Modeling of TDP-43 proteinopathy

Abstract

Described herein is the discovery that neither the nuclear localization signal (NLS) nor the prion-like domain (PLD) of TDP-43 is required for embryonic stem cell culture and differentiation into motor neurons in vitro. The ability of ES cells to express these TDP-43 mutant forms and differentiate into motor neurons that exhibit ALS-like phenotypes (whereby the TDP-43 mutant forms redistribute to the cytoplasm, aggregate, and fail to regulate splicing of cryptic exons) allows these cells to serve as a model of TDP-43 proteinopathy to examine candidate therapeutic agents that may resolve TDP-43 proteinopathy. Furthermore, these ES cells can be used to successfully generate non-human animals, such as mice, that also exhibit characteristic symptoms of ALS and can be used to examine candidate agents useful for treating TDP-43 proteinopathy.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 867,785, filed Jun. 27, 2019, the disclosure of which is incorporated herein by reference in its entirety under 35 U.S.C. § 119(e)(3).

[0002] Reference to a Sequence Listing Submitted as a Text File via EFS - Web The sequence listing described in the file 10312WO01_ST25.txt is 35 kilobytes and was created on Jun. 25, 2020, and is incorporated herein by reference.

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

Background Art

[0004] Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that affects motor neurons and ultimately causes death as a result of quadriplegia and diaphragmatic muscle impairment. An almost universal pathological finding in post - mortem examinations of ALS patient tissues 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 predominantly 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 important 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 not clear.

Summary of the Invention

Means for Solving the Problems

[0007] Provided herein are embryonic stem (ES) cells, tissues cultured therefrom (e.g., primitive ectoderm, embryoid bodies, motor neurons), and non-human animals derived therefrom that express a mutant TDP-43 polypeptide lacking a functional structural domain and may exhibit a phenotype such as ALS. Compositions and methods for making and using the same are also provided. A mutated TARDBP gene encoding a mutant TDP-43 polypeptide lacking a functional structural domain and a mutant TDP-43 polypeptide lacking a functional structural domain are also provided. Also provided are exemplary therapeutic oligonucleotides, such as 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, ES 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 apart from one or more mutations (e.g., point mutations, substitutions, replacements, insertions, deletions, etc.). In some embodiments, the wild-type TARDBP gene comprises the sequence shown in SEQ ID NO: 2 (including its degenerate variants), SEQ ID NO: 4 (including its degenerate variants), or SEQ ID NO: 6 (including its degenerate variants), which each encode a wild-type TDP-43 polypeptide comprising the amino acid sequence shown 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 in a 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) is adjacent to a coding exon, e.g., exon 3. In some embodiments, the TARDBP gene comprising a knockout mutation comprises a loxp sequence adjacent to 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 comprises (i) a substitution by a mutated TARDBP gene encoding a mutant TDP-43 polypeptide of the endogenous TARDBP gene at the endogenous TARDBP locus and (ii) 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 comprising a conditional knockout mutation at the endogenous TRADBP locus and a TARDBP gene comprising 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 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 the wild-type TDP-43 polypeptide.

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

[0015] In some embodiments, the non-human animal or non-human animal cell according to 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 control cells, and comprises an increased level of mutant TDP-43 polypeptide compared to the level of wild-type TDP-43 polypeptide in control cells, for example, comprising a higher concentration of mutant TDP-43 polypeptide found in the cytoplasm rather than the nucleus of motor neurons, and comprising a mutant TDP-43 polypeptide with increased insolubility compared to wild-type TDP-43 polypeptide cytoplasmic aggregates containing the mutant TDP-43 polypeptide, and comprising an increase in the splicing of cryptic exons and / or a decrease in the level of TDP-43 isoforms undergoing alternative splicing. In some embodiments, the non-human animal exhibits denervation of muscle tissue mainly composed of fast muscles such as the anterior tibial muscle, and / or normal innervation of muscle tissue mainly composed of slow muscles such as the intercostal muscles.

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

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

[0018] In some embodiments, the mutant TDP-43 polypeptide lacks a functional structural domain as compared to the 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 a sequence shown as SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5.

[0019] In some embodiments, the mutant 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 TARDBP gene of a non-human animal that includes a mutation, such as a point mutation, substitution, insertion, deletion, or a combination thereof. In some embodiments, the TARDBP gene of the non-human animal is shown as SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the mutated TARDBP gene is a human TARDBP gene that includes a mutation, such as a point mutation, substitution, insertion, deletion, or a combination thereof. In some embodiments, the mutated TARDBP. In some embodiments, the human TARDBP gene is shown 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) a point mutation of an amino acid in the NLS, (b) a point mutation of an amino acid in RRM1, (c) a point mutation of an amino acid 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 further comprises a sequence shown as SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, which includes (a) a point mutation of an amino acid in the NLS, (b) a point mutation of an amino acid in RRM1, (c) a point mutation of an amino acid 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 point mutation of an amino acid in the NLS includes K82A K83A, R84A, K95A, K97A, K98A, or a combination thereof; (b) the point mutation in RRM1 includes F147L and / or F149L; (c) the point mutation in RRM2 includes F194L and / or F229L; (d) the deletion of at least a portion of the nuclear export signal includes a deletion of amino acids at and between positions 239 and 250 of the wild-type TDP-43 polypeptide; and (E) the deletion of at least a portion of the prion-like domain includes a deletion of amino acids at and between positions 274 and 414 of the wild-type TDP-43 polypeptide. In some embodiments, the mutant TDP-43 polypeptide includes K82A K83A, R84A, K95A, K97A, and / or K98A compared to the wild-type TDP-43 polypeptide, and optionally, the wild-type TDP-43 polypeptide includes a sequence shown 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 in the amino acids from position 274 to position 414 of the wild-type TDP-43 polypeptide, and optionally, the wild-type TDP-43 polypeptide includes a sequence shown 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 the sequence shown 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 the sequence shown 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 in the amino acids at positions 239 to 250 compared to the wild-type TDP-43 polypeptide, and 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.

