or a combination thereof. In some embodiments, the mutated TARDBP gene is a non-human animal TARDBP gene comprising a mutation, for example, a point mutation, a substitution, an insertion, a deletion, or a combination thereof. In some embodiments, the non-human animal TARDBP gene is set forth as SEQ ID NO:2 or SEQ ID NO:4. In some embodiments, the mutated TARDBP gene is a human TARDBP gene comprising a mutation, for example, a point mutation, a substitution, an insertion, a deletion, or a combination thereof. In some embodiments, the mutated TARDBP. In some embodiments, the human TARDBP gene is set forth as SEQ ID NO:5.

[0020] In some embodiments, the mutant TDP43 polypeptide lacks a functional structural domain due to one or more of the following: (a) an amino acid point mutation in the NLS, (b) an amino acid point mutation in RRM1, (c) an amino acid point mutation in RRM2, (d) a deletion of at least a portion of the nuclear export signal, and (e) a deletion of at least a portion of the prion-like domain. For example, in some embodiments, the mutant TDP-43 polypeptide comprises a sequence set forth as SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, further comprising (a) an amino acid point mutation in the NLS, (b) an amino acid point mutation in RRM1, (c) an amino acid point mutation in RRM2, (d) a deletion of at least a portion of the nuclear export signal, and (e) a deletion of at least a portion of the prion-like domain. In some embodiments, (a) the amino acid point mutations in the NLS include K82A K83A, R84A, K95A, K97A, K98A, or a combination 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 at least a portion of the nuclear export signal deletion includes an amino acid deletion at and between positions 239 and 250 of the wild-type TDP-43 polypeptide; and (E) the at least a portion of the prion-like domain deletion includes an amino acid deletion 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, and optionally, the wild-type TDP-43 polypeptide comprises a sequence set forth as SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5. In some embodiments, the mutant TDP-43 polypeptide lacks the prion-like domain at amino acids 274 to 414 of the wild-type TDP-43 polypeptide, and optionally, the wild-type TDP-43 polypeptide comprises a sequence set forth as 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, and optionally, the wild-type TDP-43 polypeptide comprises a sequence set forth as 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, and optionally, the wild-type TDP-43 polypeptide comprises a sequence set forth as SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5. In some embodiments, the mutant TDP-43 polypeptide lacks a nuclear export signal at amino acids 239 to 250 compared to the wild-type TDP-43 polypeptide, and optionally, the wild-type TDP-43 polypeptide comprises a sequence set forth as SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5.

[0021] Also provided are mutant TDP-43 polypeptides as described herein and nucleic acid molecules encoding same. In some embodiments, the nucleic acid molecule encoding a mutant TDP-43 polypeptide as described herein further comprises, from 5' to 3': a 5' homology arm, a nucleic acid sequence encoding a mutant TDP-43 polypeptide, and a 3' homology arm, wherein the nucleic acid undergoes homologous recombination in a rodent cell. In some embodiments, the 5' and 3' homology arms are homologous to rat sequences such that the nucleic acid undergoes homologous recombination at the endogenous rat TARDBP locus, and the nucleic acid sequence encoding the mutant TDP-43 polypeptide replaces the endogenous TARDBP coding sequence. In some embodiments, the 5' and 3' homology arms are homologous to mouse sequences such that the nucleic acid undergoes homologous recombination at the endogenous mouse TARDBP locus, and the nucleic acid sequence encoding the mutant TDP-43 polypeptide replaces the endogenous TARDBP coding sequence.

[0022] Also described herein are methods for producing the non-human animals and non-human animal cells described herein. In some embodiments, the methods include engineering the genome of the non-human animal or non-human animal cell to include a mutated TARDBP gene encoding a mutant TDP43 polypeptide, where the mutant TDP-43 polypeptide lacks a functional structural domain compared to wild-type TDP-43, and optionally the wild-type TDP-43 polypeptide comprises a sequence set forth as SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5. In some embodiments, the engineering includes replacing the endogenous TARDBP gene with the mutated TARDBP gene encoding the mutant TDP-43 polypeptide as described herein. In some embodiments, the engineering further includes replacing the endogenous TARDBP gene with a TARDBP gene comprising a knockout mutation, e.g., a conditional knockout mutation. In some embodiments, the method further includes culturing the cell under conditions that eliminate expression of the TARDBP gene comprising the knockout mutation.

[0023] Also described herein are methods of using the non-human animals, non-human animal cells, non-human animal tissues, and compositions. In some embodiments, the non-human animals, non-human animal cells, non-human animal tissues, and compositions are used in methods, such as methods of identifying therapeutic candidates for the treatment of disease and / or methods of evaluating the biological function of a TDP-43 structural domain. In some embodiments of identifying therapeutic candidates, the method includes (a) contacting a non-human animal, non-human animal cell, non-human animal, or a composition (e.g., an in vitro culture) comprising a non-human animal, non-human animal cell or tissue 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 a phenotype and / or TDP-43 biological activity to the non-human animal, non-human cell or tissue that is comparable to the phenotype and / or TDP-43 biological activity of a control cell or tissue expressing a wild-type TDP-43 polypeptide.

[0024] In some embodiments of assessing the biological function of TDP-4, the method comprises (a) engineering embryonic stem (ES) cells to contain a mutated TARDBP gene encoding a mutant TDP43 polypeptide lacking a functional structural domain selected from the group consisting of a nuclear localization signal (NLS), a first RNA recognition motif (RRM1), a first RNA recognition motif (RRM2), a putative nuclear export signal (E), a prion-like domain (PLD), and combinations thereof, (b) optionally differentiating the engineered ES cells in vitro and / or obtaining a genetically engineered non-human animal from the engineered ES cells, and (c) assessing the phenotype and / or TDP-43 biological activity of the genetically engineered ES cells, the primitive ectoderm derived therefrom, the motor neurons derived therefrom, or the non-human animal derived therefrom. In some embodiments, the method of claim 39 or claim 40, wherein the phenotype is assessed by cell culture, fluorescent in situ hybridization, Western blot analysis, or a combination thereof. In some embodiments, assessing the phenotype comprises measuring the viability of the engineered ES cells, the primitive ectoderm derived therefrom, the motor neurons derived therefrom, or the non-human animals derived therefrom. In some embodiments, assessing the phenotype comprises determining the cellular location of the mutant TDP-43 polypeptide. In some embodiments, assessing the biological activity of the mutant TDP-43 polypeptide comprises measuring the splice product of a gene comprising a cryptic exon regulated by TDP-43. In some embodiments, the gene comprising a cryptic exon regulated by TDP-43 comprises Crem, Fyxd2, Clf1. In some embodiments, the biological activity of the mutant TDP-43 polypeptide comprises measuring the level of alternatively spliced ​​TDP-43.

[0025] Also described herein are oligonucleotides (e.g., antisense oligonucleotides, siRNAs, CRISPR / Cas systems, etc.) that may be useful as candidate drugs in treating TDP-43 proteinopathy. In some embodiments, the antisense oligonucleotides comprise a gapmer motif that targets the TDP-43 mRNA sequence between the 5' and 3' alternative splice sites. In some embodiments, the antisense oligonucleotides comprise a gapmer motif that targets the TDP-43 mRNA sequence between the 5' and 3' alternative splice sites, where the 5' alternative splice site correlates to 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 the 3' alternative splice site correlates to a TARDBP genomic location of chromosome 4:148,617,705. In some siRNA embodiments, the siRNA comprises a sequence that targets the TDP-43 mRNA sequence between the 5' and 3' alternative splice sites. In some embodiments, the siRNA comprises a sequence that targets the TDP-43 mRNA sequence between the 5' and 3' alternative splice sites, where the 5' alternative splice site correlates to a TARDBP genomic location selected from the group consisting of: (a) chromosome 4:148,618,647; (b) chromosome 4:148,618,665; (c) chromosome 4:148,618,674, and the 3' alternative splice site correlates to a TARDBP genomic location of chromosome 4:148,617,705. In some CRISPR / Cas system embodiments, the system comprises a Cas9 protein and at least one gRNA, where the gRNA recognizes a sequence at or near the 5' alternative splice site and / or at or near the 3' alternative splice site of the TDP-43 mRNA.In some embodiments, the CRISPR / Cas system comprises a Cas9 protein and at least one gRNA, wherein the gRNA recognizes a sequence at or near a TARDBP genomic location selected from the group consisting of: (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.

[0026] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the U.S. Patent Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0027] [Figure 1] Figure 1 provides a description (not to scale) of TDP-43, the relative location of the nuclear localization signal (NLS; amino acids 82-98), the relative location of two RNA recognition motifs (RRM1; amino acids 106-176, and RRM2; amino acids 191-262), the relative location of the putative nuclear export signal (E; amino acids 239-248), the relative location of the prion-like domain (PLD; amino acids 274-414), the amino acid substitution mutations associated with ALS, and the C-terminal fragment associated with ALS. Asterisks highlight mutations associated with FTD symptoms with or without ALS. The following mutations have also been observed in healthy individuals: A90V, S92L, N267S, G287S, G294V, G368S, S375G, A382T, I383V, N390S, and N390D. [Figure 2-1] FIG. 1 provides a diagram (not to scale) of the mouse TARDBP genomic structure showing exons 1 to 6 (rectangles), untranslated regions (unfilled rectangles), and translated regions (filled rectangles), starting with the ATG start codon. [Figure 2-2]FIG. 1 provides an amino acid sequence alignment of mouse (m)TDP-43 and human (h)TDP-43 polypeptides, the amino acid positions of the polypeptides, and the consensus sequence below the mTDP-43 and hTDP-43 sequences. In general, boxed regions within the alignment represent 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), a putative nuclear export signal (E: amino acids 239-248), and a 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 indicated by dashes in the consensus sequence. Exon junctions are also indicated as vertical lines indicating exons (EX) joined at the indicated junctions. The vertical line between amino acids 286 and 287 provides an alternative 5'-splice site (see Figure 11A). [Figure 3-1] Schematics (not to scale) of two exemplary TARDBP null alleles are provided: (1) a conditional knockout allele containing exon 3 flanked by loxP site-specific recombination recognition sites (triangles), hereafter referred to as "-", after removal of exon 3 upon cre-mediated recombination, and (2) a TARDBP null allele containing a deletion of the entire TARDBP coding sequence, hereafter referred to as "ΔCDS". The relative positions of exons 1-6 (rectangles), untranslated region (unfilled rectangles), translated region (filled rectangles), and start ATG and stop TGA codons are indicated. [Figure 3-2]FIG. 1 provides exemplary depictions (not to scale) of non-limiting mutant TDP-43 polypeptides encoded by various forms of mutated TARDBP genes. Specifically, throughout these examples and associated figures: "WT" refers to a wild-type TARDBP gene, "loxP-Ex3loxP" refers to a mutated TARDBP gene comprising exon 3 into which loxP has been introduced, "-" refers to a mutated TARDBP gene lacking a nucleotide sequence comprising the sequence of exon 3 of the wild-type TARDBP gene upon cre-mediated recombination of loxP-Ex3loxP, "ΔCDS" refers to a mutated TARDBP gene lacking the entire coding sequence of TARDBP, and "ΔNLS" refers to a mutated TARDBP gene lacking the point mutation K82A "ΔRRM1" refers to a mutated TARDBP gene encoding a mutant TDP-43 polypeptide comprising the following point mutations: F147L and F149L; "ΔRRM2" refers to a mutated TARDBP gene encoding a mutant TDP-43 polypeptide comprising the following point mutations: F194L and F229L; "ΔE" refers to a mutated TARDBP gene encoding a mutant TDP-43 polypeptide lacking amino acids 239-250 of the wild-type TDP-43 polypeptide; and "ΔPLD" refers to a mutated TARDBP gene encoding a mutant TDP-43 polypeptide lacking amino acids 274-414 of the wild-type TDP-43 polypeptide. In the case of the ΔE and ΔPLD mutant TDP-43 polypeptides, the diagonal lines represent the deleted region. [Figure 4] 1 shows the protocol used to differentiate embryonic stem (ES) cells into motor neurons. Also shown is the ability of ES cells containing a mutated TARDBP gene to maintain viability, reach the primitive ectoderm (PE) stage, and / or reach the motor neuron (MN) stage after Cre-mediated deletion (-) of exon 3 at the ES cell stage. [Diagram 5]1 shows the protocol used to assess the viability of embryonic stem cell derived motor neurons (ESMN). Also shown are results regarding the viability of ESMN containing a mutated TARDBP gene, as shown after activation of a conditional knockout allele (-). [Figure 6-1] A not-to-scale depiction of the region of TDP-43 recognized by anti-TDP-43 antibodies that recognize the N-terminus of TDP-43 (α-TDP-43N-term) and the C-terminus of TDP-43 (α-TDP-43C-term) is provided. [Figure 6-2] Western blots of cytoplasmic and nuclear fractions of cells stained with antibodies recognizing the N-terminus of TDP-43 (αTDP-43N-term) or the C-terminus of TDP-43 (αTDP-43C-term) as shown in Figure 6A are provided. Cre-mediated deletion of exon 3(-) occurred at the ES cell stage, and cells were cultured in ES medium, ADFNK medium, ADFNK medium containing retinoic acid and sonic hedgehog, and ESMN medium according to the protocol shown in Figure 4 to generate stem cell-derived motor neurons (ESMN). Cytoplasmic and nuclear fractions were isolated from TDP-43 WT / - engineered ESMN, ΔNLS / - engineered ESMN, ΔE / - engineered ESMN, ΔPLD / - engineered ESMN, or dying ΔRRM1 / - engineered cells. Graphs showing the ratio of cytoplasmic to nuclear TDP-43 in control TDP-43WT / -ESMN (●), ΔNLS / -engineered ESMN (▲), ΔRRM1 / -engineered cells (▼), or ΔPLD / -engineered ESMN (■) are also provided. [Figure 7]Provides images of fluorescence in situ hybridization at 40x magnification of engineered embryonic stem cell-derived motor neurons (ESMNs) containing a mutated TARDBP gene as indicated. Exon 3 of the mutated TARDBP gene was removed (-) at the ES cell stage, and the cells were cultured in ES medium, ADFNK medium, ADFNK medium containing retinoic acid and sonic hedgehog, and ESMN medium according to the protocol shown in Figure 4 to generate stem cell-derived motor neurons (ESMNs), after which images were captured. Cells were stained with an antibody recognizing the C-terminus of TDP-43 (αTDP-43C-term; upper panel) or an anti-MAP2 antibody and DAPI (lower panel). [Figure 8] Provides images of fluorescence in situ hybridization at 40x magnification of engineered embryonic stem cell-derived motor neurons (ESMNs) containing a mutated TARDBP gene as indicated. Exon 3 of the mutated TARDBP gene was removed (-) at the ES cell stage, and the cells were cultured in ES medium, ADFNK medium, ADFNK medium containing retinoic acid and sonic hedgehog, and ESMN medium according to the protocol shown in Figure 4 to generate embryonic stem cell-derived motor neurons (ESMNs), after which images were captured. Cells were stained with an antibody recognizing the N-terminus of TDP-43 (αTDP-43N-term; upper panel) or an anti-MAP2 antibody and DAPI (lower panel). [Figure 9-1] Western blots of sarkosyl-soluble and sarkosyl-insoluble fractions of cells stained with anti-TDP-43 antibody are provided. Cre-mediated deletion of exon 3(-) was generated at the ES cell stage, and cells were cultured in ES medium, ADFNK medium, ADFNK medium containing retinoic acid and sonic hedgehog, and ESMN medium according to the protocol shown in Figure 4 to generate embryonic stem cell-derived motor neurons (ESMN). Sarkosyl-soluble and sarkosyl-insoluble fractions were isolated from TDP-43 WT / - engineered ESMN, ΔNLS / - engineered ESMN, ΔE / - engineered ESMN, ΔPLD / - engineered ESMN, or ΔRRM1 / - engineered cells. Graphs providing the ratio of insoluble / soluble TDP-43 expressed by these ESMN are also provided. [Figure 9-2]Graphs showing the expression levels of TDP-43 mRNA (left panel; y-axis) or protein (right panel; y-axis) are provided. Cre-mediated deletion of exon 3(-) was generated at the ES cell stage, and cells were cultured in ES medium, ADFNK medium, ADFNK medium containing retinoic acid and sonic hedgehog, and ESMN medium according to the protocol shown in Figure 4 to generate embryonic stem cell-derived motor neurons (ESMN). The mRNA levels of ΔNLS / - engineered ESMN, ΔE / - engineered ESMN, ΔPLD / - engineered ESMN, or dying ΔRRM1 / - engineered cells are compared to the control (TDP-43WT / - engineered ESMN (WT / -)). [Figure 9-3] Western blots of cell lysates stained with anti-TDP-43 or anti-GAPDH antibodies are provided. Cre-mediated deletion of exon 3(-) was generated at the ES cell stage, and cells were cultured in ES medium, ADFNK medium, ADFNK medium containing retinoic acid and sonic hedgehog, and ESMN medium to generate embryonic stem cell-derived motor neurons (ESMN) according to the protocol shown in Figure 4. Cell lysates were isolated from TDP-43 WT / - engineered ESMN, ΔNLS / - engineered ESMN, ΔE / - engineered ESMN, ΔPLD / - engineered ESMN, or dying ΔRRM1 / - engineered cells after treatment with cycloheximide (CHX+) for up to 16 hours. Graphs providing the % of TDP-43 protein (y-axis) expressed by control TDP-43WT / - engineered ESMN (●), ΔNLS / - engineered ESMN (■), ΔRRM1 / - engineered cells (▲), or ΔPLD / - engineered ESMN (▼) after cycloheximide treatment (x-axis; time) are also provided. [Figure 10]We provide a representation (not to scale) of normal and cryptic exon splicing occurring in Crem, Fyxd2, and Clf1, three genes thought to be regulated by TDP-43, as well as graphs showing the levels of normal (filled bars) and aberrant (patterned and unfilled bars) splicing products. Cre-mediated deletion of exon 3(-) was generated at the ES cell stage, and cells were cultured in ES medium, ADFNK medium, ADFNK medium containing retinoic acid and sonic hedgehog, and ESMN medium according to the protocol shown in Figure 4 to generate embryonic stem cell-derived motor neurons (ESMN). The levels of cryptic exon splicing in Crem, Fyxd2, and Clf1 from ΔNLS / - engineered ESMN, ΔE / - engineered ESMN, ΔPLD / - engineered ESMN, or ΔRRM1 / - engineered cells and control (TDP-43WT / -) are shown. [Figure 11-1] FIG. 1 provides a representation (not to scale) of normal and alternative splicing events occurring in the TDP-43 gene. [Figure 11-2] A graph showing the levels of alternatively spliced ​​TDP-43 mRNA is provided. A Cre-mediated deletion of exon 3(-) was generated at the ES cell stage, and cells were cultured in ADFNK medium, ADFNK medium containing retinoic acid and sonic hedgehog, and ESMN medium to generate embryonic stem cell-derived motor neurons (ESMN) according to the protocol shown in Figure 4. The levels of alternatively spliced ​​TDP-43 mRNA from unmanipulated ES cells (WT / WT), ΔNLS / - engineered ESMN, ΔE / - engineered ESMN, ΔPLD / - engineered ESMN, or dying ΔRRM1 / - engineered cells are shown. [Figure 12]1 provides a graph showing the survival time after fertilization of 8-cell embryos injected with TDP-43- / - ES cells, TDP-43ΔNLS / - engineered ES cells, TDP-43ΔPLD / - engineered ES cells, TDP-43ΔNLS / WT engineered ES cells, TDP-43ΔPLD / WT engineered ES cells, TDP-43WT / - engineered ES cells, TDP-43loxP-Ex3-loxP / WT engineered ES cells, or wild-type TDP-43WT / WT ES cells at E3.5 (embryonic day 3.5), E10.5 (embryonic day 10.5), E15.5 (embryonic day 15.5), and P0 (postnatal day 0). [Figure 13-1] Western blots of motor neurons isolated from spinal cord tissue isolated from 16-week-old mice (n=2) are provided. Mice examined expressed (i) a mutated TARDBP gene with loxP-transduced exon 3 (loxP-Ex3-loxP), a mutated TARDBP gene with a knockout mutation in the NLS (ΔNLS), or a mutated TARDBP gene with a deletion of the prion-like domain (ΔPLD) at the endogenous TARDBP locus, and (ii) a wild-type (WT) TARDBP gene at the other TARDBP locus on the homologous chromosome. Cytoplasmic and nuclear fractions of motor neurons stained with α-TDP-43N-term or α-TDP-43C-term antibodies that recognize the N-terminus of TDP-43 or the C-terminus of TDP-43, respectively, are shown (see, e.g., FIG. 6A). Graphs showing the ratio of TDP-43 to nuclei in the cytoplasm of spinal cord tissue isolated from loxP-Ex3-loxP / WT mice (●), ΔNLS / WT mice (▲), or ΔPLD / WT mice (▼) are also provided. [Figure 13-2]Western blots of motor neurons isolated from spinal cord tissue isolated from 16-week-old mice (n=2) are provided. Mice examined expressed (i) a mutated TARDBP gene with: loxP-transduced exon 3 (loxP-Ex3-loxP), a mutated TARDBP gene with a knockout mutation in the NLS (ΔNLS), or a mutated TARDBP gene with a deletion of the prion-like domain (ΔPLD) at the endogenous TARDBP locus, and (ii) a wild-type (WT) TARDBP gene at the other TARDBP locus on the homologous chromosome. 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 are provided. [Figure 13-3] Western blots of motor neurons isolated from spinal cord tissues isolated from 16-week-old mice (n=2) are provided. Mice examined expressed (i) a mutated TARDBP gene with loxP-transduced exon 3 (loxP-Ex3-loxP), a mutated TARDBP gene with a knockout mutation in the NLS (ΔNLS), or a mutated TARDBP gene with a deletion of the prion-like domain (ΔPLD) at the endogenous TARDBP locus, and (ii) a wild-type (WT) TARDBP gene at the other TARDBP locus on the homologous chromosome. Western blots of sarkosyl-soluble and sarkosyl-insoluble fractions of cells stained with α-TDP-43N-term antibody (see, e.g., FIG. 6A) or α-TDP-43C-term antibody (see, e.g., FIG. 6A), which recognize the N-terminus of TDP-43 or the C-terminus of TDP-43, respectively, are provided. [Figure 14]Fluorescence in situ hybridization images at 40x magnification of motor neurons isolated from spinal cord tissue isolated from 16-week-old mice are presented. Mice examined expressed (i) a mutated TARDBP gene containing: loxP-transduced exon 3 (loxP-Ex3-loxP), a mutated TARDBP gene containing a knockout mutation in the NLS (ΔNLS), or a mutated TARDBP gene containing a deletion of the prion-like domain (ΔPLD) at the endogenous TARDBP locus, and (ii) a wild-type (WT) TARDBP gene at the other TARDBP locus on the homologous chromosome. Cells were stained with an antibody recognizing the N-terminus of TDP-43 (αTDP-43N-term; upper panel) or with anti-chAT and anti-NeuN antibodies (lower panel). Also shown are graphs showing the percentage of motor neurons exhibiting cytoplasmic aggregates in animals expressing wild-type TDP-43 only (●), mutant ΔNLS TDP-43 polypeptide and wild-type TDP-43 polypeptide (■), both mutant ΔNLS TDP-43 polypeptide and wild-type TDP-43 polypeptide (■), or both mutant ΔPLD TDP-43 polypeptide and wild-type TDP-43 polypeptide (▲). [Figure 15-1] Fluorescence in situ hybridization images at 10x or 40x magnification of tibialis anterior or intercostal muscle tissue isolated from 16-week-old mice were provided. Tissues were stained with antibodies recognizing synaptophysin, bungarotoxin, and / or DAPI. Arrows indicate denervated neuromuscular junctions, and asterisks indicate partially innervated neuromuscular junctions. [Figure 15-2] Graph providing the percentage of innervated neuromuscular junctions (NMJs; y-axis) in tibial anterior (TA) musculature or intercostal muscles isolated from loxP-Ex3-loxP / WT mice (●), ΔNLS / WT mice (▲), or ΔPLD / WT mice (▼). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] overview

[0029] TDP-43 is a nuclear RNA / DNA binding protein that functions primarily in RNA processing and metabolism, including RNA transcription, splicing, transport, and stability. The RNA-binding properties of TDP-43 appear to be essential for its autoregulatory activity, mediated through binding to 3'UTR sequences in its own mRNA. Ayala et al. (2011), EMBO J. 30:277-88. Following cellular stress, TDP-43 localizes to cytoplasmic stress granules and may play a role in stress granule formation. TDP-43 mislocalizes from its normal location in the nucleus to the cytoplasm, where it aggregates. Aggregated TDP-43 is ubiquitinated, hyperphosphorylated, and truncated. Furthermore, TDP-43 aggregation in the cytoplasm is a component of nearly all cases of ALS. Becker et al. (2017), Nature, 544: 367-371. 97% of ALS cases show postmortem pathology of cytoplasmic TDP-43 aggregates. The same pathology is seen in about 45% of sporadic frontotemporal lobar degeneration (FTLDU). TDP-43 was first identified as the major pathological protein in ubiquitin-positive, tau-negative inclusions in FTLDU, FTLD with motor neuron disease (FTDMND), and ALS / MND (ALS10), disorders that are now considered to represent various clinical manifestations of TDP-43 proteinopathies. Gitcho et al. (2009), Acta Neuropath. 118: 633-645. TARDBPB mutations occur in about 3% of familial ALS patients and in about 1.5% of patients with sporadic disease. Lattante et al.(2013),Hum.Mutat.34:812-26.Various mutations in the TARDBP gene are associated with ALS in less than 1% of cases. See Figure 1. As shown in Figure 1, most mutations in the TARDBP gene associated with ALS are found in the prion-like domain (PLD). Therefore, understanding all the functions played by TDP-43 may elucidate its role in neuropathologies such as ALS, FLTDU, FLTD, etc.

[0030] It is clear that TDP-43 is essential for cellular and organismal life. Depletion of TDP-43 results in embryonic lethality. Early models therefore relied on overexpression of TDP-43 or its mutant forms, or deletion of TDP-43. Various models have been created to evaluate the role of TDP-43 in ALS pathology, as reviewed in Tsao et al. (2012), Brain Res. 1462:26-39.

[0031] For example, transgenic mice overexpressing the A315T mutant form of TDP-43 developed progressive abnormalities at approximately 3-4 months of age and died at 5 months of age. Wegorzewska et al. (2009), Proc. Natl. Acad. Sci. USA. 106:18809-814. The abnormalities correlated with the presence of TDP-43 C-terminal fragments in the brain and spinal cord of these mutant mice, but no cytoplasmic TDP-43 aggregates were detected. These observations led Wegorzewska et al. to suggest that neuronal vulnerability to TDP-43-associated neurodegeneration is related to alterations in DNA / RNA-binding protein function, rather than toxic aggregation. Wegorzewska et al. (2009), supra. In contrast, in two independent studies involving overexpression of TDP-43, transgenic mice exhibited neurodegenerative hallmarks, including progressive motor dysfunction that correlated 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).