[0021] Also provided are the mutant TDP-43 polypeptides described herein and nucleic acid molecules encoding the same. In some embodiments, a 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 the mutant TDP-43 polypeptide, and a 3' homology arm, wherein the nucleic acid undergoes homologous recombination in rodent cells. In some embodiments, the 5' and 3' homology arms are homologous to a rat sequence 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 a mouse sequence 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 is a method for generating the non-human animals and non-human animal cells described herein. In some embodiments, the method comprises engineering the genome of a non-human animal or non-human animal cell to include a mutated TARDBP gene encoding a mutant TDP43 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 a sequence shown as SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5. In some embodiments, engineering comprises replacing the endogenous TARDBP gene with a mutated TARDBP gene encoding a mutant TDP-43 polypeptide as described herein. In some embodiments, engineering further comprises 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 comprises culturing the cells under conditions that eliminate expression of the TARDBP gene comprising the knockout mutation.

[0023] Also described herein is a method of using 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, e.g., methods for identifying a therapeutic candidate for treating a disease and / or methods for assessing the biological function of a TDP-43 structural domain. In some embodiments of identifying a therapeutic candidate, the method comprises (a) contacting a composition comprising a human animal, non-human animal cell, non-human animal, or non-human animal cell or tissue (e.g., in vitro culture) not as described herein with a candidate agent, (b) assessing the phenotype and / or TDP-43 bioactivity of the non-human animal, non-human cell, or tissue, and (c) identifying a candidate agent that restores to the non-human animal, non-human cell, or tissue a phenotype and / or TDP-43 bioactivity comparable to that of a control cell or tissue expressing wild-type TDP-43 polypeptide.

[0024] In some embodiments for evaluating 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) evaluating the phenotype and / or TDP-43 bioactivity 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 phenotype is evaluated by cell culture, fluorescence in situ hybridization, Western blot analysis, or a combination thereof, according to the method of claim 39 or claim 40. In some embodiments, evaluating 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. In some embodiments, evaluating the phenotype comprises determining the cellular location of the mutant TDP-43 polypeptide. In some embodiments, evaluating the bioactivity of the mutant TDP-43 polypeptide comprises measuring the splice products of genes containing cryptic exons regulated by TDP-43. In some embodiments, the genes containing cryptic exons regulated by TDP-43 include Crem, Fyxd2, Clf1. In some embodiments, the bioactivity of the mutant TDP-43 polypeptide comprises measuring the level of TDP-43 that has undergone alternative splicing.

[0025] Also described herein are oligonucleotides (e.g., antisense oligonucleotides, siRNAs, CRISPR / Cas systems, etc.) that may be useful as candidate agents for treating TDP-43 proteinopathy. In some embodiments, the antisense oligonucleotide comprises a gapmer motif that targets the TDP-43 mRNA sequence between the 5' and 3' alternative splice sites. In some embodiments, the antisense oligonucleotide comprises a gapmer motif that targets the TDP-43 mRNA sequence between the 5' and 3' alternative splice sites, wherein the 5' alternative splice site correlates with a TARDBP genomic position 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 with the TARDBP genomic position 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 comprising the sequence targets the TDP-43 mRNA sequence between the 5' and 3' alternative splice sites, wherein the 5' alternative splice site correlates with a TARDBP genomic position 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 with the TARDBP genomic position of chromosome 4:148,617,705. In some CRISPR / Cas system embodiments, the system comprises a Cas9 protein and at least one gRNA, and 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 locus 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 documents contain at least one drawing created in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the United States Patent and Trademark Office upon request and payment of the required fees.

Brief Description of the Drawings

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[0028] Overview

[0029] TDP-43 is a nuclear RNA / DNA-binding protein that functions primarily in the processing and metabolism of RNA, including transcription, splicing, transport, and stability of RNA. The RNA-binding properties of TDP-43 appear to be essential for its autoregulatory activity, which is mediated through binding to the 3’UTR sequence 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 is mislocalized from its normal nuclear location to the cytoplasm, where it aggregates. Aggregated TDP-43 is ubiquitinated, hyperphosphorylated, and truncated. Furthermore, cytoplasmic TDP-43 aggregation is a component of almost all cases of ALS. Becker et al. (2017), Nature, 544:367-371. Postmortem pathology of cytoplasmic TDP-43 aggregates is shown in 97% of ALS cases. The same lesions are seen in approximately 45% of cases of frontotemporal lobar degeneration with ubiquitin inclusions (FTLD-U). TDP-43 was first identified as the major pathological protein in ubiquitin-positive, tau-negative inclusions in FTLD-U, frontotemporal lobar degeneration with motor neuron disease (FTD-MND), and ALS / MND (ALS10), and these disorders are now considered to represent various clinical manifestations of TDP-43 proteinopathy. Gitcho et al. (2009), Acta Neuropath. 118:633-645. TARDBP mutations occur in approximately 3% of patients with familial ALS and in approximately 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 of the functions of TDP-43 may shed light on its role in neuropathologies such as ALS, FLTD-U, and FTD.

[0030] It is clear that TDP-43 is essential for the life of cells and organisms. Depletion of TDP-43 results in embryonic lethality. Therefore, initial models relied on overexpression of TDP-43 or its mutants, or deletion of TDP-43. Various models have been created to evaluate the role of TDP-43 in ALS pathology. It is 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 about 3 to 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 were correlated with the presence of the C-terminal fragment of TDP-43 in the brains and spinal cords of these mutant mice, but cytoplasmic TDP-43 aggregates were not detected. These observations led to suggest, as Wegorzewska et al. did, that the vulnerability of neurons to TDP-43-related neurodegeneration is associated with changes 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 showed neurodegenerative traits including progressive motor dysfunction 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 specific genes into mRNA, interference with mRNA translation, and promotion of nonsense-mediated mRNA decay. Ling et al. (2015), Science, 349:650-655. Since postmortem brain tissues from ALS / FTD patients show impaired suppression of cryptic exon splicing, this study suggests that TDP-43 normally acts to suppress cryptic exon splicing 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. 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, so N-terminus-driven head-to-tail oligomerization of TDP-43 is hypothesized to act to separate the aggregation-prone C-terminal domain (e.g., PLD), and thus prevent the formation of pathological aggregates. Afroz et al. (2017), Nature Communications, 8:45.

[0033] In ALS, one of the earliest pathological features is that axons retract from the neuromuscular junction and muscles become denervated. This denervation continues and results 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 neurofilaments. Motor endplates remain but eventually fragment and disappear. Recently, dose-dependent denervation was shown in mice homozygous for a knock-in TARDBP gene containing disease-related mutations. Ebstein, (2019), Cell Reports, 26:364-373.