[0032] In loss-of-function studies, ubiquitous deletion of TDP-43 using conditional knockout mutations caused mice to exhibit metabolic phenotypes and premature death. Chiang et al. (2010), Proc. Natl. Acad. Sci. USA. 107:16320-324. Depletion of TDP-43 in mouse embryonic stem cells resulted in splicing of cryptic exons of certain genes into mRNA, disruption of mRNA translation, and promotion of nonsense-mediated mRNA decay. Ling et al. (2015), Science, 349:650-655. As postmortem brain tissue from ALS / FTD patients shows impaired suppression of cryptic exon splicing, this study suggests that TDP-43 normally acts to suppress splicing of cryptic exons and maintain intron integrity, and that TDP-43 splicing defects may contribute to TDP-43 proteinopathy in certain neurodegenerative diseases. Ling et al. (2015), supra. Because point mutations in the N-terminus of TDP-43 (e.g., NLS) result in destabilization of TDP-43 oligomerization in the nucleus and loss of cryptic splicing regulation, it is hypothesized that head-to-tail oligomerization of TDP-43 driven by the N-terminus acts to separate aggregation-prone C-terminal domains (e.g., PLD), thus preventing the formation of pathological aggregates. Afroz et al. (2017), Nature Communications, 8:45.

[0033] In ALS, one of the first pathological features is the retraction of axons from the neuromuscular junction, resulting in denervation of muscles. This denervation continues to progress, resulting in loss of motor neuron cell bodies and muscle atrophy. Denervation may be observed by loss of presynaptic markers of axonal innervation: VAChT, synaptic vesicle protein 2 (SV2), synaptophysin, and neurofilament. Motor endplates remain but eventually fragment and disappear. Recently, dose-dependent denervation was demonstrated in mice homozygous for a knock-in TARDBP gene containing a disease-associated mutation. Ebstein,(2019),Cell Reports,26:364-373.

[0034] Despite the embryonic lethality of TDP-43 depletion, we show here that embryonic stem (ES) cells expressing TDP-43 mutant forms lacking functional structural domains can remain viable and may be differentiated into motor neurons (ESMNs). See Figures 4-5. These observations are unique in that ES or ESMNs as described herein express the following mutant TDP-43 polypeptides: (1) lacks a functional structural domain, e.g., lacks a functional NLS, lacks a functional RRM1, lacks a functional RRM2, lacks a functional E, or lacks a functional PLD; and (2) Expressed at normal levels from endogenous transcriptional promoters and pre-mRNA splicing signals. See, e.g., Figures 2 and 9. Using ES and ESMN as described herein, it has been shown that RRM1 is required for survival of ES cells and motor neurons derived therefrom. See Figures 4-5. Furthermore, mutant TDP-43 polypeptides (1) lacking a functional NLS or functional PLD, and (2) expressing at normal levels from the endogenous locus, have been shown to mediate two hallmarks of ALS disease in ESMN: (i) redistribution of TDP-43 from the nucleus to the cytoplasm, and (ii) Reproduce accumulation in cytoplasmic inclusion bodies, see Figures 6-8.

[0035] It is surprising that the ΔPLD mutant, i.e., TDP-43 polypeptide containing a functional NLS but lacking the PLD, aggregates in the cytoplasm. See, e.g., Afroz et al. (2017), supra. Notably, the punctate inclusions formed by the ΔPLD mutant appear to be less abundant and qualitatively different than those formed by the ΔNLS mutant, i.e., TDP-43 polypeptide lacking a functional NLS and containing the PLD. Furthermore, the ALS-like phenotype of ESMN expressing ΔPLD or ΔNLS correlates with a reduced repression of cryptic exon splicing of the gene, a splice event normally regulated by wild-type TDP-43. Figure 9. Also shown is a correlation between expression of the ΔPLD or ΔNLS mutated TARDBP gene in ESMN and a reduction in alternative splicing events involving the 3' untranslated region intron resulting in alternatively spliced ​​TDP-43 mRNA lacking the PLD domain, or a portion thereof, and sequences encoding a stop codon. See also Figure 10; Avendano-Vazquez et al. (2012), Genes & Dev. 26:1679-84; Ayala, YM. et al. (2011), EMBO J. 30:277-288. This latter observation suggests that depleting only wild-type or ALS-associated sequences resulting from normal splice events may be therapeutically useful in treating ALS associated with PLD mutations.

[0036] Mice expressing wild-type and ΔPLD or ΔNLS mutated TARDBP genes from the endogenous locus also displayed features of TDP-43 proteinopathy. Mislocalization of TDP-43 from the nucleus to the cytoplasm, phosphorylation of cytoplasmic TDP-43, and increased cytoplasmic aggregation of TDP-43 were observed in spinal motor neurons of animals expressing mutant ΔPLD or ΔNLS TDP-43 polypeptides compared to animals expressing only the wild-type protein (Figures S13A-S13B and S14). TDP-43 mutants lacking a functional NLS were insoluble, whereas TDP-43 mutants lacking the PLD were not (Figure S13C). Furthermore, in these mice expressing mutant ΔPLD or ΔNLS TDP-43 proteins, denervation of muscles composed primarily of fast-twitch fibers was observed, but not of muscles composed primarily of slow-twitch fibers (Figures S15A-S15B).

[0037] The discoveries provided herein provide methods for assessing TDP-43 mutations in viable embryonic stem (ES) cells and tissues and non-human animals derived therefrom (e.g., primitive ectoderm, motor neurons (ESMN) derived therefrom), as well as ES cells, ESMN cells, and non-human animals expressing mutant TDP-43 polypeptides lacking functional structural domains. ES cells, ESMN cells, and non-human animals (e.g., rodents, e.g., rats and mice) expressing mutant TDP-43 polypeptides lacking functional structural domains may each be used as in vitro or in vivo models of TDP-43 proteinopathy, e.g., in methods for identifying therapeutic candidates therefor.

[0038] TARDBP gene and TDP-43 polypeptide

[0039] The TARDBP gene encodes the TDP-43 polypeptide, also referred to as TAR DNA binding protein, TARDBP, 43-KD, and TDP43, and TDP-43. Nucleic acid sequences of wild-type TARDBP genes of various species and wild-type TDP-43 polypeptides encoded therefrom are well known in the art. For example, the nucleic acid and amino acid sequences of the wild-type TARDBP gene and wild-type TDP-43 polypeptide, respectively, may be found in the National Library of Medicine (NIH) National Center for Biotechnology Information (NCBI) gene database. See, for example, the website at www.ncbi.nlm.nih.gove / gene / ?term=TARDBP. In some embodiments, the wild-type mouse TARDBP gene comprises a nucleotide sequence encoding a wild-type mouse TDP-43 polypeptide comprising the amino acid sequence shown as GenBank Accession No. NP_663531 (SEQ ID NO: 1), or a variant thereof that differs therefrom due to conservative amino acid substitutions. In some embodiments, the wild-type mouse TARDBP gene comprises the nucleic acid sequence set forth as GenBank Accession No. NM_145556.4 (SEQ ID NO:2), or a variant thereof that differs therefrom due to degeneracy of the genetic code and / or conservative codon substitutions. In some embodiments, the wild-type rat TARDBP gene comprises a nucleotide sequence encoding a wild-type rat TDP-43 polypeptide comprising the amino acid sequence set forth as GenBank Accession No. NP_001011979 (SEQ ID NO:3), or a variant thereof that differs therefrom due to conservative amino acid substitutions. In some embodiments, the wild-type rat TARDBP gene comprises the nucleic acid sequence set forth as GenBank Accession No. NM_001011979.2 (SEQ ID NO:4), or a variant thereof that differs therefrom due to degeneracy of the genetic code and / or conservative codon substitutions. In some embodiments, the wild-type human TARDBP gene encodes a TDP-43 polypeptide comprising the amino acid sequence set forth as GenBank Accession No. NP_031401.1 (SEQ ID NO:5), or a variant thereof that differs therefrom due to conservative amino acid substitutions.In some embodiments, the wild-type human TARDBP gene comprises the nucleic acid sequence set forth as GenBank Accession No. NM_007375.3 (SEQ ID NO: 6), or a variant thereof that differs therefrom due to degeneracy of the genetic code and / or conservative codon substitutions.

[0040] Described herein is a mutated TARDBP gene. The mutated TARDBP gene may comprise a knockout mutation. The mutated TARDBP gene may encode a mutated TDP-43 polypeptide, where the mutated TDP-43 polypeptide lacks a functional structural domain. For example, the mutated TARDBP gene may comprise a nucleotide sequence encoding a TDP-43 structural domain comprising a point mutation, an insertion within part or all of the structural domain, and / or a deletion of part or all of the structural domain, where the point mutation, insertion, and / or deletion results in a loss of function of the structural domain, and despite the mutated TDP-43 polypeptide lacking a functional structural domain due to the mutation, the mutated TARDBP gene still encodes a TDP-43 polypeptide. The polypeptide may be referred to as a mutated TDP-43 polypeptide, where it comprises at least one wild-type TDP-43 structural domain or a mutant thereof, and / or it is specifically bound by an anti-TDP-43 antibody or an antigen-binding portion thereof. Similarly, a mutated TARDBP gene may be classified such that the mutated TARDBP gene encodes a mutant TDP-43 polypeptide, e.g., a polypeptide that includes at least one wild-type TDP-43 structural domain or a mutant thereof, and / or that may be specifically bound by an anti-TDP-43 antibody or an antigen-binding portion thereof.

[0041] The TDP-43 structural domains have been identified as a nuclear localization signal (NLS), two RNA recognition motifs (RRM1 and RRM2), a putative nuclear export signal (E), and a glycine-rich prion-like domain (PLD). See Figures 1 and 2. The wild-type TDP-43 polypeptide contains the TDP-43 NLS at amino acids 82-99, the TDP-43 RRM1 at amino acids 106-176, the TDP-43 RRM2 at amino acids 191-262, the TDP-43 E at amino acids 239-248, and the TDP-43 PLD at amino acids 274-414.

[0042] A conventional NLS sequence contains a stretch of basic amino acids, primarily lysine (K) and arginine (R) residues, and a bipartite NLS contains two clusters of these basic amino acids separated by a linker region containing about 10-13 amino acids. Amino acid substitutions and / or deletions in the basic amino acid sequence of a conventional NLS may abolish the function of the conventional NLS. McLane and Corbett, (2009), IUBMB Life, 61:697-706. The NLS of TDP-43 contains lysine and arginine residues at positions 82, 83, 84, 95, 97, and 98. A wild-type TDP-43 polypeptide modified to contain amino acid substitutions and / or deletions at positions 82, 83, 84, 95, 97, and / or 98 may lack a functional NLS. A mutant TDP-43 polypeptide lacking a functional NLS may comprise the amino acid sequence set forth in SEQ ID NO: 1 modified to include amino acid substitutions and / or deletions at positions 82, 83, 84, 95, 97, and / or 98. A mutant TDP-43 polypeptide lacking a functional NLS may comprise the amino acid sequence set forth in SEQ ID NO: 3 modified to include amino acid substitutions and / or deletions at positions 82, 83, 84, 95, 97, and / or 98. A mutant TDP-43 polypeptide lacking a functional NLS may comprise the amino acid sequence set forth in SEQ ID NO: 5 modified to include amino acid substitutions and / or deletions at positions 82, 83, 84, 95, 97, and / or 98. Thus, a mutated TARDBP gene encoding a mutant TDP-43 protein lacking a functional TDP-43 NLS may comprise a sequence encoding a TDP-43 polypeptide comprising the sequence set forth as SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5 modified to include (i) an amino acid substitution at a position selected from the group consisting of 82, 83, 84, 95, 97, and / or 98 and combinations thereof, and / or (ii) a deletion of amino acids at and between positions 82 and 98.A mutated TARDBP gene encoding a mutant TDP-43 protein lacking a functional TDP-43 NLS may comprise a nucleotide sequence encoding the amino acid sequence shown as SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5 modified to include amino acid substitutions selected from the group consisting of K82A K83A, R84A, K95A, K97A, K98A or combinations thereof. A mutated TARDBP gene encoding a mutant TDP-43 protein lacking a functional TDP-43 NLS may comprise a nucleotide sequence encoding the amino acid sequence shown as SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5 modified to include the following amino acid substitutions: K82A K83A, R84A, K95A, K97A, and K98A.

[0043] RNA binding by typical RRMs is usually achieved by contacts made between the surface of the four-stranded antiparallel β-sheet 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-strands, RNP1 (consensus K / RGF / YG / AF / YV / I / LXF / Y, where X is any amino acid) and RNP2 (consensus I / V / LF / YI / V / LXNL, where X is any amino acid), are the major mediators of RNA binding. Melamed et al. (2013), supra.

[0044] RRM1 of TDP-43, located at amino acid positions 106-176 of the wild-type TDP-43 polypeptide, contains an RNP2 consensus sequence (LIVLGL; SEQ ID NO: 7) located at amino acid positions 106-111 and an RNP1 consensus sequence (KGFGFVRF; SEQ ID NO: 8) located at amino acid positions 145-152. Previously, W113, T115, F147, F149, D169, R171, and N179 were identified as important residues for nucleic acid binding. A wild-type TDP-43 polypeptide modified to include: (i) an amino acid substitution at a position selected from the group consisting of 113, 115, 147, 149, 169, 171, 179, and any combination thereof; (ii) a deletion or substitution of any amino acid at or between positions 106-176; (iii) a deletion or substitution of any amino acid at or between positions 106-111; (iv) a deletion or substitution of any amino acid at or between positions 145-152; or (v) any combination of (i)-(iv) may lack a functional RRM1. A mutant TDP-43 polypeptide lacking functional RRM1 may comprise the sequence set forth as SEQ ID NO:1 modified to include (i) an amino acid substitution at a position selected from the group consisting of 113, 115, 147, 149, 169, 171, 179, and any combination thereof; (ii) a deletion or substitution of any amino acid at and between positions 106-176; (iii) a deletion or substitution of any amino acid at and between positions 106-111; (iv) a deletion or substitution of any amino acid at and between positions 145-152; or (v) any combination of (i)-(iv). A mutant TDP-43 polypeptide lacking functional RRM1 may comprise the sequence set forth as SEQ ID NO: 3 modified to include (i) an amino acid substitution at a position selected from the group consisting of 113, 115, 147, 149, 169, 171, 179, and any combination thereof; (ii) a deletion or substitution of any amino acid at and between positions 106-176; (iii) a deletion or substitution of any amino acid at and between positions 106-111; (iv) a deletion or substitution of any amino acid at and between positions 145-152; or (v) any combination of (i)-(iv).A mutant TDP-43 polypeptide lacking functional RRM1 may comprise the sequence set forth as SEQ ID NO:5 modified to include (i) an amino acid substitution at a position selected from the group consisting of 113, 115, 147, 149, 169, 171, 179, and any combination thereof; (ii) a deletion or substitution of any amino acid at and between positions 106-176; (iii) a deletion or substitution of any amino acid at and between positions 106-111; (iv) a deletion or substitution of any amino acid at and between positions 145-152; or (v) any combination of (i)-(iv). Thus, a mutated TARDBP gene encoding a mutant TDP-43 polypeptide lacking functional RRM1 may comprise a nucleotide sequence encoding a TDP-43 polypeptide comprising the amino acid sequence set forth as SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5 modified to include (i) an amino acid substitution at a position selected from the group consisting of 113, 115, 147, 149, 169, 171, 179, and any combination thereof; (ii) a deletion or substitution of any amino acid at and between positions 106-176; (iii) a deletion or substitution of any amino acid at and between positions 106-111; (iv) a deletion or substitution of any amino acid at and between positions 145-152; or (v) any combination of (i)-(iv). A mutated TARDBP gene encoding a mutant TDP-43 polypeptide lacking a functional RRM1 may comprise a nucleotide sequence encoding a TDP-43 polypeptide comprising the amino acid sequence shown as SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5 modified to include the following amino acid substitutions F147L and / or F149L. A mutated TARDBP gene encoding a mutant TDP-43 polypeptide lacking a functional RRM1 may comprise a nucleotide sequence encoding a TDP-43 polypeptide comprising the amino acid sequence shown as SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5 modified to include the following amino acid substitutions F147L and / or F149L.

[0045] The RRM2 of TDP-43, located at amino acid positions 191-262 of the wild-type TDP-43 polypeptide, comprises an RNP2 consensus sequence (VFVGRC; SEQ ID NO: 9) located at amino acid positions 193-198 and an RNP1 consensus sequence (RAFAFVT; SEQ ID NO: 10) located at amino acid positions 227-233. F194 and F229 may be considered important residues for nucleic acid binding. A wild-type TDP-43 polypeptide modified to include (i) an amino acid substitution at a position selected from the group consisting of 194 and / or 229, (ii) a deletion or substitution of any amino acid at and between positions 193-198, (iii) a deletion or substitution of any amino acid at and between positions 227-233, (iv) a deletion or substitution of any amino acid at and between positions 191-262, or (v) any combination of (i)-(iv) may lack a functional RRM2. A mutant TDP-43 polypeptide lacking functional RRM2 may comprise the sequence set forth as SEQ ID NO:1 modified to include (i) an amino acid substitution at a position selected from the group consisting of 194 and / or 229, (ii) a deletion or substitution of any amino acid at and between positions 193-198, (iii) a deletion or substitution of any amino acid at and between positions 227-233, (iv) a deletion or substitution of any amino acid at and between positions 191-262, or (v) any combination of (i)-(iv). A mutant TDP-43 polypeptide lacking functional RRM2 may comprise the sequence set forth as SEQ ID NO: 3 modified to include (i) an amino acid substitution at a position selected from the group consisting of 194 and / or 229, (ii) a deletion or substitution of any amino acid at and between positions 193-198, (iii) a deletion or substitution of any amino acid at and between positions 227-233, (iv) a deletion or substitution of any amino acid at and between positions 191-262, or (v) any combination of (i)-(iv).A mutant TDP-43 polypeptide lacking functional RRM2 may comprise the sequence set forth as SEQ ID NO:5 modified to include (i) an amino acid substitution at a position selected from the group consisting of 194 and / or 229, (ii) a deletion or substitution of any amino acid at and between positions 193-198, (iii) a deletion or substitution of any amino acid at and between positions 227-233, (iv) a deletion or substitution of any amino acid at and between positions 191-262, or (v) any combination of (i)-(iv). Thus, a mutated TARDBP gene encoding a mutant TDP-43 polypeptide lacking functional RRM2 may comprise a nucleotide sequence encoding a TDP-43 polypeptide comprising the amino acid sequence set forth as SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5 modified to include (i) an amino acid substitution at positions 194 and / or 229 of the wild-type TDP-43 polypeptide, (ii) a deletion or substitution of any amino acid at or between positions 191-262, or (iii) both (i) and (ii). A mutated TARDBP gene encoding a mutant TDP-43 polypeptide lacking functional RRM2 may comprise a nucleotide sequence encoding a TDP-43 polypeptide comprising the amino acid sequence set forth as SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5 modified to include the mutations F194L and / or F229L. A mutated TARDBP gene encoding a mutant TDP-43 polypeptide lacking a functional RRM2 may comprise a nucleotide sequence encoding a TDP-43 polypeptide comprising the amino acid sequence shown as SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5 modified to include F194L and F229L mutations.

[0046] The nuclear export signal of the wild-type TDP-43 polypeptide may be located at amino acid positions 239-248. The mutant TDP-43 polypeptide lacking a functional nuclear export signal may comprise the amino acid sequence shown as SEQ ID NO: 1 modified to include a deletion of any amino acid at and between positions 236-251. The mutant TDP-43 polypeptide lacking a nuclear export signal may comprise the amino acid sequence shown as SEQ ID NO: 1 modified to include a deletion of at least amino acids 239-250. The mutant TDP-43 polypeptide lacking a nuclear export signal may comprise the amino acid sequence shown as SEQ ID NO: 3 modified to include a deletion of any amino acid at and between positions 236-251. The mutant TDP-43 polypeptide lacking a nuclear export signal may comprise the amino acid sequence shown as SEQ ID NO: 3 modified to include a deletion of at least amino acids 239-250. A mutant TDP-43 polypeptide lacking a nuclear export signal may comprise the amino acid sequence shown as SEQ ID NO: 5 modified to include a deletion of any amino acids at and between positions 236-251. A mutant TDP-43 polypeptide lacking a nuclear export signal may comprise the amino acid sequence shown as SEQ ID NO: 5 modified to include a deletion of at least amino acids 239-250. Thus, a mutated TARDBP gene encoding a mutant TDP-43 polypeptide lacking a functional nuclear export signal may comprise a nucleotide sequence encoding a TDP-43 polypeptide comprising the amino acid sequence shown as SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5 modified to include a deletion of amino acids at and between positions 236-251, e.g., a deletion of amino acids at and between 239-250.

[0047] 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 set forth as SEQ ID NO: 1 modified to include a deletion of at least one or all of the amino acids at and between positions 274-414. A mutant TDP-43 polypeptide lacking a functional PLD may comprise the amino acid sequence set forth as SEQ ID NO: 3 modified to include a deletion of at least one or all of the amino acids at and between positions 274-414. A mutant TDP-43 polypeptide lacking a functional PLD may comprise the amino acid sequence set forth as SEQ ID NO: 5 modified to include a deletion of at least one or all of the amino acids at and between positions 274-414. Thus, a mutated TARDBP gene encoding a mutant TDP-43 polypeptide may comprise a nucleotide sequence encoding a TDP-43 polypeptide comprising the amino acid sequence shown as SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5 modified to include a deletion of at least one or all amino acids at and between positions 274-414.

[0048] The mutated TARDBP gene may comprise the structure shown in Figure 3 A. The mutated TARDBP gene may encode the mutant TDP-43 polypeptide shown in Figure 3A.

[0049] Methods for producing cells and non-human animals containing and expressing a mutant TARDBP gene

[0050] As outlined above, methods and compositions are provided herein that allow targeted genetic manipulation of the TARDBP locus, for example to generate cells containing a mutated TARDBP gene and / or to evaluate the biological function of the TDP-43 structural domain. In addition, it is recognized that additional targeted genetic manipulations can be performed. Such systems that allow these targeted genetic manipulations can employ a variety of components, and for ease of reference, the term "targeted genome integration system" is used herein generally to include all components required for an integration event (i.e., various nuclease agents, recognition sites, inserted DNA polynucleotides, targeting vectors, targeted genome loci, etc.).

[0051] Methods for generating a non-human animal cell expressing a mutant TDP-43 polypeptide and / or methods for assessing the biological function of a TDP-43 structural domain may include engineering the genome of the cell to contain a mutated TARDBP gene, which may encode a mutant TDP-43 polypeptide, wherein the mutant TDP-43 polypeptide lacks a functional structural domain.

[0052] Methods for generating a non-human animal cell expressing a mutant TDP-43 polypeptide and / or methods for evaluating the biological function of a TDP-43 structural domain may include engineering the genome of the cell to contain a mutated TARDBP gene, wherein the mutated TARDBP gene comprises a knockout mutation.

[0053] The methods provided herein include introducing one or more polynucleotide or polypeptide constructs that include various components of the target genome integration system into a cell. "Introducing" means presenting a sequence (polypeptide or polynucleotide) to a cell so that the sequence can be accessed inside the cell. The methods provided herein do not depend on a particular method for introducing any component of the target genome integration system into a cell, only that the polynucleotide can be accessed inside 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, transient transfection, and viral-mediated methods.

[0054] In some embodiments, the cells employed in the methods and compositions have the DNA construct stably integrated into their genome. "Stably integrated" or "stably introduced" refers to introducing a polynucleotide into a cell so that the nucleotide sequence can be integrated into the genome of the cell and inherited by its progeny. Any protocol may be used for stable integration of the DNA construct or various components of the targeted genome integration system.

[0055] Transfection protocols and protocols for introducing polypeptides or polynucleotide sequences into cells may vary. Non-limiting transfection methods include 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: W. H. Freeman and Company. pp. 96-97); dendrimers; or chemical-based transfection methods including the use of cationic polymers such as DEAE-dextran or polyethyleneimine. Non-chemical methods include electroporation, sonoporation, and optical transfection. Particle-based transfection includes the use of a gene gun, magnet-assisted transfection (Bertram, J. (2006), Current Pharmaceutical Biotechnology, 7, 277-28). Viral methods can also be used for transfection.

[0056] The cells comprising the mutated TARDBP gene can be generated using various methods disclosed herein. The engineering may include replacing the endogenous TARDBP gene with the mutated TARDBP gene encoding a mutant TDP-43 polypeptide and / or replacing the endogenous TARDBP gene with a TARDBP gene comprising a knock-out mutation, such as a conditional knock-out mutation. The engineering may include culturing the cells under conditions that preclude expression of the TARDBP gene comprising a knock-out mutation. Conditions that may preclude expression of the TARDBP gene may include expression of a recombinase protein, e.g., cre-recombinase.

[0057] Such engineering methods may include: (1) integrating a mutated TARDBP gene into a target TARDBP genomic locus of interest in a pluripotent cell of a non-human animal using the methods disclosed herein to generate an engineered pluripotent cell comprising the mutated TARDBP gene at the target TARDBP genomic locus; (2) selecting the engineered pluripotent cell having the mutated TARDBP gene at the target TARDBP genomic locus. (3) Further animals may be generated by introducing the engineered pluripotent cell into a host embryo of a non-human animal, for example at the pre-morula stage; and (4) implanting the host embryo containing the engineered pluripotent cell into a surrogate mother to generate an F0 generation derived from the engineered pluripotent cell. The non-human animal may be a non-human mammal, a rodent, a mouse, a rat, a hamster, a monkey, an agricultural mammal or livestock, or a fish or a bird.

[0058] The 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 mammalian ES cells, or domesticated mammalian ES cells. In other embodiments, the 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, hamster cells. In one embodiment, the targeted genetic engineering results in a mutated TARDBP gene.

[0059] Mouse pluripotent cells, totipotent cells, or host embryos can be derived from any mouse strain, including, for example, inbred, crossbred, and outbred strains. Exemplary mouse strains include 129 strains, C57BL strains (e.g., C57BL / 6 strains), crosses of 129 and C57BL / 6 (e.g., 50% 129 and 50% C57BL / 6), BALB / c strains, and Swiss Webster strains. Examples of 129 strains include 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 12951 / SV, 12951 / SvIm), 129S2, 129S4, 129S5, 12959 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, and 129T2 (see, e.g., Festing 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 cross between a 129 strain (e.g., a 129S6 (129 / SvEvTac) strain) and a C57BL / 6 strain, a cross between one or more of the 129 strains, or a cross between one or more of the C57BL strains. Mice can also be derived from strains other than a 129 strain.

[0060] The rat pluripotent, totipotent cells, or host embryos can be derived from any rat strain, including, for example, inbred, crossbred, 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 Fischer rat strains, such as FisherF344 and FisherF6. The rat pluripotent, totipotent cells, or host embryos can also be obtained from strains resulting from crosses of two or more of the above strains. For example, the rat pluripotent, totipotent cells, or host embryos can be derived from a strain selected from the DA strain and the ACI strain. The ACI rat strain has white belly and feet, and RT1 av1 The Dark Agouti (DA) rat strain is characterized by the presence of the black agouti haplotype. Such strains are available from a variety of sources, including Harkan Laboratories. An example of a rat ES cell line derived from ACI rats is the ACI.G1 rat ES cell line. The Dark Agouti (DA) rat strain is characterized by the presence of the agouti coat and the RT1 haplotype. av1 The rats are characterized by having a haplotype. Such rats are available from various sources, including Charles River and Harlan Laboratories. An example of a rat ES cell line derived from a DA rat is the DA.2B rat ES cell line or the DA.2C rat ES cell line. Other examples of rat strains are provided, for example, in US2014 / 0235933, US2014 / 0310828, and US2014 / 0309487, each of which is incorporated herein by reference in its entirety for all purposes.

[0061] For example, germline-transmittable rat ES cells can be obtained by culturing isolated rat ES cells on a feeder cell layer in a medium comprising N2 supplement, B27 supplement, about 50 U / mL to about 150 U / mL of leukemia inhibitory factor (LIF), and a combination of inhibitors consisting of a MEK inhibitor and a GSK3 inhibitor, where the feeder cell layer has not been engineered to express LIF, and the rat ES cells: (i) have been engineered to contain a targeted genetic modification comprising at least one insertion into the genome of the rat ES cell of a heterologous polynucleotide comprising a selectable marker and are capable of transmitting the targeted genetic modification through the germline; (ii) have a normal karyotype; (iii) lack expression of c-Myc; and (iv) form spherical, floating colonies in culture (see, e.g., US 2014-0235933 A1 and US 2014-0310828 A1, each of which is incorporated by reference in its entirety). Other examples of derivation and targeted manipulation of rat embryonic stem cells are provided, for example, 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).

[0062] Nuclear transfer techniques can also be used to generate non-human animals. Briefly, nuclear transfer methods include the steps of (1) enucleating an oocyte; (2) isolating a donor cell or nucleus that combines with the enucleated oocyte; (3) inserting the cell or nucleus into the enucleated oocyte to form a reconstituted cell; (4) implanting the reconstituted cell into the uterus of an animal to form an embryo; and (5) allowing the embryo to develop. In such methods, oocytes are typically harvested from dead animals, but may also be isolated from the oviducts and / or ovaries of living animals. Oocytes can be matured in a variety of media known to those of skill in the art prior to enucleation. Enucleation of oocytes can be performed in a number of ways well known to those of skill in the art. Insertion of a donor cell or nucleus into an enucleated oocyte to form a reconstituted cell is usually by microinjection of the donor cell under the zona pellucida prior to fusion. Fusion may be induced by application of DC electric pulses across the contact / fusion surface (electrofusion), by exposure of the cells to fusion-promoting chemicals such as polyethylene glycol, or by inactivated viruses such as Sendai virus. The reconstituted cells are usually activated by electrical and / or non-electrical means before, during, and / or after fusion of the nuclear donor and recipient oocytes. Methods of activation include electrical pulses, chemically induced shocks, penetration by sperm, increasing the level of divalent cations in the oocyte, and decreasing phosphorylation of cellular proteins in the oocyte (by kinase inhibitors). The activated reconstituted cells, or embryos, are usually cultured in media well known to those of skill 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. Pat. No. 7,612,250, each of which is incorporated herein by reference.

[0063] Other methods are provided for producing non-human animals that contain one or more genetic manipulations described herein in their germline, including: (a) manipulating a target genomic TARDBP locus of a non-human animal in a prokaryotic cell using various methods described herein; (b) selecting an engineered prokaryotic cell that contains a genetic manipulation at the target genomic locus; (c) isolating the engineered targeting vector from the genome of the engineered prokaryotic cell; (d) introducing the engineered targeting vector into a pluripotent cell of the non-human animal to generate an engineered pluripotent cell that contains an insert nucleic acid at the target TARDBP genomic locus; (e) selecting the engineered pluripotent cell; (f) introducing the engineered pluripotent cell into a host embryo of the non-human animal at a pre-morula stage; and (g) implanting the host embryo containing the engineered pluripotent cell into a surrogate mother to generate an F0 generation derived from the engineered pluripotent cell. In such methods, the targeting vector can include a large targeting vector. The non-human animal can be a non-human mammal, a rodent, a mouse, a rat, a hamster, a monkey, an agricultural mammal or a livestock mammal. The pluripotent cell can 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 livestock 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, a hamster cell. In one embodiment, the targeted genetic engineering results in a mutated TARDBP gene, for example a mutated TARDBP gene encoding a mutated TDP-43 polypeptide lacking a functional structural domain and / or a mutated TARDBP gene comprising a knockout mutation.

[0064] In a further method, the isolating step (c) further comprises (c1) linearizing the engineered targeting vector (i.e., the engineered LTVEC). In yet a further embodiment, the introducing step (d) further comprises (d1) introducing a nuclease agent into the pluripotent cells to promote homologous recombination. In one embodiment, the selection step (b) and / or (e) is carried out by applying a selectable agent to the prokaryotic or pluripotent cells as described herein. In one embodiment, the selection step (b) and / or (e) is carried out via a molecular engineering of alleles (MOA) assay as described herein.

[0065] In some embodiments, the various genetic manipulations of the target genomic loci described herein can be carried out by a series of homologous recombination reactions (BHR) in bacterial cells using LTVEC derived from bacterial artificial chromosome (BAC) DNA using VELOCIGENE® genetic engineering technology (see, e.g., U.S. Pat. No. 6,586,251 and Valenzuela, DM et al. (2003), Nature Biotechnology, 21(6):652-659, which are incorporated herein by reference in their entireties).

[0066] In some embodiments, targeted pluripotent and / or totipotent cells containing various genetic manipulations as described herein are used as insertion donor cells and introduced into a corresponding organism, e.g., from an 8-cell stage mouse embryo to the pre-morula stage, via the VELOCIMOUSE® method (see, e.g., US 7,576,259, US 7,659,442, US 7,294,754, and US 2008-0078000A1, all of which are incorporated herein by reference in their entirety). The non-human animal embryo containing the genetically engineered pluripotent and / or totipotent cells is incubated to the blastocyst stage and then implanted into a surrogate mother to generate an F0 generation. In some embodiments, targeted mammalian ES cells containing various genetic manipulations as described herein are introduced into a blastocyst stage embryo. Non-human animals carrying the genetically engineered genomic locus (i.e., the TARDBP locus) can be identified via a manipulation of alleles (MOA) assay as described herein. The resulting F0 generation non-human animals derived from the genetically engineered pluripotent and / or totipotent cells are bred with wild-type non-human animals to obtain F1 generation progeny. Following genotyping with specific primers and / or probes, the F1 non-human animals heterozygous for the genetically engineered genomic loci are bred with each other to produce F2 generation non-human animal progeny that are homozygous for the genetically engineered genomic loci.

[0067] In one embodiment, a method for producing a cell comprising a mutated TRADBP gene is provided. Such a method comprises: (a) contacting a pluripotent cell with a targeting construct comprising a mutated TARDBP gene or a mutated portion thereof flanked by 5' and 3' homology arms; wherein the targeting construct undergoes homologous recombination with the TARDBP locus in the genome of the cell to form an engineered pluripotent cell. The method for producing a non-human animal further comprises: (b) introducing the engineered pluripotent cell into a host embryo; and (c) gestating the host embryo in a surrogate mother, wherein the surrogate mother produces offspring comprising the engineered TARDBP locus, and wherein the genetic engineering results in a mutant TDP-43 polypeptide lacking a functional structural domain.

[0068] In some embodiments, cells comprising a mutated TARDBP gene may be generated by engineering ES cells to comprise a mutated TARDB gene and culturing the ES cells in vitro in a differentiation medium, in some embodiments, culturing the ES cells in vitro comprises differentiating the ES cells into primitive ectodermal cells or embryonic stem cell derived motor neurons (ESMN).

[0069] Cells and animals

[0070] The cells disclosed herein (which may be contained within a non-human animal tissue or a non-human animal) may be any type of cell that comprises a mutated TARDBP gene as disclosed herein. The cells may comprise a mutated non-human animal TARDBP gene (e.g., a mutated non-human animal TARDBP gene) or a mutated human TARDBP gene.

[0071] The cell may comprise a mutated TARDBP gene encoding a mutant TDP-43 polypeptide, wherein the mutant TDP-43 polypeptide lacks a functional structural domain, and the cell expresses the mutant TDP-43 polypeptide. For example, the cell may comprise a mutated TARDBP gene encoding a mutant TDP-43 polypeptide lacking a functional structural domain comprising a nuclear localization signal (NLS), an RNA recognition motif 1 (RRM1), an RNA recognition motif 2 (RRM2), a putative nuclear export signal (E), a prion-like domain (PLD), or a combination thereof. The cell may comprise a mutated TARDBP gene encoding a mutant TDP-43 polypeptide lacking a functional structural domain due to one or more of the following: (a) an amino acid point mutation in the NLS (e.g., K82A K83A, R84A, K95A, K97A, K98A, or a combination thereof), (b) an amino acid point mutation in RRM1 (e.g., F147L and / or F149L), (c) an amino acid point mutation in RRM2 (F194L and / or F229L), (d) a deletion of at least a portion of the nuclear export signal (e.g., a deletion of amino acids at and between positions 239 and 250 of 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 of the wild-type TDP-43 polypeptide). The cell may comprise a mutated TARDBP gene encoding a mutant TDP-43 polypeptide comprising the following mutations: K82A K83A, R84A, K95A, K97A, and K98A, wherein the mutant TDP-43 polypeptide lacks a functional NLS. The cell may comprise a mutated TARDBP gene encoding a mutant TDP-43 polypeptide comprising a deletion at amino acids 274 to 414 of the wild-type TDP-43 polypeptide, wherein the mutant TDP-43 polypeptide lacks a functional PLD. The cell may comprise a mutated TARDBP gene encoding a mutant TDP-43 polypeptide comprising point mutations F147L and F149L, wherein the mutant TDP-43 polypeptide lacks a functional RRM1.The cell may comprise a mutated TARDBP gene encoding a mutant TDP-43 polypeptide comprising point mutations F194L and F229L, wherein the mutant TDP-43 polypeptide lacks a functional RRM2.The cell may comprise a mutated TARDBP gene encoding a mutant TDP-43 polypeptide comprising a deletion of the nuclear export signal at amino acids 239 to 250 of the wild-type TDP-43 polypeptide, wherein the mutant TDP-43 polypeptide lacks a functional E.

[0072] The cell may comprise a mutated TARDBP gene comprising a knockout mutation, e.g., a conditional knockout mutation, a deletion, etc., of the entire coding sequence of the TARDBP gene. The cell may comprise a mutated TARDBP gene comprising a conditional knockout mutation, e.g., the mutated TARDBP gene may comprise a site-specific recombination recognition sequence, e.g., a loxp sequence. The cell may comprise a mutated TARDBP gene comprising a loxp sequence adjacent to an exon, e.g., exon 3, comprising a TDP-43 coding sequence. The cell may comprise a mutated TARDBP gene comprising a loxp sequence and lacking a TDP-43 coding sequence, e.g., exon 3. The cell may comprise a mutated TARDBP gene lacking the entire TDP-43 coding sequence, e.g., a mutated TARDBP gene comprising a deletion of the entire coding sequence of a TDP-43 polypeptide.

[0073] In some embodiments, the cell may comprise, for example, a mutated TRADBP gene inserted into an endogenous TARDBP locus in its germline genome. In some embodiments, the cell comprises a mutated TARDBP gene, e.g., a mutated TARDBP gene comprising a knockout mutation and / or a mutated TARDBP gene encoding a mutated TDP-43 polypeptide that replaces the endogenous TARDBP gene at the endogenous TARDBP locus. In some embodiments, the mutated TARDBP gene is operably linked to the endogenous TARDBP promoter and / or regulatory elements.

[0074] Cells may be heterozygous or homozygous for the mutated TARDBP gene. Diploid organisms have two alleles, one at each locus of a pair of homologous chromosomes. Each pair of alleles represents a genotype at a particular locus. A genotype is described as homozygous if there are two identical alleles at a particular locus, and heterozygous if the two alleles are different.

[0075] The cell may comprise (i) a replacement of the endogenous TARDBP gene at the endogenous TARDBP locus with a mutated TARDBP gene encoding a mutant TDP-43 polypeptide, and (ii) a mutated TARDBP gene comprising a knockout mutation at the other endogenous TARDBP locus of the homologous chromosome.

[0076] A cell containing a mutated TARDBP gene may express a mutant TDP-43 polypeptide encoded therein. A cell containing a mutated TARDBP gene and expressing a mutant TDP-43 polypeptide encoded therein may or may not express a wild-type TDB-43 polypeptide.

[0077] A cell comprising a mutated TARDBP gene may express a mutant TDP-43 polypeptide encoded therein and may be characterized by one or more of the following: (i) a level of mRNA transcript of the mutated TARDBP gene comparable to the level of mRNA transcript of the wild-type TARDBP gene in a control cell; (ii) an increased level of the mutant TDP-43 polypeptide compared to the level of the wild-type TDP-43 polypeptide in a control cell; (iii) the mutant TDP-43 polypeptide is found at higher concentrations in the cytoplasm of the cell than in the nucleus; (iv) the mutant TDP-43 polypeptide exhibits increased insolubility compared to the wild-type TDP-43 polypeptide; (v) cytoplasmic aggregates comprising the mutant TDP-43 polypeptide; (vi) increased splicing of cryptic exons of the gene compared to cells expressing wild-type TDP-43; and (vii) a decreased level of alternatively spliced ​​TDP-43 mRNA lacking sequences encoding the PLD of TDP-43.

[0078] The cells may be cultured in a test tube, or may be studied in vitro or in vivo. For example, the cells can be in vivo in an animal.

[0079] The cell may be a eukaryotic cell, including, for example, a fungal cell (e.g., yeast), a plant cell, an animal cell, a mammalian cell, a non-human mammalian cell, and a human cell. The term "animal" includes any member of the animal kingdom, including, for example, mammals, fish, reptiles, amphibians, birds, and insects. The mammalian cell can be, for example, a non-human mammalian cell, a rodent cell, a rat cell, a mouse cell, or a hamster cell. Other non-human mammals include, for example, non-human primates, monkeys, apes, orangutans, cats, dogs, rabbits, horses, bulls, deer, bison, sheep, livestock (e.g., bovine species such as cows, steers, etc.; ovine species such as sheep, goats, etc.; and porcine species such as pigs and hogs, etc.). Birds include, for example, chickens, turkeys, ostriches, geese, ducks, etc. Domestic and agricultural animals are also included. The term "non-human animal" excludes humans. In some embodiments, the animal can be a human or a non-human animal, including, but not limited to, a mouse, rat, rabbit, dog, cat, pig, and non-human primates, including, but not limited to, a monkey or chimpanzee. In some embodiments, the non-human animal cell is a rodent cell, e.g., a rat cell or a mouse cell.

[0080] The non-human animals can be from any genetic background. For example, suitable mice can be from 129 strains, C57BL / 6 strains, crosses of 129 and C57BL / 6, BALB / c strains, or Swiss Webster strains. In some embodiments, 129 strains 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, which is incorporated herein by reference in its entirety for all purposes. Examples of C57BL strains 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 can also be derived from crosses of the aforementioned 129 strains with the aforementioned C57BL / 6 strains (e.g., 50% 129 and 50% C57BL / 6). Similarly, suitable mice can be derived from crosses of the aforementioned 129 strains or crosses of the aforementioned BL / 6 strains (e.g., 129S6 (129 / SvEvTac) strains).

[0081] Similarly, rats can be derived from rat strains 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 strains such as FisherF344 and FisherF6. Rats can also be obtained from strains resulting from crosses of two or more of the above strains. For example, suitable rats can be derived from the DA strain or the ACI strain. The ACI rat strain has a white belly and feet, and a RT1 av1The Dark Agouti (DA) rat strain is characterized by the black agouti haplotype. Such strains are available from a variety of sources, including Harkan Laboratories. The Dark Agouti (DA) rat strain has agouti coat and RT1 av1 The rat is characterized by having a haplotype. Such rats can be obtained from various sources, including Charles River and Harlan Laboratories. Some suitable rats can be derived from inbred rat strains. See, for example, US2014 / 0235933, the entirety of which is incorporated herein by reference for all purposes.

[0082] The cells can also be in any type of undifferentiated or differentiated state. For example, the 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. Totipotent cells include undifferentiated cells that can give rise to any cell type, and pluripotent cells include undifferentiated cells that have the ability to develop into multiple differentiated cell types. 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 can contribute to any tissue of the developing embryo upon introduction into the embryo. ES cells can be derived from the inner cell mass of a blastocyst and can differentiate into cells of any of the three vertebrate germ layers (endoderm, ectoderm, and mesoderm).

[0083] The cells may also be derived from ES cells. For example, the cells can be neuronal cells (e.g., ES cell-derived motor neurons (ESMN)), primitive ectoderm-like cells, embryoid body cells, etc.

[0084] The cells provided herein can also be germ cells (e.g., sperm or oocytes). The cells can be mitotically competent or mitotically inactive, meiotically competent or meiotically inactive. Similarly, the cells can also be primary somatic cells or non-primary somatic cells. Somatic cells include any cell that is not a gamete, germ cell, gamete cell, or undifferentiated stem cell.

[0085] Suitable cells provided herein also include primary cells. Primary cells include cells or cell cultures that are directly isolated from organisms, organs, or tissues. Primary cells include cells that are not transformed and are not immortal. Primary cells include any cell obtained from an organism, organ, or tissue that has not been previously passaged in tissue culture, or that has been previously passaged in tissue culture but cannot be passaged indefinitely in tissue culture.

[0086] Other suitable cells provided herein include immortalized cells. Immortalized cells include cells from multicellular organisms that do not normally grow indefinitely, but can avoid normal cellular aging due to mutations or changes and instead continue to undergo division. Such mutations or changes can occur naturally or can be induced intentionally. Many types of immortalized cells are well known. Immortalized or primary cells include cells that are normally used to culture or express recombinant genes or recombinant proteins.

[0087] The cells provided herein also include one-cell stage embryos (i.e., fertilized oocytes or zygotes). Such one-cell stage embryos can be 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.

[0088] Methods for employing a system expressing a mutant TDP-43 polypeptide

[0089] Cells and non-human animals (and tissues or animals containing such cells) that contain a mutated TARDBP gene and express a mutant TDP-43 polypeptide that lacks a functional structural domain encoded therefrom as described herein provide models for studying the function of the TDP-43 structural domain and / or TDP-43 proteinopathy. For example, a cell or non-human animal that contains a mutated TARDBP gene and expresses a mutant TDP-43 polypeptide that lacks a functional structural domain encoded therefrom may exhibit a phenotype characteristic of TDP-43 proteinopathy. In some embodiments, for example, cells isolated from (a) embryonic stem cell derived motor neurons (ESMNs) that comprise a mutated TARDBP gene and express a mutant TDP-43 polypeptide encoded therefrom that lacks a functional structural domain, and / or (b) a non-human animal that comprises a replacement of an endogenous TARDBP gene with a mutated TARDBP gene at the endogenous TARDBP locus and expresses a mutant TDP-43 polypeptide therefrom, are characterized by: (i) a level of mRNA transcript of the mutated TARDBP gene that is comparable to the level of the mRNA transcript level of the wild-type TARDBP gene in control cells; (ii) an increased level of the mutant TDP-43 polypeptide compared to the level of the wild-type TDP-43 polypeptide in control cells. (iii) the mutant TDP-43 polypeptide is found in higher concentrations in the cytoplasm of the cell than in the nucleus; (iv) the mutant TDP-43 polypeptide exhibits increased insolubility compared to wild-type TDP-43 polypeptide; (v) cytoplasmic aggregates comprising the mutant TDP-43 polypeptide; (vi) increased splicing of cryptic exons of the gene compared to cells expressing wild-type TDP-43; and (vii) reduced levels of alternatively spliced ​​TDP-43 mRNA lacking the sequence encoding the PLD of TDP-43.

[0090] Thus, cells (and tissues or animals containing such cells) that contain a mutated TARDBP gene and express a mutant TDP-43 polypeptide lacking a functional structural domain encoded therefrom as described herein provide a system for identifying candidate therapeutic agents for treating, preventing, and / or suppressing inhibition of one or more symptoms of TDP-43 proteinopathy (e.g., cytoplasmic accumulation of mutant TDP-43 polypeptide) and / or for restoring biological function of wild-type TDP-43 polypeptide (e.g., suppression of cryptic exon splicing and / or increasing the level of alternative splicing of TDP-43 mRNA). In some embodiments, the effect of a therapeutic agent is determined by contacting a cell that contains a mutated TARDBP gene and expresses a mutant TDP-43 polypeptide lacking a functional structural domain encoded therefrom with a candidate therapeutic agent. The contacting may be performed in vitro. The contacting may include administering the candidate therapeutic agent to an animal.

[0091] In some embodiments, performing the assay includes determining the effect on the phenotype and / or genotype of a cell or animal contacted with the drug. In some embodiments, performing the assay includes determining the variability between lots of the drug (in some embodiments, performing the assay includes determining the difference between the effect on a cell or animal contacted with an administered drug described herein and a control cell or animal (e.g., expressing wild-type TDP-43)).

[0092] Exemplary parameters that may be measured in non-human animals (or in and / or using cells isolated therefrom) to assess the pharmacokinetic properties of a drug include, but are not limited to, aggregation, 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 concentrations, non-target tissue drug concentrations, transcriptional activity, and the like.

[0093] Oligonucleotides for selectively reducing full-length TDP-43 mRNA

[0094] Figure 11A illustrates the full-length TDP-43 pre-mRNA and the normal (upper panel) and alternative (lower panel) splice events occurring at its 3' end. As shown, exon 6 encodes the prion-like domain (PLD) of the full-length TDP-43 protein formed by normal splice events, and the coding sequence terminates at the end of the PLD. Two new exons (7 and 8) are formed by alternative splicing events from one of at least three alternative 5'-splice sites in exon 6 to a downstream alternative 3'-splice site, e.g., adjacent to the new exon 7. There is evidence of a second alternative splicing event from alternative exon 7 to alternative exon 8.

[0095] In mice, the alternative 5'-splice sites within or at the beginning of exon 6 described herein map to the following positions: (a) chromosome 4: 148,618,647; (b) chromosome 4: 148,618,665; (c) chromosome 4: 148,618,674. The alternative 3'-splice site of exon 7 maps to chromosome 4 position: 148,617,705. The second alternative splicing event from exon 7 to exon 8 occurs at chromosome 4: 148,617,566 to chromosome 4: 148,616,844. One skilled in the art will be able to determine similar alternative 5' and 3' splice sites in other TARDBP genes, e.g., the human TARDBP gene.

[0096] Alternative splicing from the alternative 5'-splice site in exon 6 to the downstream alternative 3'-splice site is predicted to generate an mRNA in which most of the PLD coding sequence is replaced with a sequence encoding a TDP-43 polypeptide lacking the PLD. For example, alternative splicing from any one of (a) Chrom 4:148,618,647; (b) Chrom 4:148,618,665; and (c) Chrom 4:148,618,674 to Chrom 4:148,617,705 (and any corresponding position in the human TARDBP gene) may create an mRNA in which most of the PLD coding sequence is replaced with an alternative mRNA predicted to encode a truncated form of TDP-43 lacking the PLD, in which the PLD is replaced by 18 amino acids. This second alternative splicing event does not create a new form of the TDP-43 protein because the open reading frame stops at exon 7, upstream of the 5'-splice site of exon 7.

[0097] The observation that TDP-43 lacking PLD can support viability, particularly in motor neurons, and the reduced levels of this alternatively spliced ​​TDP-43 mRNA in cells expressing ΔPLD or ΔNLS mutated TARDBP genes, together with their ALS-like phenotypes, suggest that this alternatively spliced ​​TDP-43 mRNA and its translated truncated products may not contribute to and may be protective against TDP-43 proteinopathy. Application of siRNA, antisense oligonucleotides and / or CRISPR / Cas9 systems designed to remove or inactivate TDP-43 mRNA isoforms that encode forms of the protein containing PLD may deplete mutants of TDP-43 prone to pathological aggregation while sparing alternatively spliced ​​mRNAs that result in truncated TDP-43 proteins lacking PLD. The truncated forms of TDP-43 may be resistant to pathological aggregation while still supporting cell life, particularly motor neuron viability.

[0098] Therefore, a therapeutic strategy consists of finding active antisense oligonucleotides (ASOs) or siRNAs that target only those TDP-43 mRNA sequences that contain sequences encoding PLD, for example, those mRNAs that contain sequences encoded by genomic sequences following alternative splice sites in exon 6. As a non-limiting example, ASOs or siRNAs may target mRNAs that contain sequences transcribed from the TARDBP gene after the codon(s) that encode the alternative 5' splice site that splices out the PLD domain. ASOs or siRNAs designed to target this region of TDP-43 mRNA will only recognize full-length TDP-43 mRNAs that encode TDP-43 polypeptides that include PLD, while sparing alternatively spliced ​​TDP-43 mRNAs that encode truncated and possibly protected TDP-43 polypeptides that lack PLD. In other words, such ASOs or siRNAs should not be able to recognize or enhance the degradation of alternatively spliced ​​TDP-43 mRNAs. 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 degradation of the mRNA by RNase H-mediated cleavage, e.g., via a -5-10-5 gapmer. The siRNA may promote degradation of the mRNA and / or protein synthesis by RNA interference.

[0099] Another therapeutic strategy is the application of the CRISPR / Cas system to selectively target and delete genomic sequences spanning the alternative 5' splice site within exon 6 and the downstream 3' splice site, e.g., exon 7, of the TARDBP gene. In this way, only mRNAs encoding truncated TDP-43 polypeptides lacking the PLD may be transcribed.

[0100] A. Antisense Oligonucleotides and siRNA

[0101] Antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) targeting sequences within the pre-mRNA may enhance the degradation of undesired isoforms. As designed herein, ASOs or siRNAs may be used to disrupt the TDP-43 mRNA encoding PLD while sparing alternatively spliced ​​TDP-43 mRNA. To reduce the level of only full-length TDP-43 mRNA, ASOs or siRNAs may target TDP-43 mRNA including sequences between the alternative 5' splice site in exon 6 and (ii) the downstream alternative 3' splice site, e.g., sequences encoding amino acids 287-414 of the TDP-43 polypeptide and / or any 3' untranslated region upstream of the alternative splice site. See FIG. 11A. In some embodiments, the alternative 5' splice site within exon 6 correlates to a TARDBP genomic location selected from the group consisting of: (a) mouse chromosome 4:148,618,647; (b) mouse chromosome 4:148,618,665; (c) mouse chromosome 4:148,618,674; and (d) a corresponding location in any of the human TARDBP genes. In some embodiments, the downstream alternative 3' splice site correlates to mouse chromosome 4:148,617,705 or a corresponding location in the human TARDBP gene.