[0034] Despite the embryonic lethality of TDP-43 depletion, the inventors show here that embryonic stem (ES) cells expressing a TDP-43 mutant lacking functional structural domains can survive and may differentiate 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) lacking functional structural domains, e.g., lacking a functional NLS, lacking a functional RRM1, lacking a functional RRM2, lacking a functional E, or lacking a functional PLD, and (2) expressed at normal levels from the endogenous transcription promoter and pre-mRNA splicing signals. See, for example, Figures 2 and 9. Using the ES and ESMNs described herein, it has been shown that RRM1 is required for the survival of ES cells and the motor neurons derived therefrom. See Figures 4-5. Further, expression of mutant TDP-43 polypeptides at normal levels from the endogenous locus, (1) lacking a functional NLS or a functional PLD, and (2) recapitulates two features of ALS disease in ESMNs: (i) redistribution of TDP-43 from the nucleus to the cytoplasm, and (ii) accumulation into cytoplasmic inclusions. See Figures 6-8.

[0035] It is surprising that the ΔPLD mutant, i.e., the TDP-43 polypeptide that contains a functional NLS but lacks PLD, aggregates in the cytoplasm. See, e.g., Afroz et al. (2017), supra. In particular, the punctate inclusions formed by the ΔPLD mutant appear to be less abundant and qualitatively different from the inclusions formed by the ΔNLS mutant, i.e., the TDP-43 polypeptide that lacks a functional NLS and contains PLD. Furthermore, the ALS-like phenotypes of ESMN expressing ΔPLD or ΔNLS correlate with a decrease in the suppression of cryptic exon splicing of genes whose splicing events are normally regulated by wild-type TDP-43. FIG. 9. Also shown is the correlation between the expression of the mutated TARDBP gene with ΔPLD or ΔNLS in ESMN and a decrease in alternative splicing events involving the 3' untranslated region intron that gives rise to TDP-43 mRNA that has undergone alternative splicing lacking the PLD domain, or a portion thereof and the sequence encoding the stop codon. FIG. 10; see also 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-related sequences resulting from normal splicing events may be therapeutically useful in the treatment of ALS associated with PLD mutations.

[0036] Mice expressing the wild-type TARDBP gene and the mutated TARDBP gene with ΔPLD or ΔNLS from the endogenous locus also showed the characteristics of TDP-43 proteinopathy. Compared with animals expressing only the wild-type protein, 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 the mutant TDP-43 polypeptide with ΔPLD or ΔNLS (Figures 13A - 13B, and 14). The TDP-43 mutant lacking a functional NLS was insoluble, while the TDP-43 mutant lacking PLD was not insoluble (Figure 13C). Furthermore, in these mice expressing the mutant TDP-43 protein with ΔPLD or ΔNLS, denervation of muscles mainly composed of fast muscle fibers was observed, but denervation of muscles mainly composed of slow muscle fibers was not observed (Figures 15A - B).

[0037] The discoveries provided herein not only provide methods for evaluating 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), but also ES cells, ESMN cells, and non-human animals expressing mutant TDP-43 polypeptides lacking functional structural domains. ES cells, ESMN cells, non-human animals (e.g., rodents, e.g., rats and mice) expressing mutant TDP-43 polypeptides lacking functional structural domains may be used, respectively, as in vitro or in vivo models of TDP-43 proteinopathy, for example, in methods for identifying therapeutic candidates therefor.

[0038] The TARDBP gene and the TDP-43 polypeptide

[0039] The TARDBP gene encodes a TAR DNA-binding protein, TARDBP, 43-KD, and also a TDP-43 polypeptide, also known as TDP-43. The nucleic acid sequences of wild-type TARDBP genes of various species and the wild-type TDP-43 polypeptides encoded thereby 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 Center for Biotechnology Information (NCBI) Gene Database of the National Library of Medicine (NIH), USA. For example, refer to 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 number 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 shown as GenBank accession number 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 shown as GenBank accession number 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 shown as GenBank accession number 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 acids shown as GenBank accession number 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 includes a nucleic acid sequence represented as GenBank accession number 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 include a knockout mutation. The mutated TARDBP gene may encode a mutant TDP-43 polypeptide, where the mutant TDP-43 polypeptide lacks a functional structural domain. For example, the mutated TARDBP gene may include a nucleotide sequence encoding a TDP-43 structural domain that includes a point mutation, an insertion within part or all of the structural domain, and / or a deletion of part or all, where the point mutation, insertion, and / or deletion results in a loss of function of the structural domain and, despite the mutant 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 mutant TDP-43 polypeptide, where it includes at least one wild-type TDP-43 structural domain or a variant thereof, and / or it is specifically bound by an anti-TDP-43 antibody or an antigen-binding portion thereof. Similarly, the mutated TARDBP gene may be classified as encoding a polypeptide, such as a mutant TDP-43 polypeptide, that may include at least one wild-type TDP-43 structural domain or a variant thereof, and / or is 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 NLS of TDP-43 at amino acids 82-99, the RRM1 of TDP-43 at amino acids 106-176, the RRM2 of TDP-43 at amino acids 191-262, the E of TDP-43 at amino acids 239-248, and the PLD of TDP-43 at amino acids 274-414.