[0102] Antisense oligonucleotides or siRNAs targeting TDP-43 mRNA encoding PLD may have chemically modified subunits arranged in a pattern or motif to confer properties on the antisense oligonucleotide such as enhanced inhibitory activity, increased binding affinity for the target nucleic acid, or resistance to degradation by in vivo nucleases.

[0103] Antisense oligonucleotides typically contain at least one region that has been modified to confer increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity for the target nucleic acid, and / or increased inhibitory activity. A second region of the antisense oligonucleotide may optionally serve as a substrate for the cellular endonuclease RNase H, which cleaves the RNA strand of an RNA:DNA duplex.

[0104] In certain embodiments, the antisense oligonucleotides are uniformly sugar-modified oligonucleotides. The antisense oligonucleotides may include a gapmer motif. In a gapmer, an inner region having a number of nucleotides that support RNase H cleavage is disposed between an outer region having a number of nucleotides that are chemically distinct from the nucleosides of the inner region. For antisense oligonucleotides with a gapmer motif, the gap segment generally serves as a substrate for endonuclease cleavage, while the wing segments include modified nucleosides. In certain embodiments, the regions of the gapmer are differentiated by the type of sugar moiety that comprises each distinct region. The types of sugar moieties used to differentiate the regions of the gapmer may include, in some embodiments, β-D-ribonucleosides, β-D-deoxyribonucleosides, 2'-modified nucleosides (such 2'-modified nucleosides may include 2'-MOE and 2'-O-CH3, among others), and bicyclic sugar-modified nucleosides. In certain embodiments, the wings may include several modified sugar moieties, including, for example, 2'-MOE. In certain embodiments, the wings may include some modified and unmodified sugar moieties, hi certain embodiments, the wings may include various combinations of 2'-MOE nucleosides and 2'-deoxynucleosides.

[0105] Each of the different regions may comprise uniform sugar moieties, variants, or alternating sugar moieties. A wing-gap-wing motif is often written as "XYZ," where "X" represents the length of the 5'-wing, "Y" represents the length of the gap, and "Z" represents the length of the 3'-wing. "X" and "Z" may comprise uniform sugar moieties, variant sugar moieties, or alternating sugar moieties. In certain embodiments, "X" and "Y" may comprise one or more 2'-deoxynucleosides. "Y" may comprise a 2'-deoxynucleoside. A gapmer, as used herein, written as "XYZ," has a configuration in which a gap is positioned immediately adjacent to each of the 5'-wing and 3'-wing. Thus, there are no intervening nucleotides between the 5'-wing and the gap, or between the gap and the 3'-wing. Any of the antisense compounds described herein may have a gapmer motif. In certain embodiments, "X" and "Z" are the same, while in other embodiments, they are different. In certain embodiments, "Y" is between 8 and 15 nucleosides. X, Y, or Z can be any 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 nucleosides. Thus, gapmers 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.

[0106] Antisense oligonucleotides targeted to the TDP-43 mRNA sequence encoding PLD may have a 5-10-5 gapmer motif.

[0107] Antisense oligonucleotides targeting the TDP-43 mRNA sequence encoding PLD may contain a gap-narrowed motif. Gap-narrowed antisense oligonucleotides targeting the TDP-43 mRNA may have a gap segment of 9, 8, 7, or 6 2'-deoxynucleotides located immediately adjacent to and between wing segments of 5, 4, 3, 2, or 1 chemically modified nucleosides. The chemically modified nucleoside may contain a bicyclic sugar. The bicyclic sugar may contain a 4' to 2' bridge selected from among 4'-(CH2)nO-2' bridge, where n is 1 or 2; and 4'-CH2-O-CH2-2'. The bicyclic sugar may contain a 4'-CH(CH3)-O-2' bridge. The chemical modification may include a non-bicyclic 2' modified sugar moiety, such as a 2'-O-methylethyl group or a 2'-O-methyl group. In some embodiments, the alternative 5' splice site is within exon 6, e.g., an antisense oligonucleotide comprising a gapmer motif that targets the TDP-43 mRNA sequence between the alternative 5' and 3' splice sites, where the alternative 5' splice site correlates with a TARDBP genomic location selected from the group consisting of: (a) mouse chromosome 4:148,618,647; (b) mouse chromosome 4:148,618,665; (c) mouse chromosome 4:148,618,674, and (d) the corresponding location of any of the human TARDBP genes, and the alternative 3' splice site correlates with the TARDBP genomic location of chromosome 4:148,617,705. In some embodiments, the siRNA comprises a sequence that targets the TDP-43 mRNA sequence between the alternative 5' splice site and the alternative 3' splice site, where the alternative 5' splice site is within exon 6, e.g., the alternative 5' splice site correlates with a TARDBP genomic location selected from the group consisting of: (a) mouse chromosome 4:148,618,647; (b) mouse chromosome 4:148,618,665; (c) mouse chromosome 4:148,618,674, and (d) a corresponding location of any of the human TARDBP genes, and the alternative 3' splice site correlates with a TARDBP genomic location of chromosome 4:148,617,705.

[0108] Antisense oligonucleotides or siRNAs targeting the TDP-43 mRNA sequence encoding PLD may be uniformly modified. In certain embodiments, each nucleoside is chemically modified. In certain embodiments, the chemical modification comprises a non-bicyclic 2'-modified sugar moiety. In certain embodiments, the 2'-modified sugar moiety comprises a 2'-O-methoxyethyl group. In certain embodiments, the 2'-modified sugar moiety comprises a 2'-O-methyl group.

[0109] The ASO or siRNA may 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 conjugation groups include cholesterol moieties and lipid moieties.Additional conjugation groups include carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin and dyes.

[0110] ASO or siRNA may also be modified to have one or more stabilizing groups, which are generally attached to one or both ends. Stabilizing groups include cap structures. These end modifications can protect ASO or siRNA with terminal nucleic acid from exonuclease degradation and aid in delivery and / or localization within cells. Caps can be present at the 5' end (5' cap) or 3' end (3' cap), or can be present at both ends. Cap structures are well known and include, for example, reverse deoxy abasic caps.

[0111] The ASO or siRNA may be of any length suitable for binding to a target nucleic acid (e.g., TDP-43 pre-mRNA) and having a desired effect. For example, the 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 nucleosides in length. As another example, the 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 nucleosides. Alternatively, the ASO may be about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about The ASO can be about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, or about 80 linked nucleosides. For example, the ASO can be comprised of about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 linked nucleosides. In specific examples, the ASO can be about 15 to about 25 linked nucleosides.

[0112] ASO or siRNA can be complementary and / or specifically hybridize to a target nucleic acid (e.g., TDP-43 pre-mRNA, e.g., mRNA sequence encoding PLD). ASO and target nucleic acid are complementary to each other when a sufficient number of nucleobases of the ASO can hydrogen bond with the corresponding nucleobases of the target nucleic acid to produce a desired effect. Specifically hybridizable refers to an ASO that has a sufficient degree of complementarity between the ASO and the target nucleic acid to cause a desired effect while having little or no effect on non-target nucleic acids under conditions where specific binding is desired (e.g., under physiological conditions).

[0113] 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 equal length portion of TDP-43 pre-mRNA. Alternatively, the ASO can be about 100% complementary to the equal length portion of TDP-43 pre-mRNA. The percent complementarity of the ASO with the target nucleic acid can be determined using routine methods. For example, an ASO that specifically hybridizes because 18 of the 20 nucleobases of the ASO are complementary to the target region exhibits 90% complementarity. Percent complementarity of an ASO to a region of a target nucleic acid can be routinely determined using the BLAST program (basic local sequence comparison search tool) and the well-known PowerBLAST program (see, e.g., Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656). Percent homology, percent sequence identity or percent 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 the Smith and Waterman algorithm (Adv. Appl. Math., 1981, 2, 482 489) using default settings.

[0114] Non-complementary nucleobases between the ASO or siRNA and the TDP-43 pre-mRNA may be tolerated, provided that the ASO or siRNA can remain specifically hybridized to the target nucleic acid. Furthermore, the ASO or siRNA may hybridize across one or more segments of the TDP-43 pre-mRNA such that intervening or adjacent segments are not involved in the hybridization event (e.g., loop structure, mismatch, or hairpin structure). The location of the non-complementary nucleobase may be at the 5' or 3' end of the ASO or siRNA. Alternatively, the non-complementary nucleobase or nucleobases may be at an internal position of the ASO or siRNA. When two or more non-complementary nucleobases are present, they may be contiguous (i.e., linked) or discontinuous.

[0115] B. Deletion of the genomic sequence encoding the PLD of TDP-43

[0116] As shown herein, cells remain viable despite expressing only mutant TDP-43 polypeptides lacking functional PLD. Also described herein are clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) systems, or one or more components of a CRISPR / Cas system, which may be used to delete a proteinaceous domain (or a portion thereof) of an endogenous TARDBP locus as described herein from a cell, e.g., an embryonic stem cell. The CRISPR / Cas system may delete genomic sequences at or near the 5' splice site of the short form of exon 6 and at or near the 3' splice site of exon 7 from a cell, e.g., an embryonic stem cell. Such components include, for example, a Cas protein and / or a guide RNA (gRNA), where the gRNA may comprise two separate RNA molecules, for example, a targeter RNA (e.g., a CRISPR RNA (crRNA) and an activator 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, where the gRNA recognizes a sequence at or near a TARDBP genomic location selected from the group consisting of: (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.

[0117] The CRISPR / Cas system includes transcripts and other elements involved in the expression of Cas genes or inducing the activity of Cas genes. The CRISPR / Cas system can be a type I, type II, or type III system. Alternatively, the CRISPR / Cas system can be a type V system (e.g., subtype VA or subtype VB). The sequence encoding the prion-like domain (or a portion thereof) of TDP-43 in the endogenous TARDBP locus described herein, 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 alternative exon 7), may be deleted by utilizing a CRISPR complex (including a guide RNA (gRNA) complexed with a Cas protein) for site-specific cleavage of the nucleic acid.

[0118] The CRISPR / Cas systems described herein 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, Casl0d, 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 homologs or variants thereof) and / or one or more gRNAs targeting a guide RNA (gRNA) recognition sequence. A CRISPR / Cas system as described herein may further include at least one expression construct comprising a nucleic acid encoding a Cas protein (e.g., which may be operably linked to a promoter) and / or DNA encoding a gRNA.

[0119] Site-specific binding and cleavage of the TARDBP gene by the Cas protein can occur at a location in the target DNA that is determined by both (i) the base pair complementarity between the gRNA and the target DNA and (ii) a short motif called a protospacer adjacent motif (PAM). The PAM can be adjacent to the guide RNA recognition sequence. Optionally, the guide RNA recognition sequence can be adjacent to the PAM at the 3' end. Alternatively, the guide RNA recognition sequence can be adjacent to the PAM at the 5' end. For example, the cleavage site of the Cas protein can be about 1 to about 10, or about 2 to about 5 base pairs (e.g., 3 base pairs) upstream or downstream of the PAM sequence. In some cases (e.g., when using Cas9 from S. pyogenes or a closely related Cas9), the PAM sequence of the non-complementary strand can be 5'-N1GG-3', where N1 is any DNA nucleotide and is immediately 3' to the guide RNA recognition sequence of the non-complementary strand of the target DNA. Thus, the PAM sequence of the complementary strand is 5'-CCN2-3', where N 2は It is any DNA nucleotide, immediately 5' to 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 the case of Cas9 from S. aureus, the PAM can be NNGRRT or NNGRR, where N can be A, G, C, or T, and R can be G or A.

[0120] As disclosed herein, the guide RNA may be provided in any form. In some embodiments, the gRNA may be provided in the form of RNA, either two molecules (separate crRNA and tracrRNA) or one molecule (sgRNA), and optionally in the form of a complex with a Cas protein. The gRNA may also be provided in the form of DNA encoding the gRNA. In some embodiments, the DNA encoding the gRNA may encode a single RNA molecule (sgRNA) or separate RNA molecules (e.g., separate crRNA and tracrRNA), where the separate RNA molecules may be provided as one DNA molecule or as separate DNA molecules encoding the crRNA and tracrRNA, respectively.

[0121] In one embodiment, the CRISPR / Cas system described herein comprises a Cas9 protein, or a protein derived from Cas9 from a type II CRISPR / Cas system, and / or at least one gRNA, wherein the at least one gRNA is encoded by DNA encoding a crRNA and / or a tracrRNA.

[0122] Targeted genetic engineering can be generated by contacting a cell with a Cas protein and one or more guide RNAs that hybridize to one or more guide RNA recognition sequences in a target genomic locus. At least one of the one or more guide RNAs can form a complex with the Cas protein and be directed 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. Cleavage by the Cas protein can create a double-stranded break or a single-stranded break (e.g., when the Cas protein is a nickase). The terminal sequences generated by the double-stranded break or single-stranded break can then undergo recombination.

[0123] C. Methods for Introducing Oligonucleotides

[0124] A variety of methods and compositions are provided herein to allow for the introduction of oligonucleotides into cells. Methods for introducing oligonucleotides into various cell types are known, including, for example, stable transfection, transient transfection, and viral-mediated methods.

[0125] The transfection protocol, as well as the protocol for introducing oligonucleotides into cells, may vary. Non-limiting transfection methods include liposomes; nanoparticles; calcium phosphate (Graham et al. (1973), Virology, 52(2):45-467, 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: W. H. Freeman and Company. pp. 96-97); dendrimers; or chemical-based transfection methods using cationic polymers such as DEAE-dextran or polyethyleneimine. Non-chemical methods include electroporation, sonoporation, and optical transfection. Particle-based transfection methods include the use of a gene gun, or magnet-assisted transfection (Bertram, (2006), Current Pharmaceutical Biotechnology, 7, 277-28). Viral methods can also be used for transfection.

[0126] The introduction of oligonucleotides into cells can be mediated by electroporation, intracytoplasmic injection, viral infection with adenovirus, adeno-associated virus, lentivirus, retrovirus, transfection, lipid-mediated transfection, or nucleofection.Nucleofection is an improved electroporation method that allows nucleic acid substrates to be delivered not only to the cytoplasm but also through the nuclear membrane into the nucleus.In addition, the use of nucleofection in the methods disclosed herein usually requires much fewer cells than conventional electroporation (e.g., only about 2 million cells compared to 7 million cells for conventional electroporation).In one example, nucleofection is carried out using the LONZA® NUCLEOFECTOR™ system.

[0127] Introduction of oligonucleotides into cells can also be achieved by microinjection. In zygotes (i.e., one-cell stage embryos), microinjection can be into the maternal and / or paternal pronuclei or cytoplasm. If microinjection is 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. 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.

[0128] Other methods for introducing oligonucleotide into cells include, for example, vector delivery, particle-mediated delivery, exosome-mediated delivery, lipid nanoparticle-mediated delivery, cell-penetrating peptide-mediated delivery, or implantable device-mediated delivery.Specific examples include oligonucleotide can be introduced into cells or non-human animals in carriers such as poly(lactic acid) (PLA) microspheres, poly(D,L-lactic-coglycolic acid) (PLGA) microspheres, liposomes, micelles, reverse micelles, lipid cochleates, or lipid microtubules.

[0129] Introduction of oligonucleotides can also be achieved by 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. The viruses can infect dividing cells, non-dividing cells, or both dividing and non-dividing cells. The viruses can integrate into the host genome, or alternatively, do not integrate into the host genome. Such viruses can also be engineered to have reduced immunity. The viruses can be replication-competent or replication-defective (e.g., defective in one or more genes required for additional rounds of virion replication and / or packaging). The viruses can cause 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 12 , 10 13 , 10 14 , 10 15 , and 10 16 Contains vector genomes / mL.

[0130] The ssDNA AAV genome consists of two open reading frames, Rep and Cap, flanked by two inverted terminal repeats that allow synthesis of a complementary DNA strand. When constructing an AAV transfer plasmid, the transgene is placed between the two ITRs, and Rep and Cap can be supplied in trans. In addition to Rep and Cap, AAV can require a helper plasmid containing genes from adenovirus. These genes (E4, E2a, and VA) mediate AAV replication. For example, HEK293 cells containing adenovirus genes E1+ can be transfected with the transfer plasmid, Rep / Cap, and the helper plasmid to produce infectious AAV particles. Alternatively, Rep, Cap, and adenovirus helper genes can be combined into a single plasmid. Similar packaging cells and methods can be used for other viruses, such as retroviruses.

[0131] Multiple serotypes of AAV have been identified. These serotypes differ in the type of cells they infect (i.e., their tropism), allowing preferential transduction of certain cell types. Serotypes for CNS tissue include AAV1, AAV2, AAV4, AAV5, AAV8, and AAV9. Serotypes for heart tissue include AAV1, AAV8, and AAV9. Serotypes for kidney tissue include AAV2. Serotypes for lung tissue include AAV4, AAV5, AAV6, and AAV9. Serotypes for pancreatic tissue include AAV8. Serotypes for photoreceptor cells include AAV2, AAV5, and AAV8. Serotypes for retinal pigment epithelium tissue include AAV1, AAV2, AAV4, AAV5, and AAV8. Serotypes for skeletal muscle tissue include AAV1, AAV6, AAV7, AAV8, and AAV9. Serotypes for liver tissue include AAV7, AAV8, and AAV9, especially AAV8.

[0132] Tropism can be further refined by pseudotyping, which is a mixture of capsids and genomes from different viral serotypes. For example, AAV2 / 5 refers to a virus containing a serotype 2 genome packaged in a serotype 5 capsid. Using pseudotyped viruses can improve transduction efficiency and alter tropism as well. Hybrid capsids from different serotypes can also be used to alter viral tropism. For example, AAV-DJ contains a hybrid capsid of serotype 8 and shows high infectivity across a wide range of cell types in vivo. AAV-DJ8 is another example that displays the properties of AAV-DJ but with enhanced brain uptake. AAV serotypes can also be modified through mutations. Examples of mutational modifications of AAV2 include Y444F, Y500F, Y730F, and S662V. Examples of mutational modifications of AAV3 include Y705F, Y731F, and T492V. Examples of AAV6 mutational modifications include S663V and T492V. Other pseudotyped / modified AAV variants include AAV2 / 1, AAV2 / 6, AAV2 / 7, AAV2 / 8, AAV2 / 9, AAV2.5, AAV8.2, and AAV / SASTG.

[0133] To accelerate transgene expression, self-complementary AAV (scAAV) variants can be used. Because AAV relies on the cell's DNA replication machinery to synthesize the complementary strand of the AAV's single-stranded DNA genome, transgene expression may be delayed. To address this delay, scAAV can be used that contain complementary sequences that can spontaneously anneal upon infection, eliminating the need for host cell DNA synthesis. However, single-stranded AAV (ssAAV) vectors can also be used.

[0134] The introduction of oligonucleotides can also be achieved by lipid nanoparticle (LNP)-mediated delivery. Lipid formulations can improve cellular uptake while protecting biomolecules from degradation. Lipid nanoparticles are particles that contain multiple lipid molecules physically bound to each other by intermolecular forces. These include microspheres (including unilamellar and multilamellar vesicles, e.g., liposomes), the dispersed phase in an emulsion, micelles, or the internal phase in a suspension. Such lipid nanoparticles can be used to encapsulate one or more oligonucleotides for delivery. Formulations containing cationic lipids are useful for delivering polyanions such as nucleic acids. Other lipids that can be included are neutral lipids (i.e., uncharged or zwitterionic lipids), anionic lipids, helper lipids that enhance transfection, and stealth lipids that increase the time that the nanoparticles can persist in vivo. Examples of suitable cationic lipids, neutral lipids, anionic lipids, helper lipids, and stealth lipids can be found in WO2016 / 010840A1, which is incorporated by reference in its entirety for all purposes.

[0135] In vivo administration can be by any suitable route, including, for example, parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. Systemic administration modes include, for example, oral and parenteral routes. Examples of parenteral routes include intravenous, intraarterial, intraosseous, intramuscular, intradermal, subcutaneous, intranasal, and intraperitoneal routes. A specific example is intravenous injection. Intranasal injection and intravitreal injection are other specific examples. Local modes of administration include, for example, intrathecal, intracerebroventricular, intraparenchymal (e.g., localized intraparenchymal delivery to the striatum (e.g., to the caudate or putamen), cerebral cortex, precentral gyrus, hippocampus (e.g., dentate gyrus or CA3 region), temporal cortex, amygdala, frontal cortex, thalamus, cerebellum, medulla, hypothalamus, tectum, tegmentum, or substantia nigra), intraocular, intraorbital, subconjunctival, intravitreal, subretinal, and transscleral routes. Significantly smaller amounts of the components (compared to systemic approaches) may exert an effect when administered locally (e.g., intraparenchymal or intravitreal) compared to when administered systemically (e.g., intravenously). Local modes of administration may also reduce or eliminate the occurrence of potentially toxic side effects that may occur when a therapeutically effective amount of the components is administered systemically.

[0136] One common method for promoting the uptake of reagents (e.g., antisense oligonucleotides) in cell culture involves the use of cationic lipids to transfect nucleic acids. When cationic lipids are mixed with negatively charged nucleic acids, a complex is obtained that can cross the cell membrane and release active nucleic acids into the cell cytoplasm. It is also possible to electroporate reagents (e.g., antisense oligonucleotides) into cells. This method is very effective and useful for cell lines that cannot be easily transfected by lipids.

[0137] If the cells are in vivo (e.g., in an animal), administration to the animal can be by any suitable means. For example, administration can include parenteral routes of administration, such as intraperitoneal, intravenous, and subcutaneous. Parenteral administration means administration via injection or infusion. Parenteral administration can include subcutaneous, intravenous, intramuscular, intraarterial, intraperitoneal, or intracranial administration (e.g., intrathecal or intraventricular).

[0138] In some methods, administration is by a means that the introduced reagent reaches neurons or the nervous system. This can be achieved, for example, by peripheral delivery or direct delivery to the nervous system. See, for example, Evers et al. (2015), Adv. Drug Deliv. Res. 87:90-103 (incorporated herein by reference in its entirety for all purposes).

[0139] For reagents (e.g., 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 cord barrier. One mechanism that can be used to cross the vascular barrier is receptor-mediated endocytosis. Another mechanism that can be used is a delivery system based on cell-penetrating peptides (CPPs). Different CPPs use distinct cell translocation pathways that depend on the cell type and cargo. For example, systemically delivered antisense oligonucleotides tagged with arginine-rich CPPs can cross the blood-brain barrier. Another delivery mechanism that can be used is exosomes, extracellular vesicles known to mediate cell-cell communication via the transfer of proteins and nucleic acids. For example, IV injection of exosomes transduced with short viral peptides derived from the rabies virus glycoprotein (RVG) can cross the blood-brain barrier and be delivered to the brain.

[0140] Techniques are also available that circumvent the vascular barrier via direct injection into the cerebrospinal fluid. For example, an agent (e.g., antisense oligonucleotide) can be injected intracerebroventricularly (ICV), after which the agent (e.g., antisense oligonucleotide) must pass through the ependymal cell layer that fills the ventricular system to enter the parenchyma. Intrathecal (IT) delivery refers to the delivery of an agent (e.g., antisense oligonucleotide) to the subarachnoid space of the spinal cord. From here, the agent (e.g., antisense oligonucleotide) must pass through the pia mater to enter the parenchyma. An agent (e.g., antisense oligonucleotide) can be delivered ICT or IT via an outlet catheter that is connected to an implanted reservoir. The drug is infused into the reservoir and delivered directly to the CSF. Intranasal administration is an alternative delivery route that can be used.

[0141] The scope of the present invention is defined by the claims appended hereto, and is not limited by the specific embodiments described herein; one skilled in the art reading this disclosure will recognize various modifications that may be equivalent to such described embodiments or may otherwise fall within the claims. Generally, terms follow their art-understood meanings unless otherwise specified. References cited within this specification, or relevant portions thereof, are incorporated herein by reference in their entirety.

[0142] The use of ordinal terms such as "first," "second," and "third" in the claims to modify claim elements does not, in and of itself, imply a priority, precedence, or ordering of one claim element relative to another claim element, or the temporal order in which method actions are performed, but is merely used as a label to distinguish one claim element having a particular name from another element having the same name (apart from the use of the ordinal term).

[0143] The articles "a" and "an" in the specification and claims should be understood to include plural referents unless specifically and clearly indicated to the contrary. A claim or description including "or" between one or more members of a group is deemed to be satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process, unless the contrary is indicated or otherwise clear from the context. The invention includes embodiments in which exactly one member of a group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, the invention should be understood to encompass all variations, combinations, and permutations of one or more limitations, elements, clauses, descriptive language, etc. from one or more of the enumerated claims that are introduced into another claim that relies on the same base claim (or other claims, if relevant), unless otherwise indicated or obvious to one of ordinary skill in the art that would result in a contradiction or inconsistency. When elements are presented as a list (e.g., in Markush group format or similar), it is to be understood that each subgroup of elements is also disclosed and that any element(s) may be removed from the group. In general, when the invention, or aspects of the invention, are referred to as including certain elements, features, etc., it is to be understood that an embodiment of the invention or an aspect of the invention consists of, or consists essentially of, such elements, features, etc. For purposes of brevity, those embodiments have not in every instance been literally specifically described herein. It is also to be understood that any embodiment or aspect of the invention may be expressly excluded from the claims, regardless of whether a specific exclusion is set forth in the specification.

[0144] "Control" includes the art-understood meaning of "control" being a standard against which results are compared. Typically, controls are used to enhance integrity in an experiment by isolating a variable in order to draw conclusions about such a variable. In some embodiments, a control is a reaction or assay performed simultaneously with a test reaction or assay to provide a basis for comparison. "Control" also includes "control animals." "Control animals" may have a modification as described herein, a different modification as described herein, or no modification (i.e., wild-type animals). In one experiment, the "test" (i.e., the variable being examined) is applied. In a second experiment, the "control," the variable being examined, is not applied. In some embodiments, a control is a pre-existing control (i.e., of a test or assay already performed, or of an amount or result already known). In some embodiments, a control is or includes a printed or otherwise kept record. A control may be a positive control or a negative control.

[0145] "Determining," "measuring," "assessing," "assessing," "assaying," and "analyzing" include any form of measurement, including determining whether an element is present. These terms include both quantitative and / or qualitative determinations. Assays may be relative or absolute. "Analyzing for the presence" can be determining the amount of something present and / or determining whether it is present or absent.

[0146] The terms "nucleic acid" and "polynucleotide," used interchangeably herein, include polymeric forms of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, or analogs or modifications thereof, including single-, double-, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural, or derivatized nucleotide bases.

[0147] The terms "protein", "polypeptide" and "peptide" used interchangeably herein include polymeric forms of amino acids of any length, including coded and non-coded amino acids and chemically or biochemically modified or derivatized amino acids. The term also includes modified polymers, such as polypeptides having modified peptide backbones. The term "domain" refers to any portion of a protein or polypeptide having a particular function or structure. Unless otherwise specified, any structural domain referred to herein refers to a TDP-43 structural domain.

[0148] The term "wild-type" includes entities having a structure and / or activity as found in a normal (as opposed to a mutant, diseased, altered, etc.) state or condition. Wild-type genes and polypeptides often exist in multiple alternative forms (e.g., alleles).

[0149] The term "endogenous" refers to a location, nucleic acid or amino acid sequence that is naturally found or present in a cell or animal. For example, an endogenous TARDBP sequence of a non-human animal refers to a wild-type TARDBP sequence that naturally occurs at the endogenous TARDBP locus of the non-human animal.

[0150] The term "locus" refers to the specific location of a gene (or sequence of interest), DNA sequence, sequence encoding a polypeptide, or location on a chromosome of the genome of an organism. For example, a "TARDBP locus" may refer to the specific location of the TARDBP gene, the DNA sequence of TARDBP, the sequence encoding TARDBP2, or the location of TARDBP on a chromosome of the genome of an organism where such a sequence is identified. A "TARDBP locus" may include regulatory elements of the TARDBP gene, including, for example, enhancers, promoters, 5' and / or 3' untranslated regions (UTRs), or combinations thereof.

[0151] The term "gene" refers to a DNA sequence in a chromosome that codes for a product (e.g., an RNA product and / or a polypeptide product), including coding regions interrupted by non-coding introns such that a gene corresponds to a full-length mRNA (including 5' and 3' untranslated sequences) and sequences located adjacent to the coding region at both the 5' and 3' ends. 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, silencer isolation sequences, and matrix attachment regions. These sequences may be close to the coding region of the gene (e.g., within 10 kb) or at distant sites, and they affect the level or rate of transcription and translation of the gene.

[0152] The term "allele" refers to a variant form of a gene. Some genes have a variety of different forms that are located at the same position, or locus, on a chromosome. Diploid organisms have two alleles, each at an endogenous locus on a homologous chromosome. Each pair of alleles represents a genotype at a particular locus. A genotype is described as homozygous if there are two identical alleles at a particular locus, and heterozygous if the two alleles are different.

[0153] "Operably linked" includes a juxtaposition in a relationship permitting the components described to function in their intended manner. A control sequence "operably linked" to a coding sequence is linked in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. "Operably linked" sequences include both expression control sequences contiguous with a gene of interest and expression control sequences acting in trans or at a distance to control the gene of interest. The term "expression control sequences" includes polynucleotide sequences necessary to affect the expression and processing of coding sequences to which they are linked. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequences); sequences that enhance protein stability; and, if desired, sequences that enhance protein secretion. The nature of such control sequences varies depending on the host organism. For example, in prokaryotes such control sequences generally include promoters, ribosomal binding sites, and transcription termination sequences, while in eukaryotes such control sequences usually include promoters and transcription termination sequences. The term "control sequence" is intended to include components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, e.g., leader sequences and fusion partner sequences.

[0154] "Phenotype" includes a trait, or a class or set of traits, exhibited by a cell or organism. In some embodiments, a particular phenotype may be correlated 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 expression levels, cellular localization and / or solubility / stability profiles of a protein, e.g., a mutant TDP-43 polypeptide, each of which may be determined using well-known methods such as Western blot analysis, fluorescent in situ hybridization, qualitative RT-PCR, and the like.

[0155] A "promoter" is a regulatory region of DNA that usually contains a TATA box that can direct RNA polymerase II to begin RNA synthesis at the appropriate transcription initiation site of a particular polynucleotide sequence. A promoter may further contain other regions that affect the rate of transcription initiation. The promoter sequences disclosed herein regulate the transcription of an operably linked polynucleotide. The promoter can be active in one or more cell types disclosed herein (e.g., eukaryotic cells, non-human mammalian cells, human cells, rodent cells, pluripotent cells, one-cell stage embryos, differentiated cells, or combinations thereof). The promoter can be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a temporally restricted promoter (e.g., a developmentally regulated promoter), or a spatially restricted promoter (e.g., a cell-specific or tissue-specific promoter). Examples of promoters can be found, for example, in WO2013 / 176772, which is incorporated by reference in its entirety for all purposes.

[0156] "Reference" includes a standard or control agent, cell, animal, cohort, individual, population, sample, sequence, or value to which a subject agent, cell, animal, cohort, individual, population, sample, sequence, or value is compared. In some embodiments, the reference agent, cell, animal, cohort, individual, population, sample, sequence, or value is tested and / or determined substantially simultaneously with the testing or determination of the subject agent, cell, animal, cohort, individual, population, sample, sequence, or value. 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, a reference may refer to a control. "Reference" also includes "reference cells." A "reference cell" may have a modification as described herein, a different modification as described herein, or no modification (i.e., a wild-type cell). Typically, as will be understood by one of skill in the art, a reference agent, cell, animal, cohort, individual, population, sample, sequence or value will be determined or characterized under conditions comparable to the conditions used to determine or characterize a subject agent, animal (e.g., mammal), cohort, individual, population, sample, sequence, or value.

[0157] The term "variant" refers to a nucleotide sequence that differs from a reference nucleotide sequence (e.g., by a single nucleotide) or a protein sequence that differs from a reference amino acid sequence (e.g., by a single amino acid) but retains the biological function of the reference sequence. In some embodiments, variants differ from a reference sequence due to degeneracy of the genetic code and / or conservative codon / amino acid substitutions.

[0158] The term "sequence identity" or "identity" in the context of two polynucleotide or polypeptide sequences refers to residues in the two sequences that are the same when aligned for maximum correspondence over a particular comparison window. When using percentage sequence identity in the context of proteins, non-identical residue positions often differ by conservative amino acid substitutions, where amino acid residues are replaced with other amino acid residues that have similar chemical properties (e.g., charge or hydrophobicity) and thus do not alter the functional properties of the molecule. When sequences differ by conservative substitutions, the percent sequence identity may be adjusted upwards to compensate for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity". Means for making this adjustment are well known. Typically, this involves scoring conservative substitutions as partial mismatches rather than complete mismatches, thereby increasing the percentage of sequence identity. Thus, for example, where identical amino acids are given a score of 1 and non-conservative substitutions are given a score of zero, conservative substitutions are given a score between zero and 1. Scoring of conservative substitutions is calculated, for example, as implemented in the program PC / GENE (Intelligenetics, Mountain View, California).

[0159] "Percentage of sequence identity" includes a value determined by comparing two optimally aligned sequences over a comparison window (maximum number of perfectly matched residues), where the portion of the polynucleotide sequence within the comparison window may include additions or deletions (i.e., gaps) compared to the reference sequence (no additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where there are matching nucleic acid bases or amino acid residues in both sequences 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 percentage of sequence identity. Unless otherwise specified (e.g., the shorter sequence includes a linked heterologous sequence), the comparison window is the full length of the shorter of the two sequences being compared.

[0160] Unless otherwise specified, sequence identity / similarity values ​​include values ​​obtained using GAP version 10 with the following parameters: % identity and % similarity for nucleotide sequences with a GAP weight of 50 and a length weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for amino acid sequences with a GAP weight of 2 and a length weight of 3, and the BLOSUM62 scoring matrix; or equivalent programs. "Equivalent programs" include any sequence comparison program that generates sequence comparisons that have identical nucleotide or amino acid residue matches and the same percent sequence identity for any two sequences at issue when compared to corresponding sequence comparisons generated by GAP version 10.

[0161] The term "conservative amino acid substitution" refers to the replacement of an amino acid normally present in a sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the replacement of a non-polar (hydrophobic) residue, such as isoleucine, valine, or leucine, with another non-polar residue. Similarly, examples of conservative substitutions include the replacement of one polar (hydrophilic) residue with another, such as between arginine and lysine, between glutamine and asparagine, or between glycine and serine. Furthermore, the replacement of a basic residue, such as lysine, arginine, or histidine, with another, or the replacement of one acidic residue, such as aspartic acid or glutamic acid, with another, are additional examples of conservative substitutions. Examples of non-conservative substitutions include the replacement of a polar (hydrophilic) residue, such as cysteine, glutamine, glutamic acid, or lysine, with a non-polar (hydrophobic) amino acid residue, such as isoleucine, valine, leucine, alanine, or methionine, and / or the replacement of a non-polar residue with a polar residue. Exemplary amino acid classifications are summarized in Table 1 below. Table 1. Classification of amino acids. [Table 1]

[0162] The term "in vitro" includes an artificial environment (e.g., a test tube) and processes or reactions that occur within an artificial environment. The term "in vivo" includes a natural environment (e.g., a cell or an organism or living organism) and processes or reactions that occur within a natural environment. The term "ex vivo" includes cells removed from an individual's body and processes or reactions that occur within such cells.

[0163] Non-limiting exemplary embodiments include the following: Embodiment 1. A non-human animal cell comprising a mutated TARDBP gene encoding a mutant TDP-43 polypeptide, The mutant TDP-43 polypeptide lacks a functional structural domain found in a wild-type TDP-43 polypeptide, The non-human animal or non-human animal cell expresses the mutant TDP-43 polypeptide, Optionally, the wild-type TDP-43 polypeptide comprises the sequence shown as SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5.

[0164] Embodiment 2. The non-human animal cell of embodiment 1, wherein the mutant TDP-43 polypeptide lacks a functional structural domain comprising a nuclear localization signal (NLS), an RNA recognition motif 1 (RRM1), an RNA recognition motif 2 (RRM2), a putative nuclear export signal (E), a prion-like domain (PLD), or a combination thereof.

[0165] Embodiment 3. The non-human animal cell of embodiment 1 or embodiment 2, wherein said non-human animal cell is an embryonic stem (ES) cell, an embryoid body, or an embryonic stem cell-derived motor neuron (ESMN).

[0166] Embodiment 4. The non-human animal cell of any one of the preceding embodiments, wherein said mutated TARDBP gene is a mutated TARDBP gene of said non-human animal.

[0167] Embodiment 5. The non-human animal cell according to any one of embodiments 1 to 3, wherein said mutated TARDBP gene is a mutated human TARDBP gene.

[0168] Embodiment 6. The mutant TDP-43 polypeptide is: (a) an amino acid point mutation in the NLS; (b) an amino acid point mutation in the RRM1; (c) an amino acid point mutation in the RRM2; (d) a deletion of at least a portion of the nuclear export signal; and (e) a deletion of at least a portion of the prion-like domain.

[0169] Embodiment 7. (a) the point mutations of amino acids in the NLS comprise K82A K83A, R84A, K95A, K97A, K98A, or combinations thereof; (b) the point mutation in RRM1 comprises F147L and / or F149L; (c) the point mutation in RRM2 comprises F194L and / or F229L; (d) the deletion of at least a portion of the nuclear export signal deletion comprises a deletion of amino acids at and between positions 239 and 250 of a wild-type TDP-43 polypeptide; and (e) The non-human animal cell of embodiment 6, wherein the deletion of at least a portion of the prion-like domain comprises a deletion of amino acids at and between positions 274 and 414 of the wild-type TDP-43 polypeptide.

[0170] Embodiment 8. The non-human animal cell of any one of the preceding embodiments, wherein the mutant TDP-43 polypeptide comprises K82A K83A, R84A, K95A, K97A, and K98A.

[0171] Embodiment 9. The non-human animal cell of any one of the preceding embodiments, wherein the mutant TDP-43 polypeptide lacks the prion-like domain at amino acids 274 to 414 of the wild-type polypeptide.

[0172] Embodiment 10. The non-human animal cell of any one of the preceding embodiments, wherein the mutant TDP-43 polypeptide comprises F147L and F149L.

[0173] Embodiment 11. The non-human animal cell of any one of the preceding embodiments, wherein the mutant TDP-43 polypeptide comprises F194L and F229L.

[0174] Embodiment 12. The non-human animal cell of any one of the preceding embodiments, wherein the mutant TDP-43 polypeptide lacks the nuclear export signal at amino acids 239 to 250.

[0175] Embodiment 13. The non-human animal cell of any one of the preceding embodiments, wherein said mutated TARDBP gene encoding a mutant TDP-43 polypeptide replaces the endogenous TARDBP gene at the endogenous TARDBP locus.

[0176] Embodiment 14. The non-human animal cell of embodiment 13, wherein the non-human animal cell is heterozygous for the mutated TARDBP gene encoding a mutant TDP-43 polypeptide.

[0177] Embodiment 15. The non-human animal cell of embodiment 13, wherein the non-human animal cell is homozygous for the mutated TARDBP gene encoding a mutant TDP-43 polypeptide.

[0178] Embodiment 16. The non-human animal cell according to any one of embodiments 1 to 14, wherein the non-human animal cell further comprises a TARDBP gene comprising a knockout mutation.

[0179] Embodiment 17. The non-human animal cell of embodiment 16, wherein the knockout mutation comprises a conditional knockout mutation.

[0180] Embodiment 18. The non-human animal cell of embodiment 16 or embodiment 17, wherein the knockout mutation comprises a site-specific recombination recognition sequence.

[0181] Embodiment 19. A non-human animal cell according to any one of embodiments 16 to 18, wherein the knockout mutation comprises a loxp sequence.

[0182] Embodiment 20. The non-human animal cell of embodiment 19, wherein said loxp sequences flank exon 3 of said TARDBP gene comprising a knockout mutation.

[0183] Embodiment 21. The non-human animal cell of embodiment 16, wherein the knockout mutation comprises a deletion of the entire coding sequence for the TDP-43 peptide.

[0184] Embodiment 22. The non-human animal cell is heterozygous for the engineered TARDBP locus, and (i) replacement of an endogenous TARDBP gene at an endogenous TARDBP locus on one chromosome with said mutated TARDBP gene encoding a mutant TDP-43 polypeptide; and (ii) A non-human animal cell according to any one of embodiments 16 to 21, comprising either the TARDBP gene comprising the knockout mutation at the endogenous TARDBP locus or a wild-type TARDBP gene in the other homologous chromosome.

[0185] Embodiment 23. The non-human animal cell of any one of the preceding embodiments, wherein said non-human animal cell does not express a wild-type TDP-43 polypeptide.

[0186] Embodiment 24. The non-human animal cell according to any one of 1 to 22, wherein the non-human animal cell expresses a wild-type TDP-43 polypeptide.

[0187] 25. (i) an mRNA transcript level of the mutated TARDBP gene that is comparable to the mRNA transcript level of a wild-type TARDBP gene in a control cell; (ii) an increase in the level of the mutant TDP-43 polypeptide relative to the level of a wild-type TDP-43 polypeptide in a control cell; (iii) a mutant TDP-43 polypeptide that is found, for example, at higher concentrations in the cytoplasm than in the nucleus of motor neurons; (iv) a mutant TDP-43 polypeptide that has increased insolubility compared to a wild-type TDP-43 polypeptide; (v) a cytoplasmic aggregate comprising the mutant TDP-43 polypeptide; (vi) increased splicing of cryptic exons, and / or (vii) a reduced level of an alternatively spliced ​​TDP-43 form.

[0188] Embodiment 26. A non-human animal cell comprising (i) a conditional knockout mutation of the TARDBP gene at an endogenous TARDBP locus in one chromosome and (ii) a deletion of the entire TARDBP coding sequence at the endogenous TARDBP locus in the other homologous chromosome.

[0189] Embodiment 27. The non-human animal cell of any one of the preceding embodiments, wherein said cell is an embryonic stem (ES) cell, a primitive ectodermal cell, or a motor neuron derived from a motor neuron (ESMN).

[0190] Embodiment 28 The non-human animal cell of any one of the preceding embodiments, wherein said non-human animal cell is a rodent cell.

[0191] Embodiment 29 The non-human animal cell of any one of the preceding embodiments, wherein said non-human animal cell is a rat cell.

[0192] Embodiment 30. A non-human animal cell according to any one of embodiments 1 to 28, wherein the non-human animal cell is a mouse cell.

[0193] Embodiment 31 The non-human animal cell of any one of the preceding embodiments, wherein said non-human animal cell is cultured in vitro.

[0194] Embodiment 32 A non-human animal tissue comprising a non-human animal cell according to any one of the preceding embodiments.

[0195] Embodiment 33. A composition comprising a non-human animal cell or tissue according to any one of the preceding embodiments.

[0196] Embodiment 34. A method for producing a non-human animal or a non-human animal cell that expresses a mutant TDP-43 polypeptide, comprising manipulating the genome of said non-human animal or a non-human animal cell to contain a mutated TARDBP gene encoding said mutant TDP-43 polypeptide, wherein said mutant TDP-43 polypeptide lacks a functional structural domain compared to wild-type TDP-43, and optionally said wild-type TDP-43 polypeptide comprises the sequence set forth as SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5.

[0197] Embodiment 35. The method of embodiment 34, wherein manipulating comprises replacing the endogenous TARDBP gene with the mutated TARDBP gene encoding the mutant TDP-43 polypeptide.

[0198] Embodiment 36 The method of embodiment 34 or embodiment 35, wherein the manipulating further comprises replacing the endogenous TARDBP gene with a TARDBP gene comprising a knockout mutation.

[0199] Embodiment 37 The method of embodiment 36, wherein the knockout mutation comprises a conditional knockout mutation.

[0200] Embodiment 38 The method of embodiment 37, further comprising culturing said cells under conditions that eliminate expression of said TARDBP gene comprising a knockout mutation.

[0201] Embodiment 39. A method for identifying therapeutic candidates for the treatment of a disease, comprising: (a) contacting a non-human animal cell or tissue according to any one of embodiments 1 to 31, or a composition according to embodiment 32, with a candidate agent; (b) assessing the phenotype and / or TDP-43 biological activity of the non-human cells or tissues; and (c) identifying the candidate agent that restores a phenotype and / or TDP-43 biological activity to a non-human cell or tissue that is comparable to the phenotype and / or TDP-43 biological activity of a control cell or tissue expressing a wild-type TDP-43 polypeptide.

[0202] Embodiment 40. A method for evaluating the biological function of a TDP-43 structural domain, comprising: (a) engineering embryonic stem (ES) cells to contain a mutated TARDBP gene encoding a mutant TDP-43 polypeptide lacking a functional structural domain selected from the group consisting of a nuclear localization signal (NLS), a first RNA recognition motif (RRM1), a first RNA recognition motif (RRM2), a putative nuclear export signal (E), a prion-like domain (PLD), and combinations thereof; (b) optionally differentiating the engineered ES cells in vitro and / or obtaining a genetically engineered non-human animal from the engineered ES cells; and (c) evaluating the phenotype and / or TDP-43 biological activity of the genetically engineered ES cells, the primitive ectoderm derived therefrom, the motor neurons derived therefrom, or the non-human animal derived therefrom.

[0203] Embodiment 41 The method of embodiment 39 or embodiment 40, wherein the phenotype is assessed by cell culture, fluorescent in situ hybridization, Western blot analysis, or a combination thereof.

[0204] Embodiment 42. The method of any one of embodiments 39 to 41, wherein assessing the phenotype comprises measuring the viability of the genetically engineered ES cells, the primitive ectoderm derived therefrom, the motor neurons derived therefrom, or the non-human animal derived therefrom.

[0205] Embodiment 43. The method of any one of embodiments 39 to 42, wherein assessing the phenotype comprises determining the cellular location of the mutant TDP-43 polypeptide.

[0206] Embodiment 44. The method of any one of embodiments 39 to 43, wherein assessing the biological activity of the mutant TDP-43 polypeptide comprises measuring a splice product of a gene that contains a cryptic exon regulated by TDP-43.

[0207] Embodiment 45. The method of embodiment 44, wherein the genes containing cryptic exons regulated by TDP-43 include Crem, Fyxd2, and Clf1.

[0208] Embodiment 46. The method of any one of embodiments 39 to 45, wherein assessing the biological activity of the mutant TDP-43 polypeptide comprises measuring the level of alternatively spliced ​​TDP-43.

[0209] Embodiment 47. An antisense oligonucleotide comprising a gapmer motif that targets a TDP-43 mRNA sequence that encodes the PLD of the TDP-43 polypeptide and / or includes untranslated sequences downstream of exon 6 and upstream of exon 7, Optionally, the TDP-mRNA comprises a sequence between an alternative 5' splice site in exon 6 and a downstream alternative 3' splice site; Optionally, the antisense oligonucleotide wherein the alternative 5' splice site correlates to a TARDBP genomic location selected from the group consisting of: (a) mouse chromosome 4:148,618,647; (b) mouse chromosome 4:148,618,665; (c) mouse chromosome 4:148,618,674, and (d) a corresponding location in any of the human TARDBP genes, and / or the alternative 3' splice site correlates to a TARDBP genomic location of chromosome 4:148,617,705.

[0210] Embodiment 48. A siRNA comprising a sequence encoding the PLD of the TDP-43 polypeptide and / or targeting the TDP-43 mRNA sequence including the untranslated sequence downstream of exon 6 and upstream of exon 7, Optionally, the TDP-mRNA sequence is between an alternative 5' splice site in exon 6 and a downstream alternative 3' splice site; Optionally, the siRNA, wherein the alternative 5' splice site correlates to a TARDBP genomic location selected from the group consisting of: (a) mouse chromosome 4:148,618,647; (b) mouse chromosome 4:148,618,665; (c) mouse chromosome 4:148,618,674, and (d) any corresponding location in the human TARDBP gene, and / or the alternative 3' splice site correlates to the TARDBP genomic location of chromosome 4:148,617,705.

[0211] Embodiment 49. A CRISPR / Cas system comprising a Cas9 protein and at least one gRNA, wherein the gRNA recognizes a sequence at or near a sequence encoding an alternative splice site that results in an alternative mRNA encoding a truncated TDP-43 polypeptide lacking the PLD; Optionally, the alternative splice sites include an alternative 5' splice site within exon 6 and a downstream alternative 3' splice site; Optionally, the alternative 5' splice site correlates with a TARDBP genomic location selected from the group consisting of: (a) mouse chromosome 4:148,618,647; (b) mouse chromosome 4:148,618,665; (c) mouse chromosome 4:148,618,674, and (d) a corresponding location of any of the human TARDBP genes, and / or the alternative 3' splice site correlates with a TARDBP genomic location of chromosome 4:148,617,705, of the CRISPR / Cas system.

[0212] Embodiment 50. A non-human animal comprising the embryonic stem cells of embodiment 2.

[0213] Embodiment 51. A non-human animal comprising a mutated TARDBP gene encoding a mutant TDP-43 polypeptide, The mutant TDP-43 polypeptide lacks a functional structural domain compared to a wild-type TDP-43 polypeptide; and The non-human animal expresses the mutant TDP-43 polypeptide. Optionally, the non-human animal, wherein the wild-type TDP-43 polypeptide comprises the sequence shown as SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5.

[0214] Embodiment 52. The non-human animal of embodiment 51, wherein the mutant TDP-43 polypeptide lacks a functional structural domain comprising a nuclear localization signal (NLS), an RNA recognition motif 1 (RRM1), an RNA recognition motif 2 (RRM2), a putative nuclear export signal (E), a prion-like domain (PLD), or a combination thereof.

[0215] Embodiment 53. A non-human animal according to embodiment 51 or embodiment 52, wherein said mutated TARDBP gene is a mutated TARDBP gene of said non-human animal.

[0216] Embodiment 54. A non-human animal according to any one of embodiments 51 to 53, wherein said mutated TARDBP gene is a mutated human TARDBP gene.

[0217] Embodiment 55. The mutant TDP-43 polypeptide is: (a) an amino acid point mutation in the NLS; (b) an amino acid point mutation in the RRM1; (c) an amino acid point mutation in the RRM2; (d) a deletion of at least a portion of the nuclear export signal; and (e) a deletion of at least a portion of the prion-like domain.

[0218] EMBODIMENT 56. (a) the point mutations of amino acids in the NLS comprise K82A K83A, R84A, K95A, K97A, K98A, or combinations thereof; (b) the point mutation in RRM1 comprises F147L and / or F149L; (c) the point mutation in RRM2 comprises F194L and / or F229L; (d) the deletion of at least a portion of the nuclear export signal deletion comprises a deletion of amino acids at and between positions 239 and 250 of a wild-type TDP-43 polypeptide; and (e) The non-human animal of embodiment 55, wherein said deletion of at least a portion of the prion-like domain comprises a deletion of amino acids at and between positions 274 and 414 of the wild-type TDP-43 polypeptide.

[0219] Embodiment 57. The non-human animal of any one of embodiments 51 to 56, wherein the mutant TDP-43 polypeptide comprises K82A K83A, R84A, K95A, K97A, and K98A.

[0220] Embodiment 58. The non-human animal of any one of embodiments 51 to 57, wherein the mutant TDP-43 polypeptide lacks the prion-like domain at amino acids 274 to 414 of the wild-type polypeptide.

[0221] Embodiment 59. The non-human animal of any one of embodiments 51 to 58, wherein the mutant TDP-43 polypeptide comprises F147L and F149L.

[0222] Embodiment 60. The non-human animal of any one of embodiments 51 to 59, wherein the mutant TDP-43 polypeptide comprises F194L and F229L.

[0223] Embodiment 61. The non-human animal of any one of embodiments 51 to 60, wherein the mutant TDP-43 polypeptide lacks the nuclear export signal at amino acids 239 to 250.

[0224] Embodiment 62. A non-human animal according to any one of embodiments 51 to 61, wherein said mutated TARDBP gene encoding a mutant TDP-43 polypeptide replaces the endogenous TARDBP gene at the endogenous TARDBP locus.

[0225] Embodiment 63. The non-human animal of embodiment 62, wherein the non-human animal is heterozygous for the mutated TARDBP gene encoding a mutant TDP-43 polypeptide.

[0226] Embodiment 64. The non-human animal of any one of embodiments 51 to 63, wherein the non-human animal further comprises a TARDBP gene comprising a knockout mutation.

[0227] Embodiment 65. The non-human animal of embodiment 64, wherein the knockout mutation comprises a conditional knockout mutation.

[0228] Embodiment 66 The non-human animal of embodiment 64 or embodiment 65, wherein the knockout mutation comprises a site-specific recombination recognition sequence.

[0229] Embodiment 67. A non-human animal according to any one of embodiments 64 to 66, wherein the knockout mutation comprises a loxp sequence.

[0230] Embodiment 68. The non-human animal of embodiment 67, wherein said loxp sequences flank exon 3 of said TARDBP gene comprising a knockout mutation.

[0231] Embodiment 69. The non-human animal of embodiment 64, wherein the knockout mutation comprises a deletion of the entire coding sequence for the TDP-43 peptide.