[0042] Conventional NLS sequences contain stretches of basic amino acids, mainly lysine (K) and arginine (R) residues. A bipartite NLS contains two clusters of these basic amino acids separated by a linker region of about 10 to 13 amino acids. Amino acid substitutions and / or deletions in the basic amino acid sequence of a conventional NLS may abrogate 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 contain the amino acid sequence set forth in SEQ ID NO: 1 modified to contain 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 contain the amino acid sequence set forth in SEQ ID NO: 3 modified to contain 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 contain the amino acid sequence set forth in SEQ ID NO: 5 modified to contain amino acid substitutions and / or deletions at positions 82, 83, 84, 95, 97, and / or 98. Accordingly, a mutated TARDBP gene encoding a mutant TDP-43 protein lacking the NLS of functional TDP-43 may contain a sequence encoding a TDP-43 polypeptide that contains (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, and is modified as set forth in SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5.The mutated TARDBP gene encoding a mutant TDP-43 protein lacking the functional NLS of TDP-43 may comprise a nucleotide sequence encoding an amino acid sequence shown as SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5 modified to include an amino acid substitution selected from the group consisting of K82A K83A, R84A, K95A, K97A, K98A, or combinations thereof. The mutated TARDBP gene encoding a mutant TDP-43 protein lacking the functional NLS of TDP-43 may comprise a nucleotide sequence encoding an 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] Typical RNA binding by a canonical RRM is generally achieved by contacts between the surface of the four-stranded antiparallel β-sheet of the canonical RRM and single-stranded RNA. Melamed et al. (2013), RNA, 19:1537-1551. Two highly conserved motifs in the central two β-strands, RNP1 (consensus K / R-G-F / Y-G / A-F / Y-V / I / L-X-F / Y, X is any amino acid) and RNP2 (consensus I / V / L-F / Y-I / V / L-X-N-L, 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 to 176 of the wild-type TDP-43 polypeptide contains an RNP2 consensus sequence (LIVLGL; SEQ ID NO: 7) located at amino acid positions 106 to 111 and an RNP1 consensus sequence (KGFGFVRF; SEQ ID NO: 8) located at amino acid positions 145 to 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) any amino acid deletion or substitution at and between positions 106 to 176, (iii) any amino acid deletion or substitution at and between positions 106 to 111, (iv) any amino acid deletion or substitution at and between positions 145 to 152, or (v) any combination of (i) to (iv) may lack a functional RRM1. A mutant TDP-43 polypeptide lacking a functional RRM1 may include a sequence shown 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) any amino acid deletion or substitution at and between positions 106 to 176, (iii) any amino acid deletion or substitution at and between positions 106 to 111, (iv) any amino acid deletion or substitution at and between positions 145 to 152, or (v) any combination of (i) to (iv). A mutant TDP-43 polypeptide lacking a functional RRM1 may include a sequence shown 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) any amino acid deletion or substitution at and between positions 106 to 176, (iii) any amino acid deletion or substitution at and between positions 106 to 111, (iv) any amino acid deletion or substitution at and between positions 145 to 152, or (v) any combination of (i) to (iv).A mutant TDP-43 polypeptide lacking functional RRM1 may comprise a sequence shown 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 to 176; (iii) a deletion or substitution of any amino acid at and between positions 106 to 111; (iv) a deletion or substitution of any amino acid at and between positions 145 to 152; or (v) any combination of (i) to (iv). Accordingly, a mutated TARDBP gene encoding a mutant TDP-43 polypeptide lacking functional RRM1 may comprise a nucleotide sequence encoding a TDP-43 polypeptide comprising an amino acid sequence shown 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 to 176; (iii) a deletion or substitution of any amino acid at and between positions 106 to 111; (iv) a deletion or substitution of any amino acid at and between positions 145 to 152; or (v) any combination of (i) to (iv). A mutated TARDBP gene encoding a mutant TDP-43 polypeptide lacking functional RRM1 may comprise a nucleotide sequence encoding a TDP-43 polypeptide comprising an amino acid sequence shown as SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5 modified to include a mutation of F147L and / or F149L. A mutated TARDBP gene encoding a mutant TDP-43 polypeptide lacking functional RRM1 may comprise a nucleotide sequence encoding a TDP-43 polypeptide comprising an 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 to 262 of the wild-type TDP-43 polypeptide contains an RNP2 consensus sequence (VFVGRC; SEQ ID NO: 9) located at amino acid positions 193 to 198 and an RNP1 consensus sequence (RAFAFVT; SEQ ID NO: 10) located at amino acid positions 227 to 233. F194 and F229 may be regarded 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 194 and / or 229, (ii) any amino acid deletion or substitution at and between positions 193 to 198, (iii) any amino acid deletion or substitution at and between positions 227 to 233, (iv) any amino acid deletion or substitution at and between positions 191 to 262, or (v) any combination of (i) to (iv) may lack a functional RRM2. A mutant TDP-43 polypeptide lacking a functional RRM2 may include a sequence shown 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) any amino acid deletion or substitution at and between positions 193 to 198, (iii) any amino acid deletion or substitution at and between positions 227 to 233, (iv) any amino acid deletion or substitution at and between positions 191 to 262, or (v) any combination of (i) to (iv). A mutant TDP-43 polypeptide lacking a functional RRM2 may include a sequence shown 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) any amino acid deletion or substitution at and between positions 193 to 198, (iii) any amino acid deletion or substitution at and between positions 227 to 233, (iv) any amino acid deletion or substitution at and between positions 191 to 262, or (v) any combination of (i) to (iv).A mutant TDP-43 polypeptide lacking functional RRM2 may comprise a sequence shown 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 to 198, (iii) a deletion or substitution of any amino acid at and between positions 227 to 233, (iv) a deletion or substitution of any amino acid at and between positions 191 to 262, or (v) any combination of (i) to (iv). Accordingly, a mutated TARDBP gene encoding a mutant TDP-43 polypeptide lacking functional RRM2 may comprise a nucleotide sequence encoding a TDP-43 polypeptide comprising an amino acid sequence shown as SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5 modified to include (i) an amino acid substitution at position 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 to 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 an amino acid sequence shown 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 functional RRM2 may comprise a nucleotide sequence encoding a TDP-43 polypeptide comprising an amino acid sequence shown as SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5 modified to include the mutations F194L and F229L.

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

[0047] The prion-like domain (PLD) of the wild-type TDP-43 polypeptide may be located at amino acids 274 to 414. A mutant TDP-43 polypeptide lacking a functional PLD may include an amino acid sequence shown as SEQ ID NO: 1 modified to include at least one or all amino acid deletions at and between positions 274 to 414. A mutant TDP-43 polypeptide lacking a functional PLD may include an amino acid sequence shown as SEQ ID NO: 3 modified to include at least one or all amino acid deletions at and between positions 274 to 414. A mutant TDP-43 polypeptide lacking a functional PLD may include an amino acid sequence shown as SEQ ID NO: 5 modified to include at least one or all amino acid deletions at and between positions 274 to 414. Thus, the mutated TARDBP gene encoding the mutant TDP-43 polypeptide may include a nucleotide sequence encoding a TDP-43 polypeptide that includes an amino acid sequence shown as SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5 modified to include at least one or all amino acid deletions at and between positions 274 to 414.