[0232] Embodiment 70. The non-human animal is heterozygous for the engineered TARDBP locus, and (i) replacement of an endogenous TARDBP gene at the endogenous TARDBP locus on one chromosome with said mutated TARDBP gene encoding a mutant TDP-43 polypeptide; and (ii) A non-human animal according to any one of embodiments 64 to 69, comprising either the TARDBP gene comprising the knockout mutation or a wild-type TARDBP gene at the endogenous TARDBP locus in the other homologous chromosome.

[0233] Embodiment 71. A non-human animal according to any one of embodiments 50 to 70, wherein the non-human animal expresses a wild-type TDP-43 polypeptide.

[0234] EMBODIMENT 72. (i) an mRNA transcript level of the mutated TARDBP gene that is comparable to the mRNA transcript level of the wild-type TARDBP gene in a control animal; (ii) an increase in the level of the mutant TDP-43 polypeptide relative to the level of a wild-type TDP-43 polypeptide in a control animal; (iii) a mutant TDP-43 polypeptide that is found, for example, at higher concentrations in the cytoplasm than in the nucleus of motor neurons; (iv) a mutant TDP-43 polypeptide that has increased insolubility compared to a wild-type TDP-43 polypeptide; (v) a cytoplasmic aggregate comprising the mutant TDP-43 polypeptide; (vi) increased splicing of cryptic exons; (vii) reduced levels of alternatively spliced ​​forms of TDP-43; (viii) denervation of muscle tissue that is primarily composed of fast twitch muscle, such as the tibialis anterior; and / or (ix) normal innervation of muscle tissue composed primarily of slow-twitch muscles, such as the intercostal muscles.

[0235] Embodiment 73. A non-human animal comprising a TARDBP gene that comprises a conditional knockout mutation at the endogenous TRADBP locus, and a TARDBP gene at the other endogenous TARDBP locus of the homologous chromosome that comprises a deletion of the entire TARDBP coding sequence.

[0236] Embodiment 74. A non-human animal according to any one of embodiments 50 to 73, wherein the non-human animal is a rodent.

[0237] Embodiment 75. A non-human animal according to any one of embodiments 50 to 74, wherein the non-human animal is a rat.

[0238] Embodiment 76. A non-human animal according to any one of embodiments 50 to 74, wherein the non-human animal is a mouse.

[0239] Embodiment 77. A method for identifying therapeutic candidates for the treatment of a disease, comprising: (a) contacting a non-human animal according to any one of embodiments 50 to 76 with a candidate agent; (b) assessing the phenotype and / or TDP-43 biological activity of the non-human animal; and and (c) identifying said candidate agent that restores the non-human phenotype and / or TDP-43 biological activity.

[0240] Embodiment 78. A mutant TDP-43 polypeptide comprising: (a) point mutation of an amino acid in an NLS; (b) an amino acid point mutation in RRM1; (c) an amino acid point mutation in RRM2; (d) deletion of at least a portion of the nuclear export signal; and (e) a deletion of at least a portion of the prion-like domain.

[0241] 79. (a) the point mutations of amino acids in the NLS comprise K82A K83A, R84A, K95A, K97A, K98A, or combinations thereof; (b) the point mutation in RRM1 comprises F147L and / or F149L; (c) the point mutation in RRM2 comprises F194L and / or F229L; (d) the deletion of at least a portion of the nuclear export signal deletion comprises a deletion of amino acids at and between positions 239 and 250 of a wild-type TDP-43 polypeptide; and (e) The mutant TDP-43 polypeptide of embodiment 78, wherein the deletion of at least a portion of the prion-like domain comprises a deletion of amino acids at and between positions 274 and 414 of a wild-type TDP-43 polypeptide.

[0242] Embodiment 80. A mutant TDP-43 polypeptide according to embodiment 78 or embodiment 79, comprising a K82A mutation, a K83A mutation, a R84A mutation, a K95A mutation, a K97A mutation, and / or a K98A mutation.

[0243] Embodiment 81. A mutant TDP-43 polypeptide according to any one of embodiments 78 to 80, comprising a deletion of the prion-like domain at amino acids 274 to 414 of the wild-type polypeptide.

[0244] Embodiment 82. A mutant TDP-43 polypeptide according to any one of embodiments 78 to 81, wherein the mutant TDP-43 polypeptide comprises an F147L mutation and / or an F149L mutation.

[0245] Embodiment 83. A mutant TDP-43 polypeptide according to any one of embodiments 78 to 82, wherein the mutant TDP-43 polypeptide comprises an F194L mutation and / or an F229L mutation.

[0246] Embodiment 84. The mutant TDP-43 polypeptide of any one of embodiments 78 to 83, wherein the mutant TDP-43 polypeptide lacks the nuclear export signal at amino acids 239 to 250.

[0247] Embodiment 85. A nucleic acid comprising a nucleic acid sequence encoding a mutant TDP-43 polypeptide according to any one of embodiments 78 to 84.

[0248] Embodiment 86. The nucleic acid of embodiment 85, further comprising, from 5' to 3': a 5' homology arm, the nucleic acid sequence encoding the mutant TDP-43 polypeptide, and a 3' homology arm, wherein the nucleic acid undergoes homologous recombination in a rodent cell.

[0249] Embodiment 87. The nucleic acid of embodiment 86, wherein the 5' and 3' homology arms are homologous to rat sequences such that the nucleic acid undergoes homologous recombination at the endogenous rat TARDBP locus such that the nucleic acid sequence encoding the mutant TDP-43 polypeptide replaces the endogenous TARDBP coding sequence.

[0250] Embodiment 88. The nucleic acid of embodiment 86, wherein the 5' and 3' homology arms are homologous to mouse sequences such that the nucleic acid undergoes homologous recombination at the endogenous mouse TARDBP locus such that the nucleic acid sequence encoding the mutant TDP-43 polypeptide replaces the endogenous TARDBP coding sequence.

[0251] Embodiment 89. A method for selectively reducing TDP-43 mRNA encoding a TDP-43 polypeptide comprising a PLD while sparing an alternative TDP-43 mRNA encoding a truncated TDP-43 lacking the PLD in a cell, comprising: (i) an antisense oligonucleotide comprising a gapmer motif that targets a TDP-43 mRNA sequence that encodes the PLD of the TDP-43 polypeptide and / or includes the untranslated sequence downstream of exon 6 and upstream of exon 7; (ii) an siRNA comprising a sequence encoding the PLD of the TDP-43 polypeptide and / or targeting the TDP-43 mRNA sequence including the untranslated sequence downstream of exon 6 and upstream of exon 7; and / or (iii) introducing into the cell a CRISPR / Cas system comprising a Cas9 protein and at least one gRNA, wherein the gRNA recognizes a sequence at or near a sequence encoding an alternative splice site that results in an alternative mRNA encoding a truncated TDP-43 polypeptide lacking the PLD.

[0252] EMBODIMENT 90. (i) the antisense oligonucleotide is the ASO of embodiment 47; (ii) the siRNA is the siRNA of embodiment 48; and / or (iii) The method of embodiment 89, wherein the CRISPR / Cas system is the CRISPR / Cas system of embodiment 49.

[0253] Embodiment 91. The method of embodiment 89 or 90, wherein the cell is present in vivo. [Table 4-1] [Table 4-2] EXAMPLES

[0254] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention.

[0255] Example 1: Generation of embryonic stem cells expressing a mutated TARDBP gene Because TDP-43 is essential for viability, embryonic stem (ES) cells may be generated that contain a conditional knockout in a first endogenous TDP-43 allele and a mutation in another, second endogenous TDP-43 allele such that wild-type TDP-43 from the first endogenous allele sustains ES cell viability until activation, after which the effect of a mutant TDP-43 polypeptide expressed from the second allele may be ascertained.

[0256] To assess the biological, biochemical, and / or pathogenic roles played by various TDP-43 structural domains, mouse ES cells were engineered to contain (i) a conditional knockout mutation at the endogenous TARDBP locus and (ii) a mutated TARDBP gene at the other TARDBP locus on the homologous chromosome encoding a mutant TDP-43 polypeptide in which one of the five structural domains—nuclear localization signal (NLS), RNA recognition motif 1 (RRM1), RNA recognition motif 2 (RRM2), putative nuclear export signal (E), or prion-like domain (PLD)—has been altered or deleted in a manner predicted to abolish function (see Figure 3). Cells harboring a mutated TARDBP gene(s) and expressing mutant TDP-43 polypeptides lacking a functional NLS, RRM1, RRM2, E or PLD were analyzed for both (1) phenotype and (2) biological activity of the mutant TDP-43 polypeptides as described in Examples 2 and 3, respectively.

[0257] The conditional allele was designed based on a previously published study showing that deletion of TDP-43 exon 3 does not produce a functional protein. Chiang et al. (2010), Proc. Natl. Acad. Sci. USA. 107:16320-324. Exon 3 of the endogenous mouse TARDBP gene was loxP-transfected at loxP sites. After Cre-mediated recombination, a deletion of genomic coordinates chr4:147995844-147996841 was achieved. ES cells containing the loxP-transfected exon 3 were further engineered with a mutated TARDBP gene as described herein. As a control, mouse ES cells engineered with a conditional knockout mutation in one allele and a deletion from the start codon to the stop codon of the second exon in the other allele (genomic coordinates chr4:147992370-147999471) were also generated.

[0258] Example 2: Phenotypic analysis of cells expressing a mutated TARDBP gene The phenotypes of the embryonic stem (ES) cells generated in Example 1, the primitive ectoderm derived therefrom, or the motor neurons (ESMN) derived therefrom were analyzed by assessing cell viability and the localization and stability of mutant TDP-43 polypeptides.

[0259] Notably, ES cells expressing mutant TDP-43 polypeptides lacking a functional NLS or a functional PLD were viable; however, cells expressing mutant TDP-43 polypeptides lacking a functional PLD appeared to have reduced fitness (Figure 4). Neither ES cells nor ESMN expressing mutant TDP-43 polypeptides lacking functional RRM1 or RRM2 remained viable (Figures 4 and 5).

[0260] Mutant TDP-43 polypeptides lacking a functional NLS redistributed from the nucleus to the cytoplasm in ESMN, where mutant TDP-43 accumulated in many large aggregate-like inclusions reminiscent of ALS pathology (Figures 6-8). Lack of a functional NLS caused extensive cytoplasmic aggregation of mutant TDP-43 polypeptides, accompanied by loss of nuclear staining (Figures 7-8). Mutant TDP-43 polypeptides lacking a functional PLD also redistributed to the cytoplasm in ESMN, where they accumulated in punctate inclusions that appeared less abundant and qualitatively different than those produced by mutant TDP-43 polypeptides lacking a functional NLS (Figures 6-8). Deletion of the PLD caused maximal mislocalization of mutant TDP-43 polypeptides to the cytoplasm, although nuclear staining was retained (Figures 7-8).

[0261] Mutant TDP-43 polypeptides lacking a functional NLS or PLD showed increased solubility of mutant TDP-44. Figure 9A. The solubility of mutant TDP-43 polypeptides lacking a functional E or RRM1 was unchanged compared to wild-type TDP-43 polypeptide. Figure 9A. There was no difference in mRNA expression levels for any of the mutant TDP-43 polypeptides, but increased protein levels were observed for mutant TDP-43 polypeptides lacking a functional NLS, PLD, or RRM1. Figure 9B. Since the mRNA expression levels of these mutant TDP-43 polypeptides were comparable to that of wild-type TDP-43, the increased protein levels could be due to increased stability of the mutant TDP-43 polypeptides. Figure 9C.

[0262] The materials and methods used to analyze the phenotype of cells expressing mutant TDP-43 polypeptides lacking functional structural domains are described below.

[0263] cell culture

[0264] The ability of mutant TDP-43 protein, as the only form of protein expressed by the cells, to support the viability of embryonic stem (ES) cells and their derived motor neurons (ESMNs) was examined by differentiation in culture. ES cells were cultured in embryonic stem cell medium (ESM; DMEM + 15% fetal bovine serum + penicillin / streptomycin + glutamine + non-essential amino acids + nucleosides + β-mercaptoethanol + sodium pyruvate + LIF) for 2 days, during which the medium was changed daily. One hour before trypsinization, the ES medium was replaced with 7 mL of ADFNK medium (advanced DMEM / F12 + neurobasal medium + 10% knockout serum + penicillin / streptomycin + glutamine + β-mercaptoethanol). The ADFNK medium was aspirated 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 2 days. The cells were cultured for an additional 4 days in ADFNK supplemented with retinoic acid (RA), smoothogonist, and purmorphamine to obtain limb-like motor neurons (ESMN). Dissociated motor neurons were plated in embryonic stem cell-derived motor neuron medium (ESMN; neurobasal medium + 2% horse serum + B27 + glutamine + penicillin / streptomycin + β-mercaptoethanol + 10 ng / mL GDNF, BDNF, CNTF) and allowed to mature. The conditional knockout allele was activated using Cre recombinase delivered via electroporation at the ES cell stage (Figures 4, 6-9) or 7 days after plating (Figure 5).

[0265] Subcellular localization of mutant TDP-43 polypeptides

[0266] The subcellular localization of TDP-43 mutants was analyzed using an antibody recognizing the N-terminus of the TDP-43 polypeptide (α-TDP-43N-term) and an antibody recognizing the C-terminal prion-like domain of the TDP-43 polypeptide (α-TDP-43C-term) (Proteintech, Rosemont, IL). Soluble cytoplasmic protein extracts were prepared by incubating MN derived from ES cells for 10 min on ice 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). The cells were then passed five times through a 27-gauge syringe. After centrifugation at 4000 rpm for 5 min at 4 °C, the protein supernatant containing the soluble cytoplasmic extract was collected. Insoluble nuclear protein extracts were prepared by resuspending the pellet in an equal volume of RBS-100 buffer (10 mM Tris-HCl, pH 7.4, 2.5 mM MgCl2, 100 mM NaCl, 0.1% NP-40) supplemented with proteases and phosphatases. An equal volume of 2x SDS sample buffer was added to each fraction, and 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 for TDP-43 using α-TDP-43-N-term or α-TDP-43-C-term antibodies, the latter of which does not recognize the PLD deletion mutant. Densitometry was performed using ImageJ. Figure 6B. Cytoplasmic / nuclear TDP-43 ratios were plotted and statistically analyzed using GraphPad for Prism. Figure 6B; Figure 9B, right panel.

[0267] Fluorescence in situ hybridization (FISH)

[0268] ES cell-derived MNs were placed on polyornithine / laminin-coated coverslips and cultured for 7 days. Coverslips were immersion fixed in ice-cold 4% PFA for 15 min and washed with 1× PBS. Cells were blocked with 5% normal donkey serum diluted in Tris-buffered saline (pH 7.4) containing 0.2% TritonX-100 (TBS-T) and incubated overnight at 4°C with primary antisera (TDP-43 C-term and MAP2) diluted in TBS-T with 5% normal donkey serum. After washing with TBS-T, cells were incubated with species-specific secondary antibodies conjugated to Alexa488 and Alexa568 (1:1,000; Life Technologies, Carlsbad, CA, USA) for 1 h at room temperature. After washing with TBS-T, the stained tissue coverslips were mounted on microscope slides in Floromount (Southern Biotech, Birmingham, AL, USA) and imaged at 40x magnification using a Leica 710LSM confocal microscope (Figures 7 and 8).

[0269] Solubility of mutant TDP-43 polypeptides

[0270] This protocol was adapted from Jo et al. (2014), Nature Communications, 5:3496. 500ul of ice-cold solubility buffer (0.1M MES (pH 7), 1mM EDTA, 0.5mM MgSO4, 1M sucrose) containing 50mM N-ethylmaleimide (NEM), 1mM NaF, 1mM Na3VO4, 1mM PMSF, and 10ug / mL each of aprotinin, leupeptin, and pepstatin. Cells were passed 3-5 times through a 21-gauge needle, followed by 3-5 times through a 23-gauge needle. Equal amounts of homogenate were then collected from each sample and centrifuged at 50,000×g for 20 minutes at 4°C, and the remainder was stored at -80°C. The supernatant was removed and each pellet was resuspended in 700 ul 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 sarkosine (Sarkosyl) and protease inhibitors (1 mM PMSF, 50 mM NEM, and 10 ug / mL each of aprotinin, leupeptin, and pepstatin), vortexed for 1 min at RT, and incubated overnight at 4° C. with end-over-end rotation. Samples were then centrifuged at 200,000×g at 12° C. for 30 min, and the supernatant was collected as the Sarkosyl-soluble fraction. The pellet was resuspended in 700ul of RAB buffer and passed through a 26 gauge needle 3-5 times to completely disperse the pellet, creating the sarkosyl-insoluble fraction. Equal amounts of the sarkosyl-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 amounts of each fraction were then loaded onto a 14% SDS gel and electrophoresed at 225V for 50 minutes, followed by western blotting for TDP-43. Densitometry was performed using ImageJ. Figure 9A. The ratios of soluble:insoluble TDP-43 were plotted and statistically analyzed using GraphPad for Prism. Figure 9A.

[0271] Expression levels of mutant TDP-43 polypeptides

[0272] Expression levels of TDP-43 mutants were analyzed by Western blot analysis as described herein. Messenger RNA levels in this example were performed by quantitative polymerase chain reaction.

[0273] Total RNA from each sample was extracted and reverse transcribed using primers spanning the junction of conventional exons 4 and 5 and a probe detecting a region of the mouse TDP-43 locus. DROSHA qPCR was performed using probes and primers from a readily available kit.

[0274] Specifically, RNA was isolated from embryonic stem cell derived motor neurons (ESMN) as described in Example 1.

[0275] Direct-zol according to the manufacturer's protocol (Zymo Research) Total RNA was isolated using the RNA Miniprep plus kit. 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). qRT-PCR reactions contained 2 μL of RNA and 8 μL of a mixture containing RT-PCR master mix, ROX dye, RT mix, and 20× gene-specific primer-probe mix in a final volume of 10 μL.

[0276] Unless otherwise stated, 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 optical 384-well plates with an RT step of 45° C. for 10 min followed by 95° C. for 10 min and 45 cycles of two-step cycling at 95° C. for 5 s and 60° C. for 30 s. The sequences of primers and probes used in the analysis (pan assay) are provided in Table 2 below. Table 2 [Table 2]

[0277] Solubility of mutant TDP-43 polypeptides

[0278] ES cell colonies were dissociated after 2 days and cultured in ADFNK medium. The medium was changed on day 2 and supplemented with retinoic acid (100 nM-2 μM) (Sigma) and sonic hedgehog (Shh-N; 300 nM) (Curis Inc.), and embryoid bodies (EBs) were cultured for 4 days. Day 4 embryoid bodies were treated with cycloheximide (100 μg / mL) to block new protein synthesis. The medium was changed every 4 hours and fresh cycloheximide was added. Cell lysates were harvested at the indicated time points and analyzed by immunoblotting with TDP-43 and GAPDH antibodies. Figure 9C. Example 3: Analysis of TDP-43 biological activity by TDP-43 mutants

[0279] Cryptic exons often have GU-rich TDP-43 binding sites, and TDP-43 has been shown to repress the recognition of cryptic exons, thereby promoting normal splicing. Loss of TDP-43 results in loss of normal mRNA and protein levels of regulated genes. TDP-43 also binds to the 3' end of its own transcript in a negative feedback autoregulatory loop to maintain TDP-43 levels. The biological activity of mutant TDP-43 polypeptides lacking functional structural domains was examined by assessing the ability of the mutant TDP-43 polypeptides to continue to repress splicing of cryptic exons and / or their participation in the autoregulatory loop.

[0280] ESMNs heterozygous for wild-type TARDBP gene or mutated TARDBP gene encoding mutant TDP-43 polypeptide lacking functional RRM1, NLS, or PLD were analyzed for the expression products of three genes containing cryptic exons whose splicing is known to be suppressed by wild-type TDP-43: Crem, Fyxd2, and Clf1. Figure 10. Normally spliced ​​Crem, Fyxd2, and Clf1 products were found in all ESMNs expressing mutant TDP-43 polypeptide lacking functional RRM1, NLS, or PLD, and normally spliced ​​products were found in amounts comparable to ESMNs expressing wild-type TDP-43 polypeptide. Figure 10. However, splicing of the cryptic exons was increased in ESMNs expressing mutant TDP-43 polypeptide lacking functional RRM1, NLS, or PLD compared to ESMNs expressing wild-type TDP-43 polypeptide. Figure 10. This data suggests that mutant TDP-43 polypeptides lacking functional RRM1, NLS, or PLD are unable to suppress splicing of the cryptic exons of the Crem, Fyxd2, and Clf1 genes.

[0281] ESMN heterozygous for wild-type TARDBP gene or mutated TARDBP gene encoding mutant TDP-43 polypeptide lacking functional NLS, RRM1, RRM2, E, or PLD were analyzed for the levels of alternatively spliced ​​TDP-43 mRNA. FIG. 11B. Compared to control ESMN expressing wild-type TDP-43 polypeptide, ESMN expressing mutant TDP-43 polypeptide lacking functional NLS, RRM1, E, or PLD showed reduced levels of alternatively spliced ​​TDP-43 mRNA. FIG. 11B. ESMN expressing mutant TDP-43 polypeptide lacking functional E showed equivalent levels of alternatively spliced ​​TDP-43 mRNA. FIG. 11B. This data, together with that provided in Example 2 showing that ESMN expressing mutant TDP-43 forms lacking functional NLS or PLD exhibit an ALS phenotype ( FIG. 5 ), suggests that strategies to reduce levels of normally spliced ​​TDP-43 mRNA while sparing alternatively spliced ​​TDP-43 mRNA may be therapeutic for TDP-43-related pathologies.

[0282] The materials and methods used to analyze the phenotype of cells expressing mutant TDP-43 polypeptides lacking functional structural domains are described below.

[0283] Quantitative polymerase chain reaction

[0284] Total RNA from each sample was extracted and reverse transcribed using primers flanking splice regions and probes detecting regions of the gene locus (Crem, Fxyd2, Clf1, TDP-43) investigated. Detectable regions for Crem, Fxyd2, and Clf1 genes investigated included regions spanning the junction of mouse sequences of normal and cryptic exons of each investigated gene. Detectable regions for TDP-43 region investigated included regions spanning alternative splice regions. DROSHA qPCR was performed using probes and primers from a readily available kit.

[0285] Specifically, RNA was isolated from embryonic stem cell-derived motor neurons (ESMN) 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). 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). qRT-PCR reactions contained 2 μL of RNA and 8 μL of a mixture containing RT-PCR master mix, ROX dye, RT mix, and 20× gene-specific primer-probe mix to a final volume of 10 μL.

[0286] Unless otherwise stated, 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 optical 384-well plates with a RT step of 45°C for 10 min followed by a RT step of 95°C for 10 min and 50 cycles of two-step cycling at 95°C for 5 s and 60°C for 30 s.

[0287] qRT-PCR to assess productive Crem splicing from exon 1 to exon 2 was performed with primers comprising the nucleotide sequences shown as SEQ ID NO: 14 and SEQ ID NO: 15, and a primer comprising the nucleotide sequence shown as SEQ ID NO: 16. Crem exon 1 to cryptic exon splicing was assessed using primers comprising the nucleotide sequences shown as SEQ ID NO: 17 and SEQ ID NO: 18, and a primer comprising the nucleotide sequence shown as SEQ ID NO: 19. Crem cryptic exon to exon 2 splicing was assessed using primers comprising the nucleotide sequences shown as SEQ ID NO: 20 and SEQ ID NO: 21, and a primer comprising the nucleotide sequence shown as SEQ ID NO: 22.

[0288] qRT-PCR to assess productive Fyxd2 splicing from exon 3 to exon 4 was performed with primers comprising the nucleotide sequences shown as SEQ ID NO: 23 and SEQ ID NO: 24, and a primer comprising the nucleotide sequence shown as SEQ ID NO: 25. Fyxd2 exon 3 to cryptic exon splicing was assessed with primers comprising the nucleotide sequences shown as SEQ ID NO: 26 and SEQ ID NO: 27, and a primer comprising the nucleotide sequence shown as SEQ ID NO: 28. Fyxd2 cryptic exon to exon 4 splicing was assessed with primers comprising the nucleotide sequences shown as SEQ ID NO: 29 and SEQ ID NO: 30, and a primer comprising the nucleotide sequence shown as SEQ ID NO: 31.

[0289] qRT-PCR of productive Crlf1 splice products was performed with primers comprising the nucleotide sequences shown as SEQ ID NO:32 and SEQ ID NO:33, and a primer comprising the nucleotide sequence shown as SEQ ID NO:34. Splicing from exon 1 to the cryptic exon of Crlf1 was assessed using primers comprising the nucleotide sequences shown as SEQ ID NO:35 and SEQ ID NO:36, and a primer comprising the nucleotide sequence shown as SEQ ID NO:37. Splicing from the cryptic exon to exon 2 of Crlf1 was assessed using primers comprising the nucleotide sequences shown as SEQ ID NO:38 and SEQ ID NO:39, and a primer comprising the nucleotide sequence shown as SEQ ID NO:40.

[0290] Alternatively spliced ​​TDP-43 mRNA lacking the sequence encoding the PLD domain was assessed using primers containing the nucleotide sequences shown as SEQ ID NO:41 and SEQ ID NO:42, and a primer containing the nucleotide sequence shown as SEQ ID NO:43.

[0291] The sequences of the primers and probes used in each qPCR analysis (normal splicing and cryptic splicing) in this example are provided in Table 3 below. Table 3 [Table 3-1] [Table 3-2]

[0292] Example 4: Generation of mice expressing mutated TDP-43 proteins Although deletion of TDP-43 results in embryonic lethality, embryonic stem cells expressing only mutant ΔNLS TDP-43 or mutant ΔPLD TDP-43 genes from the endogenous TARDBP locus are viable and may differentiate into motor neurons in vitro. This data raises the possibility that embryonic stem cells expressing mutant TDP-43 polypeptides lacking functional structural domains from the endogenous TARDBP locus may be viable and useful for creating animal models of TDP-43 proteinopathy. For example, such embryonic stem cells may be used to generate non-human animals, such as mice, expressing mutant TDP-43 proteins lacking functional structural domains to investigate the role of the TDP-43 structural domain in normal and pathological biological processes.

[0293] To generate embryos or animals expressing mutant TDP-43 proteins 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), in which targeted ES cells containing (i) a conditionally loxP-transfected exon 3 (loxP-Ex3-loxP), a null allele (-) of a Cre-mediated deletion of loxP-transfected exon 3, a TARDBP gene containing an NLS knockout mutation (ΔNLS), a deletion of the prion-like domain (ΔPLD), or a wild-type TARDBP gene (WT) at the endogenous TARDBP locus (see FIG. 3A ), and (ii) a wild-type (WT) TARDBP gene or a null allele (-) of a Cre-mediated deletion of loxP-transfected exon 3 at the other TARDBP locus on the homologous chromosome were injected into non-confluent 8-cell Swiss Webster embryos. After fertilization, the survival rate of the embryos was examined and their ability to produce live-born F0 mice was assessed.