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

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

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

[0051] Methods for generating non-human animal cells that express a mutant TDP-43 polypeptide and / or for evaluating the biological function of the TDP-43 structural domain may include manipulating the genome of a cell to include a mutated TARDBP gene. The mutated TARDBP gene may encode a mutant TDP-43 polypeptide, wherein the mutant TDP-43 polypeptide lacks a functional structural domain.

[0052] Methods for generating non-human animal cells that express a mutant TDP-43 polypeptide and / or for evaluating the biological function of the TDP-43 structural domain may include manipulating the genome of a cell to include a mutated TARDBP gene, wherein the mutated TARDBP gene includes a knockout mutation.

[0053] The methods provided herein involve introducing into a cell one or more polynucleotide or polypeptide constructs that include various components of a target genome integration system. "Introducing" means presenting the sequence (polypeptide or polynucleotide) to the cell such that the sequence can access the interior of the cell. The methods provided herein do not depend on a particular method for introducing any component of the target genome integration system into the cell, as long as the polynucleotide can access the interior of at least one cell. Methods for introducing polynucleotides into various cell types are known in the art and include, but are not limited to, stable transfection methods, transient transfection methods, and virus-mediated methods.

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

[0055] The transfection protocol and the protocol for introducing the polypeptide or polynucleotide sequence 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 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 gene guns, magnet-assisted transfection (Bertram, J. (2006), Current Pharmaceutical Biotechnology, 7, 277-28). Viral methods can also be used for transfection.

[0056] Cells containing the mutated TARDBP gene can be generated using the various methods disclosed herein. Manipulation may include replacing the endogenous TARDBP gene with a mutated TARDBP gene encoding a mutant TDP-43 polypeptide and / or replacing the endogenous TARDBP gene with a TARDBP gene containing a knockout mutation such as a conditional knockout mutation. Manipulation may also include culturing the cells under conditions that exclude the expression of the TARDBP gene containing the knockout mutation. Conditions that may exclude the expression of the TARDBP gene may include the expression of a recombinase protein, such as cre-recombinase.

[0057] Such a method of operation may include: (1) incorporating a mutated TARDBP gene into a target TARDBP genomic locus of pluripotent cells of a non-human animal using the method disclosed herein, to generate a genetically engineered pluripotent cell comprising the mutated TARDBP gene mutated at the target TARDBP genomic locus; and (2) selecting a genetically engineered pluripotent cell having the mutated TARDBP gene mutated at the target TARDBP genomic locus. (3) introducing the genetically engineered pluripotent cell into a host embryo of a non-human animal, for example, at a stage prior to the morula; and (4) implanting the host embryo containing the genetically engineered pluripotent cell into a surrogate mother to generate an F0 generation derived from the genetically engineered pluripotent cell, whereby an animal may be further generated. The non-human animal can be a non-human mammal, rodent, mouse, rat, hamster, monkey, agricultural mammal or livestock, or fish or 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 manipulation 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, outbred, and non-inbred strains. Examples of mouse strains include the 129 strain, C57BL strains (e.g., C57BL / 6 strain), crosses of 129 and C57BL / 6 (e.g., 50% 129 and 50% C57BL / 6), BALB / c strain, and Swiss Webster strain. Examples of 129 strains include 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, and 129T2 (see, for example, 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. The mouse can be a cross of the aforementioned 129 strain (e.g., 129S6 (129 / SvEvTac) strain) and the aforementioned C57BL / 6 strain, a cross of one or more of the aforementioned 129 strains, or a cross of one or more of the aforementioned C57BL strains. The mouse can also be derived from a strain other than the 129 strain.

[0060] The rat pluripotent cells, totipotent cells, or host embryos can be derived from any rat strain, including, for example, inbred strains, outbred strains, and non-inbred strains. Examples of rat strains include the ACI rat strain, Dark Agouti (DA) rat strain, Wistar rat strain, LEA rat strain, Sprague Dawley (SD) rat strain, or Fischer rat strains such as Fisher F344 or Fisher F6. The rat pluripotent cells, totipotent cells, or host embryos can also be obtained from a strain derived from the cross of two or more of the above strains. For example, the rat pluripotent cells, totipotent cells, or host embryos can be derived from a strain selected from the DA strain and the ACI strain. The ACI rat strain is characterized by having a white belly and feet, and a black agouti with the RT1 av1 haplotype. Such strains are available from various sources, including Harkan Laboratories. An example of a rat ES cell line derived from the ACI rat is the ACI.G1 rat ES cell. The Dark Agouti (DA) rat strain is characterized by having an agouti coat and the RT1 av1 haplotype. Such rats are available from various sources, including Charles River and Harlan Laboratories. Examples of rat ES cell lines derived from the DA rat are 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 hereby incorporated by reference in its entirety for all purposes.

[0061] For example, germline-transmissible rat ES cells can be obtained by culturing rat ES cells isolated on a feeder cell layer in a medium containing N2 supplement, B27 supplement, leukemia inhibitory factor (LIF) at about 50 U / mL to about 150 U / mL, and a combination of inhibitors consisting of a MEK inhibitor and a GSK3 inhibitor, wherein the feeder cell layer is not engineered to express LIF, and the rat ES cells are: (i) engineered to include targeted gene manipulation including at least one insertion of a heterologous polynucleotide containing a selectable marker into the genome of the rat ES cells and capable of transmitting the targeted gene manipulation through the germline; (ii) have a normal karyotype; (iii) lack the expression of c-Myc; and (iv) form spherical floating colonies in culture (see, e.g., US 2014-0235933 A1 and US 2014-0310828 A1, which are hereby incorporated by reference in their entireties). Other examples of the derivation and targeted manipulation of rat embryonic stem cells are provided, for example, by 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, the method of nuclear transfer includes the steps of: (1) enucleating an oocyte; (2) isolating a donor cell or nucleus that binds to the enucleated oocyte; (3) inserting the cell or nucleus into the enucleated oocyte to form a reconstructed cell; (4) transplanting the reconstructed cell into the uterus of an animal to form an embryo; and (5) enabling the development of the embryo. In such a method, oocytes are generally harvested from dead animals, but may also be separated from the oviduct and / or ovary of a living animal. The oocytes can be matured in various media known to those skilled in the art prior to enucleation. Enucleation of the oocytes can be carried out by many methods well known to those skilled in the art. Inserting a donor cell or nucleus into the enucleated oocyte to form a reconstructed cell is usually by microinjecting the donor cell under the zona pellucida prior to fusion. Fusion may be induced by applying a DC electrical pulse across the contact / fusion surface (electrical fusion), by exposing the cells to a fusion-promoting chemical such as polyethylene glycol, or by an inactivated virus such as Sendai virus. The reconstructed cells are usually activated by electrical and / or non-electrical means during, and / or after, fusion of the nuclear donor and recipient oocyte. Methods of activation include electrical pulses, chemically induced shocks, penetration by sperm, an increase in the level of divalent cations in the oocyte, and a decrease in the phosphorylation of cellular proteins in the oocyte (by a kinase inhibitor). The activated reconstructed cells, or embryos, are usually cultured in media well known to those skilled in the art and then transferred to the uterus of an animal. See, for example, US20080092249, WO / 1999 / 005266A2, US20040177390, WO / 2008 / 017234A1, and U.S. Patent No. 7,612,250, each of which is incorporated herein by reference.