[0294] Consistent with previous experiments, functional TDP-43 protein (TDP-43- / - Embryos lacking the TDP-43 protein (TDP-43) were not viable and did not survive beyond the E3.5 stage (Figure 12). Similarly, embryos lacking the TDP-43 protein (TDP-43 ΔNLS / - ) or a TDP-43 protein lacking a functional PLD (TDP-43 ΔPLD / - Embryos expressing only the TARDBP locus survived longer but were not viable (Figure 12). Expression of wild-type TDP-43 protein from one allele of the TARDBP locus resulted in the expression of a TDP-43 protein lacking a functional NLS (TDP-43) from the other allele of the chromosome. ΔNLS / - ) or a TDP-43 protein lacking a functional PLD (TDP-43 ΔPLD / - )-expressing embryos were rescued (Figure 12).

[0295] Live-born F0 mice were successfully produced from 8-cell stage Swiss Webster embryos injected with ES cells containing (i) the wild-type gene (WT), a Cre-mediated deletion of loxP-transfected exon 3, loxP-transfected exon 3 (loxP-Ex3-loxP), NLS knockout mutation (ΔNLS), and prion-like domain deletion (ΔPLD) at the endogenous TARDBP locus (see FIG. 3A ), and (iii) the wild-type (WT) TARDBP gene at the other TARDBP locus on the homologous chromosome.

[0296] Example 4: Phenotypic analysis of mice expressing mutant TDP-43 polypeptides lacking functional structural domains The phenotype of the animals generated in Example 3 was analyzed by assessing the localization, phosphorylation state, and solubility of the TDP-43 polypeptide in spinal cord tissue or motor neurons isolated from the animals. In addition, the denervation or innervation of the muscles of the animals was also determined.

[0297] Cytoplasmic and nuclear fractions of motor neurons derived from the spinal cord of 16-week-old mice were assessed by Western blot analysis with: (1) an antibody that recognizes the N-terminus of the wild-type TDP-43 protein and therefore binds to wild-type TDP-43, ΔNLS TDP-43, and ΔPLD TDP-43; (2) an antibody that recognizes the C-terminus of the wild-type TDP-43 protein and therefore binds to wild-type TDP-43 and ΔNLS TDP-43 but not ΔPLD TDP-4; or (3) an antibody that recognizes TDP-43 in its phosphorylated form.

[0298] As shown in Figures 13A-13C, wild-type and ΔNLS mutant TDP-43 proteins were detected at the expected size of approximately 43 Kd, and the ΔPLD mutant was detected at the expected size of approximately 30 Kd. Similar to ESMN analyzed in Example 2, mutant TDP-43 polypeptides lacking functional NLS or PLD were redistributed from the nucleus to the cytoplasm of spinal cord tissue even in the presence of wild-type TDP-43 protein. Figure 13A. Phosphorylated TDP-43 polypeptides of approximately 43 Kd were detected in the cytoplasm of motor neurons derived from the spinal cord of mice expressing mutant ΔNLS or ΔPLD polypeptides, but not in mice expressing only wild-type TDP-43 polypeptides. Figure 13B. Phosphorylated TDP-43 in the nuclei of motor neurons remained undetectable in all samples examined. Figure 13B. Since the phosphorylation site is at amino acid positions 409 / 410, it is not surprising that phosphorylated TDP-43 polypeptides lacking functional PLD were not detected. Figure 13B. Spinal motor neurons of 16-week-old mice expressing ΔNLS mutant TDP-43 protein containing a functional mutation in the NLS domain showed increased levels of insoluble TDP-43 protein overall. Figure 13C. Mice expressing ΔPLD mutant TDP-43 protein did not appear to have increased TDP-43 protein solubility. The ΔPLD mutant appears to be soluble, as it was not detected in the insoluble fraction. Figure 13C.

[0299] A subset of motor neurons from mice expressing ΔNLS mutant TDP-43 protein containing a functional mutation in the ΔNLS domain or ΔPLD TDP-43 mutant protein lacking a functional PLD displayed extensive cytoplasmic TDP-43 aggregation. Figure 14. Cytoplasmic aggregation was detected less frequently in motor neurons from mice expressing ΔPLD mutant protein compared to mice expressing mutant TDP-43 polypeptide lacking a functional NLS. Figure 14.

[0300] Since denervation is one of the first pathological features that appear in ALS, muscles containing mainly fast (tibialis anterior) or slow (intercostal) twitch fibers were analyzed for denervation. Mislocalization of TDP-43 resulted in partially innervated endplates (*) and denervation of muscles containing mainly fast but no slow twitch fibers (arrows). Figure 15A-15B.

[0301] The data presented herein suggest that the animals described herein may be valuable disease models for ALS. In typical ALS patients, distal fast fatigable (FF) motor units are affected earliest, and neurogenic changes in muscles can be observed before motor neuron loss. Similarly, motor neuron loss in SOD1 G93A mice, the most widely used "ALS" model, is also preceded by denervation of skeletal muscles with early and preferential involvement of FF motor units. In contrast, proximal muscles, such as the intercostal muscles and diaphragm, which are innervated primarily by slow-twitch fibers, are generally spared until the end—and denervation of these muscles is fatal. As the disease progresses, denervation of the intercostal muscles may be expected.

[0302] The materials and methods used to analyze the phenotype of mice expressing both (a) a mutant TDP-43 polypeptide lacking a functional NLS or PLD and (b) a wild-type TDP-43 polypeptide are described below.

[0303] Detection of intracellular localization and phosphorylation of mutant TDP-43 polypeptides

[0304] The subcellular localization of TDP-43 mutants was analyzed using an antibody recognizing the N-terminus of the TDP-43 polypeptide (α-TDP-43N-term) and an antibody recognizing the C-terminal prion-like domain of the TDP-43 polypeptide (α-TDP-43C-term) (Proteintech, Rosemont, IL). Soluble cytoplasmic protein extracts were prepared by incubating whole spinal cord tissues for 10 min on ice 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 passed five times through a 27-gauge syringe. After centrifugation at 4000 rpm for 5 min at 4 °C, the protein supernatant containing the soluble cytoplasmic extract was collected. Insoluble nuclear protein extracts were prepared by resuspending the pellet in an equal volume of RBS-100 buffer (10 mM Tris-HCl, pH 7.4, 2.5 mM MgCl2, 100 mM NaCl, 0.1% NP-40) supplemented with proteases and phosphatases. An equal volume of 2x SDS sample buffer was added to each fraction, and 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 for TDP-43 using α-TDP-43-N-term antibody (Figure 13A), α-TDP-43-C-term antibody (Figure 13A), or α-phospho-TDP-43 antibody (Figure 13B) that detects phosphorylation of TDP-43 at amino acids 409 / 410 (Cosmo Bio USA; Catalog No. CAC-TIP-PTD-M01). Neither the α-TDP-43-C-term nor the α-phospho-TDP-43 antibodies recognize the PLD deletion mutant. Densitometry was performed using ImageJ (Figures 13A and 13B). The ratio of cytoplasmic / nuclear TDP-43 was plotted and statistically analyzed using GraphPad for Prism (Figure 13A, lower panel).

[0305] Fluorescence in situ hybridization (FISH)

[0306] Spinal cords were separated from the vertebral column, immersion fixed in 4% PFA overnight (or 1 h for FUS immunostaining) and washed with 1x PBS. Spinal cord segments were embedded in 4% low melting point agarose (Promega) and serial transverse sections (70 μM) were cut using a vibratome (Leica VT 1000S) and suspended. Suspended spinal cord sections were blocked with 5% normal donkey serum diluted in Tris-buffered saline (pH 7.4) with 0.2% TritonX-100 (TBS-T) and incubated overnight at room temperature in primary antisera diluted in TBS-T with 5% normal donkey serum. 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 with species-specific secondary antibodies conjugated to Alexa 488, 555, 647 (1:1,000; Life Technologies, Carlsbad, CA, USA), Cy3 or Cy5 (dilution 1:500; Jackson Immunoresearch Labs, West Grove, PA, USA) for 4 h at room temperature. After washing with TBS-T, stained tissue sections were mounted on microscope slides in Floromount G (Southern Biotech, Birmingham, AL, USA) and imaged using an LSM510 confocal microscope at 40x magnification and 1.5 zoom (Figure 14).

[0307] Solubility of mutant TDP-43 polypeptides

[0308] This protocol was adapted from Jo et al. (2014), Nature Communications, 5:3496. 500ul of ice-cold solubility buffer (0.1M MES (pH 7), 1mM EDTA, 0.5mM MgSO4, 1M sucrose) containing 50mM N-ethylmaleimide (NEM), 1mM NaF, 1mM Na3VO4, 1mM PMSF, and 10ug / mL each of aprotinin, leupeptin, and pepstatin. Cells from spinal cord tissue from 16-week-old mice were lysed by passing 3-5 times through a 21-gauge needle, followed by 3-5 times through a 23-gauge needle. Equal amounts of homogenate were then collected from each sample and centrifuged at 50,000×g for 20 minutes at 4°C, and the remainder was stored at -80°C. The supernatant was removed and each pellet was resuspended in 700 ul 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 sarkosine (Sarkosyl) and protease inhibitors (1 mM PMSF, 50 mM NEM, and 10 ug / mL each of aprotinin, leupeptin, and pepstatin), vortexed for 1 min at RT, and incubated overnight at 4° C. with end-over-end rotation. Samples were then centrifuged at 200,000×g at 12° C. for 30 min, and the supernatant was collected as the Sarkosyl-soluble fraction. The pellet was resuspended in 700ul of RAB buffer and passed through a 26-gauge needle 3-5 times to completely disperse the pellet, creating the sarkosyl-insoluble fraction. Equal amounts of the sarkosyl-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 amounts of each fraction were then loaded onto a 14% SDS gel and electrophoresed at 225V for 50 minutes, followed by Western blotting for TDP-43. Densitometry was performed using ImageJ (Figure 13C). The ratios of soluble:insoluble TDP-43 were plotted and statistically analyzed using GraphPad for Prism (Figure 13C).

[0309] Denervation test

[0310] For muscle analysis, tibialis anterior (TA) and intercostal muscles were dissected, postfixed for 2 h by immersion in 4% PFA, and washed with 1× phosphate-buffered saline, pH 7.4 (PBS). Muscles were then equilibrated in a sucrose gradient (10%-20%-30% sucrose in 0.1 M phosphate buffer, pH 7.4), embedded in OCT compound (Sakura, Torrance, CA), and frozen at −20°C. Serial sections (30 μm thick) were cut using a freezing microtome (Leica CM 3050S). Muscle cryosections (30 μm) were stained with an antibody against synaptophysin (invitrogen) to identify presynaptic terminals and with Alexa488-conjugated α-BTX (invitrogen) to detect postsynaptic acetylcholine receptors. Images were acquired using a Zeiss Pascal LSM510 confocal microscope with ×10 and ×40 objectives. The NMJ innervation percentage (%) was determined by dividing the total number of overlapping areas between VAChT and α-BTX signals (total number of innervated endplates) by the number of areas α-BTX signal (total number of endplates). (Item 1) A non-human animal cell comprising a mutated TARDBP gene encoding a mutant TDP-43 polypeptide, The mutant TDP-43 polypeptide lacks a functional structural domain, including a nuclear localization signal (NLS), an RNA recognition motif 1 (RRM1), an RNA recognition motif 2 (RRM2), a putative nuclear export signal (E), a prion-like domain (PLD), or a combination thereof, found in a wild-type TDP-43 polypeptide; and The non-human animal cell expresses the mutant TDP-43 polypeptide, Optionally, the wild-type TDP-43 polypeptide comprises the sequence shown as SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5. (Item 2) The non-human animal cell according to item 1, wherein the non-human animal cell is an embryonic stem (ES) cell, an embryoid body, or an embryonic stem cell-derived motor neuron (ESMN). (Item 3) 3. The non-human animal cell according to item 1 or 2, wherein the mutated TARDBP gene is a mutated TARDBP gene of the non-human animal. (Item 4) 3. The non-human animal cell according to any one of items 1 to 2, wherein the mutated TARDBP gene is a mutated human TARDBP gene. (Item 5) The mutant TDP-43 polypeptide is: (a) an amino acid point mutation in the NLS; (b) an amino acid point mutation in the RRM1; (c) an amino acid point mutation in the RRM2; (d) a deletion of at least a portion of the nuclear export signal; and (e) a deletion of at least a portion of the prion-like domain. (Item 6) (a) the point mutations of amino acids in the NLS comprise K82A K83A, R84A, K95A, K97A, K98A, or combinations thereof; (b) the point mutation in RRM1 comprises F147L and / or F149L; (c) the point mutation in RRM2 comprises F194L and / or F229L; (d) the deletion of at least a portion of the nuclear export signal deletion comprises a deletion of amino acids at and between positions 239 and 250 of a wild-type TDP-43 polypeptide; and (e) The non-human animal cell according to item 5, wherein the deletion of at least a portion of the prion-like domain comprises a deletion of amino acids at and between positions 274 and 414 of the wild-type TDP-43 polypeptide. (Item 7) 2. The non-human animal cell of any one of the preceding items, wherein the mutant TDP-43 polypeptide comprises K82A K83A, R84A, K95A, K97A, and K98A. (Item 8) 2. The non-human animal cell of any one of the preceding items, wherein the mutant TDP-43 polypeptide lacks the prion-like domain at amino acids 274 to 414 of the wild-type polypeptide. (Item 9) 2. The non-human animal cell according to any one of the preceding items, wherein the mutant TDP-43 polypeptide comprises F147L and F149L. (Item 10) 2. The non-human animal cell according to any one of the preceding items, wherein the mutant TDP-43 polypeptide comprises F194L and F229L. (Item 11) 2. The non-human animal cell of any one of the preceding items, wherein the mutant TDP-43 polypeptide lacks the nuclear export signal at amino acids 239 to 250. (Item 12) 2. The non-human animal cell according to any one of the preceding items, wherein said mutated TARDBP gene encoding a mutant TDP-43 polypeptide replaces the endogenous TARDBP gene at the endogenous TARDBP locus. (Item 13) 14. The non-human animal cell according to item 13, wherein the non-human animal cell is heterozygous for the mutated TARDBP gene encoding a mutant TDP-43 polypeptide. (Item 14) 14. The non-human animal cell according to item 13, wherein the non-human animal cell is homozygous for the mutated TARDBP gene encoding a mutant TDP-43 polypeptide. (Item 15) 14. The non-human animal cell according to any one of items 1 to 13, wherein the non-human animal cell further comprises a TARDBP gene comprising a knockout mutation. (Item 16) 17. The non-human animal cell of item 16, wherein the knockout mutation comprises a conditional knockout mutation. (Item 17) 17. The non-human animal cell of claim 15 or 16, wherein the knockout mutation comprises a site-specific recombination recognition sequence. (Item 18) 18. The non-human animal cell according to any one of items 15 to 17, wherein the knockout mutation comprises a loxp sequence. (Item 19) 19. The non-human animal cell of item 18, wherein the loxp sequences flank exon 3 of the TARDBP gene that contains a knockout mutation. (Item 20) 17. The non-human animal cell of item 16, wherein the knockout mutation comprises a deletion of the entire coding sequence of the TDP-43 peptide. (Item 21) the non-human animal cell is heterozygous for the engineered TARDBP locus; and (i) replacement of an endogenous TARDBP gene at an endogenous TARDBP locus on one chromosome with said mutated TARDBP gene encoding a mutant TDP-43 polypeptide; and (ii) The non-human animal cell according to any one of items 15 to 20, comprising either the TARDBP gene comprising the knockout mutation at the endogenous TARDBP locus or a wild-type TARDBP gene in the other homologous chromosome. (Item 22) 2. The non-human animal cell of any one of the preceding items, wherein the non-human animal cell does not express a wild-type TDP-43 polypeptide. (Item 23) 22. The non-human animal cell according to any one of items 1 to 21, wherein the non-human animal cell expresses a wild-type TDP-43 polypeptide. (Item 24) (i) an mRNA transcript level of the mutated TARDBP gene that is comparable to the mRNA transcript level of a wild-type TARDBP gene in a control cell; (ii) an increase in the level of the mutant TDP-43 polypeptide relative to the level of a wild-type TDP-43 polypeptide in a control cell; (iii) a mutant TDP-43 polypeptide that is found, for example, at higher concentrations in the cytoplasm than in the nucleus of motor neurons; (iv) a mutant TDP-43 polypeptide that has increased insolubility compared to a wild-type TDP-43 polypeptide; (v) a cytoplasmic aggregate comprising the mutant TDP-43 polypeptide; (vi) increased splicing of cryptic exons, and / or (vii) a reduced level of an alternatively spliced ​​TDP-43 form. (Item 25) (i) a conditional knockout mutation of the TARDBP gene at the endogenous TARDBP locus on one chromosome; (ii) a deletion of the entire TARDBP coding sequence at the endogenous TARDBP locus in the other homologous chromosome. (Item 26) 2. The non-human animal cell of any one of the preceding items, wherein the cell is an embryonic stem (ES) cell, a primitive ectodermal cell, or a motor neuron derived from a motor neuron (ESMN). (Item 27) 2. The non-human animal cell of any one of the preceding claims, wherein the non-human animal cell is a rodent cell. (Item 28) 2. The non-human animal cell according to any one of the preceding items, wherein the non-human animal cell is a rat cell. (Item 29) 28. The non-human animal cell according to any one of items 1 to 27, wherein the non-human animal cell is a mouse cell. (Item 30) 2. The non-human animal cell of any one of the preceding items, wherein the non-human animal cell is cultured in vitro. (Item 31) A non-human animal tissue comprising a non-human animal cell according to any one of the preceding items. (Item 32) A composition comprising a non-human animal cell or tissue according to any one of the preceding items. (Item 33) A method for producing a non-human animal or a non-human animal cell that expresses a mutant TDP-43 polypeptide, comprising manipulating the genome of the non-human animal or the non-human animal cell to contain a mutated TARDBP gene encoding the mutant TDP-43 polypeptide, wherein the mutant TDP-43 polypeptide lacks a functional structural domain compared to wild-type TDP-43, and optionally, the wild-type TDP-43 polypeptide comprises the sequence set forth as SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5. (Item 34) 34. The method of claim 33, wherein manipulating comprises replacing an endogenous TARDBP gene with the mutated TARDBP gene encoding the mutant TDP-43 polypeptide. (Item 35) 35. The method of claim 33 or 34, wherein the manipulating further comprises replacing the endogenous TARDBP gene with a TARDBP gene comprising a knockout mutation. (Item 36) 36. The method of claim 35, wherein the knockout mutation comprises a conditional knockout mutation. (Item 37) 37. The method of claim 36, further comprising culturing the cells under conditions that preclude expression of the TARDBP gene comprising a knockout mutation. (Item 38) 1. A method for identifying therapeutic candidates for the treatment of a disease, comprising: (a) contacting a non-human animal cell or tissue according to any one of items 1 to 31 or a composition according to item 32 with a candidate drug; (b) assessing the phenotype and / or TDP-43 biological activity of the non-human cells or tissues; and (c) identifying the candidate agent that restores a phenotype and / or TDP-43 biological activity to the non-human cell or tissue that is comparable to the phenotype and / or TDP-43 biological activity of a control cell or tissue expressing a wild-type TDP-43 polypeptide. (Item 39) 1. A method for assessing a biological function of a TDP-43 structural domain, comprising: (a) engineering embryonic stem (ES) cells to contain a mutated TARDBP gene encoding a mutant TDP-43 polypeptide lacking a functional structural domain selected from the group consisting of a nuclear localization signal (NLS), a first RNA recognition motif (RRM1), a first RNA recognition motif (RRM2), a putative nuclear export signal (E), a prion-like domain (PLD), and combinations thereof; (b) optionally, differentiating the engineered ES cells in vitro and / or obtaining a genetically engineered non-human animal from the engineered ES cells; and (c) evaluating the phenotype and / or TDP-43 biological activity of the genetically engineered ES cells, the primitive ectoderm derived therefrom, the motor neurons derived therefrom, or the non-human animal derived therefrom. (Item 40) 40. The method of claim 38 or 39, wherein the phenotype is assessed by cell culture, fluorescent in situ hybridization, Western blot analysis, or a combination thereof. (Item 41) 41. The method of any one of items 38 to 40, wherein assessing the phenotype comprises measuring the viability of the engineered ES cell, the primitive ectoderm derived therefrom, the motor neuron derived therefrom, or the non-human animal derived therefrom. (Item 42) 42. The method according to any one of items 38 to 41, wherein the evaluating the phenotype comprises determining the location of the mutant TDP-43 polypeptide in a cell. (Item 43) 43. The method according to any one of items 38 to 42, wherein evaluating the biological activity of the mutant TDP-43 polypeptide comprises measuring a splice product of a gene comprising a cryptic exon regulated by TDP-43. (Item 44) 44. The method of claim 43, wherein the genes containing cryptic exons regulated by TDP-43 include Crem, Fyxd2, and Clf1. (Item 45) 45. The method according to any one of items 38 to 44, wherein evaluating the biological activity of the mutant TDP-43 polypeptide comprises measuring a level of alternatively spliced ​​TDP-43. (Item 46) An antisense oligonucleotide comprising a gapmer motif that targets a TDP-43 mRNA sequence that encodes the PLD of the TDP-43 polypeptide and / or includes the untranslated sequence downstream of exon 6 and upstream of exon 7. (Item 47) A siRNA comprising a sequence that encodes the PLD of the TDP-43 polypeptide and / or targets a TDP-43 mRNA sequence including the untranslated sequence downstream of exon 6 and upstream of exon 7. (Item 48) A CRISPR / Cas system comprising a Cas9 protein and at least one gRNA, wherein the gRNA recognizes a sequence at or near a sequence encoding an alternative splice site that results in an alternative mRNA encoding a truncated TDP-43 polypeptide lacking the PLD. (Item 49) A non-human animal comprising the embryonic stem cell according to item 2. (Item 50) A non-human animal comprising a mutated TARDBP gene encoding a mutant TDP-43 polypeptide, The mutant TDP-43 polypeptide lacks a functional structural domain, including a nuclear localization signal (NLS), an RNA recognition motif 1 (RRM1), an RNA recognition motif 2 (RRM2), a putative nuclear export signal (E), a prion-like domain (PLD), or a combination thereof, found in a wild-type TDP-43 polypeptide; and Optionally, the non-human animal, wherein the wild-type TDP-43 polypeptide comprises the sequence shown as SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5. (Item 51) 51. The non-human animal of item 50, wherein the mutated TARDBP gene is a mutated TARDBP gene of the non-human animal. (Item 52) 52. The non-human animal of item 50 or item 51, wherein the mutated TARDBP gene is a mutated human TARDBP gene. (Item 53) The mutant TDP-43 polypeptide is: (a) an amino acid point mutation in the NLS; (b) an amino acid point mutation in the RRM1; (c) an amino acid point mutation in the RRM2; (d) a deletion of at least a portion of the nuclear export signal; and (e) a deletion of at least a portion of the prion-like domain. (Item 54) (a) the point mutations of amino acids in the NLS comprise K82A K83A, R84A, K95A, K97A, K98A, or combinations thereof; (b) the point mutation in RRM1 comprises F147L and / or F149L; (c) the point mutation in RRM2 comprises F194L and / or F229L; (d) the deletion of at least a portion of the nuclear export signal deletion comprises a deletion of amino acids at and between positions 239 and 250 of a wild-type TDP-43 polypeptide; and (e) The non-human animal of item 53, wherein the deletion of at least a portion of the prion-like domain comprises a deletion of amino acids at and between positions 274 and 414 of the wild-type TDP-43 polypeptide. (Item 55) 55. The non-human animal according to any one of items 50 to 54, wherein the mutant TDP-43 polypeptide comprises K82A K83A, R84A, K95A, K97A, and K98A. (Item 56) 56. The non-human animal according to any one of items 50 to 55, wherein the mutant TDP-43 polypeptide lacks the prion-like domain at amino acids 274 to 414 of the wild-type polypeptide. (Item 57) 57. The non-human animal according to any one of items 50 to 56, wherein the mutant TDP-43 polypeptide comprises F147L and F149L. (Item 58) 58. The non-human animal according to any one of items 50 to 57, wherein the mutant TDP-43 polypeptide comprises F194L and F229L. (Item 59) 59. The non-human animal according to any one of items 50 to 58, wherein the mutant TDP-43 polypeptide lacks the nuclear export signal at amino acids 239 to 250. (Item 60) 60. The non-human animal according to any one of items 50 to 59, wherein said mutated TARDBP gene encoding a mutant TDP-43 polypeptide replaces the endogenous TARDBP gene at the endogenous TARDBP locus. (Item 61) 61. The non-human animal of item 60, wherein the non-human animal is heterozygous for the mutated TARDBP gene encoding a mutant TDP-43 polypeptide. (Item 62) 62. The non-human animal of any one of items 50 to 61, wherein the non-human animal further comprises a TARDBP gene comprising a knockout mutation. (Item 63) 63. The non-human animal of item 62, wherein the knockout mutation comprises a conditional knockout mutation. (Item 64) 64. The non-human animal of claim 62 or 63, wherein the knockout mutation comprises a site-specific recombination recognition sequence. (Item 65) 65. The non-human animal according to any one of items 62 to 64, wherein the knockout mutation comprises a loxp sequence. (Item 66) 66. The non-human animal of item 65, wherein the loxp sequences flank exon 3 of the TARDBP gene that contains a knockout mutation. (Item 67) 63. The non-human animal of item 62, wherein the knockout mutation comprises a deletion of the entire coding sequence of the TDP-43 peptide. (Item 68) the non-human animal is heterozygous for the engineered TARDBP locus, and (i) replacement of an endogenous TARDBP gene at the endogenous TARDBP locus on one chromosome with said mutated TARDBP gene encoding a mutant TDP-43 polypeptide; and (ii) The non-human animal according to any one of items 62 to 67, comprising either the TARDBP gene comprising the knockout mutation or a wild-type TARDBP gene at the endogenous TARDBP locus in the other homologous chromosome. (Item 69) 69. The non-human animal according to any one of items 49 to 68, wherein the non-human animal expresses a wild-type TDP-43 polypeptide. (Item 70) (i) an mRNA transcript level of the mutated TARDBP gene that is comparable to the mRNA transcript level of the wild-type TARDBP gene in a control animal; (ii) an increase in the level of the mutant TDP-43 polypeptide relative to the level of a wild-type TDP-43 polypeptide in a control animal; (iii) a mutant TDP-43 polypeptide that is found, for example, at higher concentrations in the cytoplasm than in the nucleus of motor neurons; (iv) a mutant TDP-43 polypeptide that has increased insolubility compared to a wild-type TDP-43 polypeptide; (v) a cytoplasmic aggregate comprising the mutant TDP-43 polypeptide; (vi) increased splicing of cryptic exons; (vii) reduced levels of alternatively spliced ​​forms of TDP-43; (viii) denervation of muscle tissue that is primarily composed of fast twitch muscle, such as the tibialis anterior; and / or (ix) normal innervation of muscle tissue composed mainly of slow-twitch muscles, such as intercostal muscles. (Item 71) A non-human animal comprising: (i) a conditional knockout mutation of the TARDBP gene at the endogenous TARDBP locus on one chromosome; and (ii) a deletion of the entire TARDBP coding sequence at the endogenous TARDBP locus on the other homologous chromosome. (Item 72) 72. The non-human animal according to any one of items 49 to 71, wherein the non-human animal is a rodent. (Item 73) The non-human animal according to any one of Items 49 to 72, wherein the non-human animal is a rat. (Item 74) The non-human animal according to any one of Items 49 to 72, wherein the non-human animal is a mouse. 1. A method for identifying therapeutic candidates for the treatment of a disease, comprising: (a) contacting a non-human animal according to any one of items 49 to 74 with a candidate drug; (b) assessing the phenotype and / or TDP-43 biological activity of the non-human animal; and and (c) identifying the candidate agent that restores the non-human phenotype and / or TDP-43 biological activity. (Item 76) A mutant TDP-43 polypeptide comprising: (a) point mutation of an amino acid in an NLS; (b) an amino acid point mutation in RRM1; (c) an amino acid point mutation in RRM2; (d) deletion of at least a portion of the nuclear export signal; and (e) a deletion of at least a portion of the prion-like domain. (Item 77) (a) the point mutations of amino acids in the NLS comprise K82A K83A, R84A, K95A, K97A, K98A, or combinations thereof; (b) the point mutation in RRM1 comprises F147L and / or F149L; (c) the point mutation in RRM2 comprises F194L and / or F229L; (d) the deletion of at least a portion of the nuclear export signal deletion comprises a deletion of amino acids at and between positions 239 and 250 of a wild-type TDP-43 polypeptide; and (e) The mutant TDP-43 polypeptide of item 76, wherein the deletion of at least a portion of the prion-like domain comprises a deletion of amino acids at and between positions 274 and 414 of a wild-type TDP-43 polypeptide. (Item 78) 78. The mutant TDP-43 polypeptide according to claim 76 or 77, comprising a K82A mutation, a K83A mutation, a R84A mutation, a K95A mutation, a K97A mutation, and / or a K98A mutation. (Item 79) 79. The mutant TDP-43 polypeptide according to any one of items 76 to 78, comprising a deletion of the prion-like domain at amino acids 274 to 414 of the wild-type polypeptide. (Item 80) 80. The mutant TDP-43 polypeptide according to any one of items 76 to 79, wherein the mutant TDP-43 polypeptide comprises an F147L mutation and / or an F149L mutation. (Item 81) 81. The mutant TDP-43 polypeptide according to any one of items 76 to 80, wherein the mutant TDP-43 polypeptide comprises an F194L mutation and / or an F229L mutation. (Item 82) 82. The mutant TDP-43 polypeptide according to any one of items 76 to 81, wherein the mutant TDP-43 polypeptide lacks the nuclear export signal at amino acids 239 to 250. (Item 83) 83. A nucleic acid comprising a nucleic acid sequence encoding the mutant TDP-43 polypeptide according to any one of items 76 to 82. (Item 84) 84. The nucleic acid of item 83, further comprising, from 5' to 3': a 5' homology arm, the nucleic acid sequence encoding the mutant TDP-43 polypeptide, and a 3' homology arm, wherein the nucleic acid undergoes homologous recombination in a rodent cell. (Item 85) 85. The nucleic acid of item 84, wherein the 5' and 3' homology arms are homologous to rat sequences such that the nucleic acid undergoes homologous recombination at the endogenous rat TARDBP locus and the nucleic acid sequence encoding the mutant TDP-43 polypeptide replaces the endogenous TARDBP coding sequence. (Item 86) 85. The nucleic acid of item 84, wherein the 5' and 3' homology arms are homologous to mouse sequences such that the nucleic acid undergoes homologous recombination at the endogenous mouse TARDBP locus and the nucleic acid sequence encoding the mutant TDP-43 polypeptide replaces the endogenous TARDBP coding sequence. (Item 87) TDP-43 encoding a TDP-43 polypeptide containing the PLD, while preserving alternative TDP-43 mRNA encoding a truncated TDP-43 lacking the PLD in cells A method for selectively reducing mRNA, comprising: (i) an antisense oligonucleotide comprising a gapmer motif that targets a TDP-43 mRNA sequence that encodes the PLD of the TDP-43 polypeptide and / or includes the untranslated sequence downstream of exon 6 and upstream of exon 7; (ii) an siRNA comprising a sequence encoding the PLD of the TDP-43 polypeptide and / or targeting the TDP-43 mRNA sequence including the untranslated sequence downstream of exon 6 and upstream of exon 7; and / or (iii) introducing into the cell a CRISPR / Cas system comprising a Cas9 protein and at least one gRNA, wherein the gRNA recognizes a sequence at or near a sequence encoding an alternative splice site that results in an alternative mRNA encoding a truncated TDP-43 polypeptide lacking the PLD. (Item 88) The subject matter first described, disclosed or illustrated in this patent application encompasses any embodiment or any applicable category of items, e.g., products, processes, or uses, characterized by the following: non-human animals expressing mutant TDP-43, methods of making non-human animals, nucleic acids for use in said methods of making non-human animals, cells for use in said methods of making non-human animals, uses of said non-human animals so made, and cells derived from said non-human animals and / or cells expressing mutant TDP-43 polypeptides, mutant TDP-43 polypeptides and nucleic acids encoding mutant polypeptides, antisense oligonucleotides, or siRNAs, CRISPR / Cas systems. The present invention provides, for example, the following items. (Item A1) A non-human animal cell comprising a mutated TARDBP gene encoding a mutant TDP-43 polypeptide, The mutant TDP-43 polypeptide lacks a functional structural domain, including a nuclear localization signal (NLS), an RNA recognition motif 1 (RRM1), an RNA recognition motif 2 (RRM2), a putative nuclear export signal (E), a prion-like domain (PLD), or a combination thereof, found in a wild-type TDP-43 polypeptide; and The non-human animal cell expresses the mutant TDP-43 polypeptide, Optionally, the wild-type TDP-43 polypeptide comprises the sequence shown as SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5. (Item A2) The non-human animal cell according to item A1, wherein the non-human animal cell is an embryonic stem (ES) cell, an embryoid body, or an embryonic stem cell-derived motor neuron (ESMN). (Item A3) The non-human animal cell according to item A1 or item A2, wherein the mutated TARDBP gene is a mutated TARDBP gene of the non-human animal. (Item A4) The non-human animal cell according to any one of items A1 to A2, wherein the mutated TARDBP gene is a mutated human TARDBP gene. (Item A5) The mutant TDP-43 polypeptide is: (a) an amino acid point mutation in the NLS; (b) an amino acid point mutation in the RRM1; (c) an amino acid point mutation in the RRM2; (d) a deletion of at least a portion of the nuclear export signal; and (e) a deletion of at least a portion of the prion-like domain. (Item A6) (a) the point mutations of amino acids in the NLS comprise K82A K83A, R84A, K95A, K97A, K98A, or combinations thereof; (b) the point mutation in RRM1 comprises F147L and / or F149L; (c) the point mutation in RRM2 comprises F194L and / or F229L; (d) the deletion of at least a portion of the nuclear export signal deletion comprises a deletion of amino acids at and between positions 239 and 250 of a wild-type TDP-43 polypeptide; and (e) The non-human animal cell according to item A5, wherein the deletion of at least a portion of the prion-like domain comprises a deletion of amino acids at and between positions 274 and 414 of the wild-type TDP-43 polypeptide. (Item A7) 2. The non-human animal cell of any one of the preceding paragraphs A, wherein the mutant TDP-43 polypeptide comprises K82A K83A, R84A, K95A, K97A, and K98A. (Item A8) 2. The non-human animal cell of any one of the preceding paragraphs A, wherein the mutant TDP-43 polypeptide lacks the prion-like domain at amino acids 274 to 414 of the wild-type polypeptide. (Item A9) The non-human animal cell according to any one of the preceding paragraphs A, wherein the mutant TDP-43 polypeptide comprises F147L and F149L. (Item A10) The non-human animal cell according to any one of the preceding paragraphs A, wherein the mutant TDP-43 polypeptide comprises F194L and F229L. (Item A11) 2. The non-human animal cell of any one of the preceding paragraphs A, wherein the mutant TDP-43 polypeptide lacks the nuclear export signal at amino acids 239 to 250. (Item A12) The non-human animal cell according to any one of the preceding paragraphs A, wherein said mutated TARDBP gene encoding a mutant TDP-43 polypeptide replaces the endogenous TARDBP gene at the endogenous TARDBP locus. (Item A13) The non-human animal cell according to item A13, wherein the non-human animal cell is heterozygous for the mutated TARDBP gene encoding a mutant TDP-43 polypeptide. (Item A14) The non-human animal cell according to item A13, wherein the non-human animal cell is homozygous for the mutated TARDBP gene encoding a mutant TDP-43 polypeptide. (Item A15) The non-human animal cell according to any one of items A1 to A13, wherein the non-human animal cell further comprises a TARDBP gene comprising a knockout mutation. (Item A16) The non-human animal cell according to item A16, wherein the knockout mutation comprises a conditional knockout mutation. (Item A17) The non-human animal cell according to item A15 or A16, wherein the knockout mutation comprises a site-specific recombination recognition sequence. (Item A18) The non-human animal cell according to any one of items A15 to A17, wherein the knockout mutation comprises a loxp sequence. (Item A19) The non-human animal cell according to item A18, wherein the loxp sequences flank exon 3 of the TARDBP gene comprising a knockout mutation. (Item A20) The non-human animal cell according to item A16, wherein the knockout mutation comprises a deletion of the entire coding sequence of the TDP-43 peptide. (Item A21) the non-human animal cell is heterozygous for the engineered TARDBP locus; and (i) replacement of an endogenous TARDBP gene at an endogenous TARDBP locus on one chromosome with said mutated TARDBP gene encoding a mutant TDP-43 polypeptide; and (ii) The non-human animal cell according to any one of items A15 to A20, comprising either the TARDBP gene comprising the knockout mutation at the endogenous TARDBP locus or a wild-type TARDBP gene in the other homologous chromosome. (Item A22) 2. The non-human animal cell of any one of the preceding paragraphs A, wherein the non-human animal cell does not express a wild-type TDP-43 polypeptide. (Item A23) The non-human animal cell according to any one of items A1 to A21, wherein the non-human animal cell expresses a wild-type TDP-43 polypeptide. (Item A24) (i) an mRNA transcript level of the mutated TARDBP gene that is comparable to the mRNA transcript level of a wild-type TARDBP gene in a control cell; (ii) an increase in the level of the mutant TDP-43 polypeptide relative to the level of a wild-type TDP-43 polypeptide in a control cell; (iii) a mutant TDP-43 polypeptide that is found, for example, at higher concentrations in the cytoplasm than in the nucleus of motor neurons; (iv) a mutant TDP-43 polypeptide that has increased insolubility compared to a wild-type TDP-43 polypeptide; (v) a cytoplasmic aggregate comprising the mutant TDP-43 polypeptide; (vi) increased splicing of cryptic exons, and / or (vii) a reduced level of an alternatively spliced ​​TDP-43 form. (Item A25) (i) a conditional knockout mutation of the TARDBP gene at the endogenous TARDBP locus on one chromosome; (ii) a deletion of the entire TARDBP coding sequence at the endogenous TARDBP locus in the other homologous chromosome. (Item A26) The non-human animal cell of any one of the preceding paragraphs A, wherein the cell is an embryonic stem (ES) cell, a primitive ectodermal cell, or an embryonic stem cell derived motor neuron (ESMN). (Item A27) 2. The non-human animal cell of any one of the preceding paragraphs A, wherein the non-human animal cell is a rodent cell. (Item A28) 2. The non-human animal cell according to any one of the preceding paragraphs A, wherein the non-human animal cell is a rat cell. (Item A29) The non-human animal cell according to any one of items A1 to A27, wherein the non-human animal cell is a mouse cell. (Item A30) 2. The non-human animal cell of any one of the preceding paragraphs A, wherein the non-human animal cell is cultured in vitro. (Item A31) A non-human animal tissue comprising a non-human animal cell according to any one of the preceding paragraphs A. (Item A32) A composition comprising a non-human animal cell or tissue according to any one of the preceding paragraphs A. (Item A33) A method for producing a non-human animal or a non-human animal cell that expresses a mutant TDP-43 polypeptide, comprising manipulating the genome of the non-human animal or the non-human animal cell to contain a mutated TARDBP gene encoding the mutant TDP-43 polypeptide, wherein the mutant TDP-43 polypeptide lacks a functional structural domain compared to wild-type TDP-43, and optionally, the wild-type TDP-43 polypeptide comprises the sequence set forth as SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5. (Item A34) The method according to item A33, wherein manipulating comprises replacing an endogenous TARDBP gene with the mutated TARDBP gene encoding the mutant TDP-43 polypeptide. (Item A35) The method according to item A33 or item A34, wherein the manipulating further comprises replacing the endogenous TARDBP gene with a TARDBP gene comprising a knockout mutation. (Item A36) The method of claim A35, wherein the knockout mutation comprises a conditional knockout mutation. (Item A37) The method according to item A36, further comprising culturing the cells under conditions that preclude expression of the TARDBP gene comprising a knockout mutation. (Item A38) 1. A method for identifying therapeutic candidates for the treatment of a disease, comprising: (a) contacting a non-human animal cell or tissue according to any one of items A1 to A31 or a composition according to item A32 with a candidate drug; (b) assessing the phenotype and / or TDP-43 biological activity of the non-human cells or tissues; and (c) identifying the candidate agent that restores a phenotype and / or TDP-43 biological activity to the non-human cell or tissue that is comparable to the phenotype and / or TDP-43 biological activity of a control cell or tissue expressing a wild-type TDP-43 polypeptide. (Item A39) 1. A method for assessing a biological function of a TDP-43 structural domain, comprising: (a) engineering embryonic stem (ES) cells to contain a mutated TARDBP gene encoding a mutant TDP-43 polypeptide lacking a functional structural domain selected from the group consisting of a nuclear localization signal (NLS), a first RNA recognition motif (RRM1), a first RNA recognition motif (RRM2), a putative nuclear export signal (E), a prion-like domain (PLD), and combinations thereof; (b) optionally, differentiating the engineered ES cells in vitro and / or obtaining a genetically engineered non-human animal from the engineered ES cells; and (c) evaluating the phenotype and / or TDP-43 biological activity of the genetically engineered ES cells, the primitive ectoderm derived therefrom, the motor neurons derived therefrom, or the non-human animal derived therefrom. (Item A40) The method according to item A38 or item A39, wherein the phenotype is assessed by cell culture, fluorescent in situ hybridization, Western blot analysis, or a combination thereof. (Item A41) The method according to any one of paragraphs A38 to A40, wherein assessing the phenotype comprises measuring the viability of the engineered ES cell, the primitive ectoderm derived therefrom, the motor neuron derived therefrom, or the non-human animal derived therefrom. (Item A42) The method according to any one of items A38 to A41, wherein the evaluating the phenotype comprises determining the location of the mutant TDP-43 polypeptide in a cell. (Item A43) The method according to any one of items A38 to A42, wherein evaluating the biological activity of the mutant TDP-43 polypeptide comprises measuring a splice product of a gene comprising a cryptic exon regulated by TDP-43. (Item A44) The method according to item A43, wherein the genes containing cryptic exons regulated by TDP-43 include Crem, Fyxd2, and Clf1. (Item A45) The method according to any one of items A38 to A44, wherein evaluating the biological activity of the mutant TDP-43 polypeptide comprises measuring the level of alternatively spliced ​​TDP-43. (Item A46) An antisense oligonucleotide comprising a gapmer motif that targets a TDP-43 mRNA sequence that encodes the PLD of the TDP-43 polypeptide and / or includes the untranslated sequence downstream of exon 6 and upstream of exon 7. (Item A47) A siRNA comprising a sequence that encodes the PLD of the TDP-43 polypeptide and / or targets a TDP-43 mRNA sequence including the untranslated sequence downstream of exon 6 and upstream of exon 7. (Item A48) A CRISPR / Cas system comprising a Cas9 protein and at least one gRNA, wherein the gRNA recognizes a sequence at or near a sequence encoding an alternative splice site that results in an alternative mRNA encoding a truncated TDP-43 polypeptide lacking the PLD. (Item A49) A non-human animal comprising the embryonic stem cell according to item A2. (Item A50) A non-human animal comprising a mutated TARDBP gene encoding a mutant TDP-43 polypeptide, The mutant TDP-43 polypeptide lacks a functional structural domain, including a nuclear localization signal (NLS), an RNA recognition motif 1 (RRM1), an RNA recognition motif 2 (RRM2), a putative nuclear export signal (E), a prion-like domain (PLD), or a combination thereof, found in a wild-type TDP-43 polypeptide; and Optionally, the non-human animal, wherein the wild-type TDP-43 polypeptide comprises the sequence shown as SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5. (Item A51) The non-human animal according to item A50, wherein said mutated TARDBP gene is a mutated TARDBP gene of said non-human animal. (Item A52) The non-human animal according to item A50 or item A51, wherein said mutated TARDBP gene is a mutated human TARDBP gene. (Item A53) The mutant TDP-43 polypeptide is: (a) an amino acid point mutation in the NLS; (b) an amino acid point mutation in the RRM1; (c) an amino acid point mutation in the RRM2; (d) a deletion of at least a portion of the nuclear export signal; and (e) a deletion of at least a portion of the prion-like domain. (Item A54) (a) the point mutations of amino acids in the NLS comprise K82A K83A, R84A, K95A, K97A, K98A, or combinations thereof; (b) the point mutation in RRM1 comprises F147L and / or F149L; (c) the point mutation in RRM2 comprises F194L and / or F229L; (d) the deletion of at least a portion of the nuclear export signal deletion comprises a deletion of amino acids at and between positions 239 and 250 of a wild-type TDP-43 polypeptide; and (e) The non-human animal according to item A53, wherein the deletion of at least a portion of the prion-like domain comprises a deletion of amino acids at and between positions 274 and 414 of the wild-type TDP-43 polypeptide. (Item A55) The non-human animal according to any one of A50 to A54, wherein the mutant TDP-43 polypeptide comprises K82A K83A, R84A, K95A, K97A, and K98A. (A56) The non-human animal according to any one of items A50 to A55, wherein the mutant TDP-43 polypeptide lacks the prion-like domain at amino acids 274 to 414 of the wild-type polypeptide. (Item A57) The non-human animal according to any one of items A50 to A56, wherein the mutant TDP-43 polypeptide comprises F147L and F149L. (Item A58) The non-human animal according to any one of items A50 to A57, wherein the mutant TDP-43 polypeptide comprises F194L and F229L. (Item A59) The non-human animal according to any one of items A50 to A58, wherein the mutant TDP-43 polypeptide lacks the nuclear export signal at amino acids 239 to 250. (Item A60) The non-human animal according to any one of items A50 to A59, wherein said mutated TARDBP gene encoding a mutant TDP-43 polypeptide replaces the endogenous TARDBP gene at the endogenous TARDBP locus. (Item A61) The non-human animal according to item A60, wherein the non-human animal is heterozygous for the mutated TARDBP gene encoding a mutant TDP-43 polypeptide. (Item A62) The non-human animal according to any one of items A50 to A61, wherein the non-human animal further comprises a TARDBP gene comprising a knockout mutation. (Item A63) The non-human animal of item A62, wherein the knockout mutation comprises a conditional knockout mutation. (Item A64) The non-human animal according to item A62 or item A63, wherein the knockout mutation comprises a site-specific recombination recognition sequence. (Item A65) The non-human animal according to any one of items A62 to A64, wherein the knockout mutation comprises a loxp sequence. (Item A66) The non-human animal of item A65, wherein the loxp sequences flank exon 3 of the TARDBP gene containing a knockout mutation. (Item A67) The non-human animal of item A62, wherein the knockout mutation comprises a deletion of the entire coding sequence of the TDP-43 peptide. (Item A68) the non-human animal is heterozygous for the engineered TARDBP locus, and (i) replacement of an endogenous TARDBP gene at the endogenous TARDBP locus on one chromosome with said mutated TARDBP gene encoding a mutant TDP-43 polypeptide; and (ii) The non-human animal according to any one of items A62 to A67, comprising either the TARDBP gene comprising the knockout mutation or a wild-type TARDBP gene at the endogenous TARDBP locus in the other homologous chromosome. (Item A69) The non-human animal according to any one of items A49 to A68, wherein the non-human animal expresses a wild-type TDP-43 polypeptide. (Item A70) (i) an mRNA transcript level of the mutated TARDBP gene that is comparable to the mRNA transcript level of the wild-type TARDBP gene in a control animal; (ii) an increase in the level of the mutant TDP-43 polypeptide relative to the level of a wild-type TDP-43 polypeptide in a control animal; (iii) a mutant TDP-43 polypeptide that is found, for example, at higher concentrations in the cytoplasm than in the nucleus of motor neurons; (iv) a mutant TDP-43 polypeptide that has increased insolubility compared to a wild-type TDP-43 polypeptide; (v) a cytoplasmic aggregate comprising the mutant TDP-43 polypeptide; (vi) increased splicing of cryptic exons; (vii) reduced levels of alternatively spliced ​​forms of TDP-43; (viii) denervation of muscle tissue that is primarily composed of fast twitch muscle, such as the tibialis anterior; and / or (ix) normal innervation of muscle tissue that is primarily composed of slow-twitch muscles, such as intercostal muscles. (Item A71) A non-human animal comprising: (i) a conditional knockout mutation of the TARDBP gene at the endogenous TARDBP locus on one chromosome; and (ii) a deletion of the entire TARDBP coding sequence at the endogenous TARDBP locus on the other homologous chromosome. (Item A72) The non-human animal according to any one of items A49 to A71, wherein the non-human animal is a rodent. (Item A73) The non-human animal according to any one of items A49 to A72, wherein the non-human animal is a rat. (Item A74) The non-human animal according to any one of items A49 to A72, wherein the non-human animal is a mouse. (Item A75) 1. A method for identifying therapeutic candidates for the treatment of a disease, comprising: (a) contacting a non-human animal according to any one of paragraphs A49 to A74 with a candidate drug; (b) assessing the phenotype and / or TDP-43 biological activity of the non-human animal; and and (c) identifying the candidate agent that restores the non-human phenotype and / or TDP-43 biological activity. (Item A76) A mutant TDP-43 polypeptide comprising: (a) point mutation of an amino acid in an NLS; (b) an amino acid point mutation in RRM1; (c) an amino acid point mutation in RRM2; (d) deletion of at least a portion of the nuclear export signal; and (e) a deletion of at least a portion of the prion-like domain. (Item A77) (a) the point mutations of amino acids in the NLS comprise K82A K83A, R84A, K95A, K97A, K98A, or combinations thereof; (b) the point mutation in RRM1 comprises F147L and / or F149L; (c) the point mutation in RRM2 comprises F194L and / or F229L; (d) the deletion of at least a portion of the nuclear export signal deletion comprises a deletion of amino acids at and between positions 239 and 250 of a wild-type TDP-43 polypeptide; and (e) The mutant TDP-43 polypeptide according to item A76, wherein the deletion of at least a portion of the prion-like domain comprises a deletion of amino acids at and between positions 274 and 414 of the wild-type TDP-43 polypeptide. (Item A78) A mutated TDP-43 polypeptide according to item A76 or item A77, comprising a K82A mutation, a K83A mutation, a R84A mutation, a K95A mutation, a K97A mutation, and / or a K98A mutation. (Item A79) The mutant TDP-43 polypeptide according to any one of items A76 to A78, comprising a deletion of the prion-like domain at amino acids 274 to 414 of the wild-type polypeptide. (Item A80) The mutant TDP-43 polypeptide according to any one of items A76 to A79, wherein the mutant TDP-43 polypeptide comprises a F147L mutation and / or a F149L mutation. (Item A81) The mutant TDP-43 polypeptide according to any one of items A76 to A80, wherein the mutant TDP-43 polypeptide comprises a F194L mutation and / or a F229L mutation. (Item A82) The mutant TDP-43 polypeptide according to any one of items A76 to A81, wherein the mutant TDP-43 polypeptide lacks the nuclear export signal at amino acids 239 to 250. (Item A83) A nucleic acid comprising a nucleic acid sequence encoding the mutant TDP-43 polypeptide according to any one of items A76 to A82. (Item A84) The nucleic acid according to item A83, further comprising, from 5' to 3': a 5' homology arm, the nucleic acid sequence encoding the mutant TDP-43 polypeptide, and a 3' homology arm, wherein the nucleic acid undergoes homologous recombination in a rodent cell. (Item A85) The nucleic acid of item A84, wherein the 5' and 3' homology arms are homologous to rat sequences such that the nucleic acid undergoes homologous recombination at the endogenous rat TARDBP locus and the nucleic acid sequence encoding the mutant TDP-43 polypeptide replaces the endogenous TARDBP coding sequence. (Item A86) The nucleic acid of item A84, wherein the 5' and 3' homology arms are homologous to mouse sequences such that the nucleic acid undergoes homologous recombination at the endogenous mouse TARDBP locus and the nucleic acid sequence encoding the mutant TDP-43 polypeptide replaces the endogenous TARDBP coding sequence. (Item A87) TDP-43 encoding a TDP-43 polypeptide containing the PLD, while preserving alternative TDP-43 mRNA encoding a truncated TDP-43 lacking the PLD in cells A method for selectively reducing mRNA, comprising: (i) an antisense oligonucleotide comprising a gapmer motif that targets a TDP-43 mRNA sequence that encodes the PLD of the TDP-43 polypeptide and / or includes the untranslated sequence downstream of exon 6 and upstream of exon 7; (ii) an siRNA comprising a sequence encoding the PLD of the TDP-43 polypeptide and / or targeting the TDP-43 mRNA sequence including the untranslated sequence downstream of exon 6 and upstream of exon 7; and / or (iii) introducing into the cell a CRISPR / Cas system comprising a Cas9 protein and at least one gRNA, wherein the gRNA recognizes a sequence at or near a sequence encoding an alternative splice site that results in an alternative mRNA encoding a truncated TDP-43 polypeptide lacking the PLD. (Item A88) The subject matter first described, disclosed or illustrated in this patent application encompasses any embodiment or any applicable category of items, e.g., products, processes, or uses, characterized by the following: non-human animals expressing mutant TDP-43, methods of making non-human animals, nucleic acids for use in said methods of making non-human animals, cells for use in said methods of making non-human animals, uses of said non-human animals so made, and cells derived from said non-human animals and / or cells expressing mutant TDP-43 polypeptides, mutant TDP-43 polypeptides and nucleic acids encoding mutant polypeptides, antisense oligonucleotides, or siRNAs, CRISPR / Cas systems.

Claims

[Claim 1] The invention described in the specification.

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