[0063] (a) Operating the 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 includes a genetic manipulation at the target genomic locus; (c) Isolating the genetically engineered targeting vector from the genome of the engineered prokaryotic cell; (d) Introducing the genetically engineered targeting vector into pluripotent cells of a non - human animal to generate genetically engineered pluripotent cells that contain an inserted nucleic acid at the target TARDBP genomic locus; (e) Selecting the genetically engineered pluripotent cells; (f) At a stage prior to the blastocyst, introducing the genetically engineered pluripotent cells into a host embryo of a non - human animal; (g) Transplanting the host embryo containing the genetically engineered pluripotent cells into a surrogate mother to generate an F0 generation derived from the genetically engineered pluripotent cells. Another method for producing a non - human animal that includes one or more genetic manipulations described herein in the germline is provided. In such a method, 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 domesticated mammal. 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 can be 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, human cells, rodent cells, rat cells, mouse cells, hamster cells. In one embodiment, the targeted genetic manipulation results in a mutated TARDBP gene, for example, a mutated TARDBP gene encoding a mutant TDP - 43 polypeptide lacking a functional structural domain and / or a mutated TARDBP gene containing a knockout mutation.

[0064] In a further method, the isolation step (c) further comprises (c1) linearizing the genetically engineered target-directed vector (i.e., the genetically engineered LTVEC). In a further embodiment, the introduction 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 performed 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 performed via an allelic manipulation (MOA) assay as described herein.

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

[0066] In some embodiments, the targeted pluripotent and / or totipotent cells, including the various genetic manipulations as described herein, are used as donor cells for insertion and introduced via the VELOCIMOUSE® method (see, e.g., US7,576,259, US7,659,442, US7,294,754, and US2008-0078000A1, which are hereby incorporated by reference in their entireties) into the corresponding organism, e.g., from an 8-cell stage mouse embryo to the pre-morula stage. The non-human animal embryos containing the genetically engineered pluripotent and / or totipotent cells are incubated until the blastocyst stage and then implanted into a surrogate mother to generate the F0 generation. In some embodiments, the targeted mammalian ES cells, including the various genetic manipulations as described herein, are introduced into blastocyst stage embryos. Non-human animals having the genetically engineered genomic locus (i.e., the TARDBP locus) can be identified via the allelic manipulation (MOA) assay as described herein. The resulting F0 generation non-human animals derived from the genetically engineered pluripotent and / or totipotent cells are mated with wild-type non-human animals to obtain F1 generation progeny. Following genotyping with specific primers and / or probes, F1 non-human animals that are heterozygous for the genetically engineered genomic locus are mated with each other to produce progeny of F2 generation non-human animals that are homozygous for the genetically engineered genomic locus.

[0067] In one embodiment, a method of producing a cell containing a mutated TRADBP gene is provided. Such a method includes (a) contacting a pluripotent cell with a targeting construct containing 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 a manipulated pluripotent cell. The method of producing a non-human animal further includes (b) introducing the manipulated pluripotent cell into a host embryo; and (c) gestating the host embryo in a surrogate mother, wherein the surrogate mother gives birth to offspring containing the manipulated TARDBP locus, and the genetic manipulation results in a mutant TDP-43 polypeptide lacking a functional structural domain.

[0068] In some embodiments, a cell containing a mutated TARDBP gene may be produced by manipulating ES cells to contain the mutated TARDB gene and culturing the ES cells in vitro in a differentiation medium. In some embodiments, culturing the ES cells in vitro includes differentiating the ES cells into primitive ectoderm cells or embryonic stem cell-derived motor neurons (ESMN).

[0069] Cells and Animals

[0070] The cells (which may be included in non-human animal tissues or non-human animals) disclosed herein may be any type of cell containing a mutated TARDBP gene as disclosed herein. The cells may contain a mutated non-human animal TARDBP gene (e.g., a mutated TARDBP gene of a non-human animal) or a mutated human TARDBP gene.

[0071] The cell may contain a mutated TARDBP gene encoding a mutant TDP-43 polypeptide, where the mutant TDP-43 polypeptide lacks a functional structural domain and the cell expresses the mutant TDP-43 polypeptide. For example, the cell may contain a mutated TARDBP gene encoding a mutant TDP-43 polypeptide lacking a functional structural domain that includes a nuclear localization signal (NLS), RNA recognition motif 1 (RRM1), RNA recognition motif 2 (RRM2), putative nuclear export signal (E), prion-like domain (PLD), or a combination thereof. The cell may contain a mutated TARDBP gene encoding a mutant TDP-43 polypeptide lacking a functional structural domain due to one or more of the following: (a) point mutations in amino acids in the NLS (e.g., K82A K83A, R84A, K95A, K97A, K98A or combinations thereof), (b) point mutations in amino acids in RRM1 (e.g., F147L and / or F149L), (c) point mutations in amino acids in RRM2 (F194L and / or F229L), (d) deletion of at least a portion of the nuclear export signal (e.g., deletion of amino acids at and between positions 239 and 250 of the wild-type TDP-43 protein), and (e) deletion of at least a portion of the prion-like domain (e.g., deletion of amino acids at and between positions 274 and 414 of the wild-type TDP-43 polypeptide). The cell may contain a mutated TARDBP gene encoding a mutant TDP-43 polypeptide containing the mutations K82A K83A, R84A, K95A, K97A, and K98A, where the mutant TDP-43 polypeptide lacks a functional NLS. The cell may contain a mutated TARDBP gene encoding a mutant TDP-43 polypeptide containing a deletion in the amino acids from position 274 to 414 of the wild-type TDP-43 polypeptide, where the mutant TDP-43 polypeptide lacks a functional PLD. The cell may contain a mutated TARDBP gene encoding a mutant TDP-43 polypeptide containing the point mutations F147L and F149L, where the mutant TDP-43 polypeptide lacks a functional RRM1.The cell may contain a mutated TARDBP gene encoding a mutant TDP-43 polypeptide containing point mutations F194L and F229L, wherein the mutant TDP-43 polypeptide lacks a functional RRM2. The cell may contain a mutated TARDBP gene encoding a mutant TDP-43 polypeptide containing a deletion of a 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 contain a mutated TARDBP gene containing a knockout mutation of the entire coding sequence of the TARDBP gene, such as a conditional knockout mutation, deletion, etc. The cell may contain a mutated TARDBP gene containing a conditional knockout mutation. For example, the mutated TARDBP gene may contain a site-specific recombination recognition sequence, such as a loxp sequence. The cell may contain a mutated TARDBP gene containing a loxp sequence adjacent to an exon containing the TDP-43 coding sequence, such as exon 3. The cell may contain a mutated TARDBP gene containing a loxp sequence and lacking the TDP-43 coding sequence, such as exon 3. The cell may contain a mutated TARDBP gene lacking the entire TDP-43 coding sequence, such as a mutated TARDBP gene containing a deletion of the entire coding sequence of the TDP-43 polypeptide.

[0073] In some embodiments, the cell may contain, for example, a mutated TRADBP gene inserted at the endogenous TARDBP locus in its germline genome. In some embodiments, the cell contains a mutated TARDBP gene, such as a mutated TARDBP gene containing a knockout mutation, and / or a mutated TARDBP gene encoding a mutant 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 an endogenous TARDBP promoter and / or regulatory element.

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

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

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

[0077] Cells containing the mutated TARDBP gene may express the mutant TDP-43 polypeptide encoded thereby, and may be characterized by one or more of the following: (i) the level of the mRNA transcript of the mutated TARDBP gene at a level comparable to the mRNA transcription level of the wild-type TARDBP gene in control cells; (ii) an increase in the level of the mutant TDP-43 polypeptide as compared to the level of the wild-type TDP-43 polypeptide in control cells; (iii) the mutant TDP-43 polypeptide is found at a higher concentration in the cytoplasm than in the nucleus of the cell; (iv) the mutant TDP-43 polypeptide shows an increase in insolubility as compared to the wild-type TDP-43 polypeptide; (v) cytoplasmic aggregates containing the mutant TDP-43 polypeptide; (vi) an increase in the splicing of cryptic exons of genes as compared to cells expressing wild-type TDP-43; (vii) a decrease in the level of TDP-43 mRNA that has undergone alternative splicing lacking the sequence encoding the PLD of TDP-43.

[0078] The cells may be cultured in vitro and may be examined in vitro or in vivo. For example, the cells can be in vivo within an animal.

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

[0080] The non-human animals can be derived from any genetic background. For example, suitable mice can be derived from the 129 strain, C57BL / 6 strain, a cross of 129 and C57BL / 6, BALB / c strain, or Swiss Webster strain. In some embodiments, the 129 strain includes 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 hereby incorporated by reference in its entirety for all purposes. Examples of the C57BL strain include C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaL_wN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr, and C57BL / Ola. Suitable mice can also be derived from a cross of the aforementioned 129 strain and the aforementioned C57BL / 6 strain (e.g., 50% 129 and 50% C57BL / 6). Similarly, suitable mice can be derived from a cross of the aforementioned 129 strain or a cross of the aforementioned BL / 6 strain (e.g., 129S6 (129 / SvEvTac) strain).

[0081] Similarly, rats can be derived from rat strains including, for example, the ACI rat strain, Dark Agouti (DA) rat strain, Wistar rat strain, LEA rat strain, Sprague Dawley (SD) rat strain, or Fischer rat strains such as Fisher F344 and Fisher F6. Rats can also be obtained from strains derived from a cross 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 RT1 av1Characterized by having a black agouti with a haplotype. Such strains are available from various sources including Harkan Laboratories. The Dark Agouti (DA) rat strain has an agouti coat and RT1 av1 Characterized by having a haplotype. Such rats are available from various sources including Charles River and Harlan Laboratories. Some suitable rats can be derived from inbred rat strains. See, for example, US2014 / 0235933, which is hereby incorporated by reference in its entirety for all purposes.

[0082] The cells can also be in any type of undifferentiated or differentiated state. For example, the cells can be totipotent cells, pluripotent cells (e.g., human pluripotent cells, or non-human pluripotent cells such as mouse embryonic stem (ES) cells or rat ES cells), or non-pluripotent cells. Totipotent cells include undifferentiated cells 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-like cells such as ES cells or induced pluripotent stem (iPS) cells. ES cells include totipotent or pluripotent cells derived from an embryo 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 can also be derived from ES cells. For example, the cells can be neuron cells (e.g., ES cell-derived motor neurons (ESMN)), primitive ectoderm-like cells, embryoid body cells, and the like.

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

[0085] Suitable cells provided herein also include primary cells. Primary cells include cells or cell cultures directly isolated from an organism, organ, or tissue. Primary cells include cells that are not transformed and not immortal. Primary cells include any cells obtained from an organism, organ, or tissue that have not been previously passaged in tissue culture or that have 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 derived from multicellular organisms that normally do not proliferate indefinitely but, due to a mutation or change, avoid normal cellular aging and instead can 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 cells or primary cells include cells commonly 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 derived from any genetic background (e.g., for mice, BALB / c, C57BL / 6, 129, or combinations thereof), can be fresh, or can be frozen, and can be derived from natural reproduction or in vitro fertilization.

[0088] Methods of employing a system for 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 lacking the functional structural domain encoded therefrom as described herein provide a model 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 lacking the functional structural domain encoded therefrom may exhibit a phenotype characteristic of TDP-43 proteinopathy. In some embodiments, for example, (a) embryonic stem cell-derived motor neurons (ESMNs) that contain a mutated TARDBP gene and express a mutant TDP-43 polypeptide lacking the functional structural domain encoded therefrom, and / or (b) cells isolated from a non-human animal that contains a substitution of the endogenous TARDBP gene with a mutated TARDBP gene at the endogenous TARDBP locus and expresses a mutant TDP-43 polypeptide therefrom may be characterized by one or more of the following: (i) levels of mRNA transcripts of the mutated TARDBP gene comparable to the levels of mRNA transcripts of the wild-type TARDBP gene in control cells; (ii) an increase in the 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 at a higher concentration in the cytoplasm than in the nucleus of the cell; (iv) the mutant TDP-43 polypeptide shows an increase in insolubility compared to the wild-type TDP-43 polypeptide; (v) cytoplasmic aggregates containing the mutant TDP-43 polypeptide; (vi) an increase in the splicing of cryptic exons of genes compared to cells expressing wild-type TDP-43; (vii) a decrease in the level of TDP-43 mRNA that has undergone alternative splicing and lacks 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 are for identifying therapeutic candidate agents for treating, preventing, and / or suppressing one or more symptoms of TDP-43 proteinopathy (e.g., cytoplasmic accumulation of mutant TDP-43 polypeptide), and / or for restoring the biological function of wild-type TDP-43 polypeptide (e.g., suppressing 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 cells expressing a mutant TDP-43 polypeptide that contains a mutated TARDBP gene and lacks a functional structural domain encoded therefrom with the therapeutic candidate agent. The contacting may be performed in vitro. The contacting may include administering the therapeutic candidate agent to an animal.

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

[0092] Exemplary parameters that may be measured in non-human animals (or in cells isolated therefrom and / or using the same) to evaluate 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 titer, drug metabolism, gene expression array, metabolic activity, mitochondrial activity, oxidative stress, phagocytosis, protein biosynthesis, proteolysis, protein secretion, stress response, target tissue drug concentration, non-target tissue drug concentration, transcriptional activity, and the like.

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

[0094] Figure 11A illustrates the pre-mRNA of full-length TDP-43 and the normal (upper panel) and alternative (lower panel) splicing events that occur at its 3' end. As shown, exon 6 encodes the prion-like domain (PLD) of the full-length TDP-43 protein formed in a normal splicing event, and its coding sequence terminates at the end of the PLD. Two new exons (7 and 8) are formed by alternative splicing events from at least one of the three alternative 5'-splice sites within exon 6 to an alternative 3'-splice site downstream, for example, an alternative splicing event adjacent to 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 the position on chromosome 4: 148,617,705. The second alternative splicing event from exon 7 to exon 8 occurs from chromosome 4: 148,617,566 to chromosome 4: 148,616,844. One of ordinary skill in the art will be able to determine similar alternative 5' and 3' splice sites in other TARDBP genes, such as the human TARDBP gene.

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

[0097] The observation that TDP-43 lacking PLD can support viability, particularly in motor neurons, and the decrease in the level of TDP-43 mRNA undergoing this alternative splicing in cells expressing a mutated TARDBP gene with ΔPLD or ΔNLS, along with their ALS-like phenotypes, suggest that the TDP-43 mRNA undergoing this alternative splicing and its translated truncated products may not contribute to TDP-43 proteinopathy and may be preventive against TDP-43 proteinopathy. Application of siRNAs, antisense oligonucleotides, and / or CRISPR / Cas9 systems designed to remove or inactivate TDP-43 mRNA isoforms encoding forms of the protein containing PLD may deplete variants of TDP-43 prone to pathological aggregation while preserving the mRNA undergoing alternative splicing that gives rise to a truncated TDP-43 protein lacking PLD. The truncated form of TDP-43 may further support cell life, particularly the viability of motor neurons, and may be resistant to pathological aggregation.

[0098] Therefore, the treatment strategy consists of finding active antisense oligonucleotides (ASOs) or siRNAs that target only those TDP-43 mRNA sequences that contain the sequence encoding PLD, for example, those mRNAs that are encoded by the genomic sequence following the alternative splice site within exon 6. As a non-limiting example, the ASO or siRNA may target an mRNA that contains a sequence transcribed from the TARDBP gene after the codon(s) encoding an alternative 5' splice site that removes the PLD domain by splicing. An ASO or siRNA designed to target this region of the TDP-43 mRNA recognizes only the full-length TDP-43 mRNA encoding the TDP-43 polypeptide containing PLD, while sparing the alternatively spliced TDP-43 mRNA encoding the truncated, protected TDP-43 polypeptide lacking PLD. In other words, such an ASO or siRNA should not be able to recognize or enhance the degradation of alternatively spliced TDP-43 mRNA. The ASO or siRNA may target a TDP-43 mRNA sequence encoding any 3' untranslated region upstream of amino acids 287-414 of the TDP-43 polypeptide or the 3' alternative splice site of exon 7. The ASO may promote the degradation of mRNA by RNase H-mediated cleavage, for example, via a -5-10-5 gapmer. The siRNA may promote the degradation of mRNA and / or protein synthesis by RNA interference.

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

[0100] A. Antisense Oligonucleotides and siRNAs

[0101] Antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) that target sequences within pre-mRNAs may enhance the degradation of unwanted isoforms. As designed herein, the ASO or siRNA may be used to disrupt the TDP-43 mRNA encoding PLD while preserving the alternatively spliced TDP-43 mRNA. To reduce only the level of full-length TDP-43 mRNA, the ASO or siRNA may target a TDP-43 mRNA comprising a sequence between (ii) a selected 3' splice site downstream from a selected 5' splice site within exon 6, e.g., a sequence encoding amino acids 287-414 of the TDP-43 polypeptide and / or a TDP-43 mRNA comprising any 3' untranslated region upstream of the selected splice site. See FIG. 11A. In some embodiments, the selected 5' splice site within exon 6 correlates to a TARDBP genomic position 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 position of the human TARDBP gene. In some embodiments, the downstream selected 3' splice site correlates to mouse chromosome 4:148,617,705 or the corresponding position of the human TARDBP gene.

[0102] An antisense oligonucleotide or siRNA targeting the TDP-43 mRNA encoding PLD may have chemically modified subunits arranged in a pattern or motif to confer properties to 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 modified to confer increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity for the target nucleic acid, and / or increased inhibitory activity. Optionally, a second region of the antisense oligonucleotide may serve as a substrate for the cellular endonuclease RNase H that cleaves the RNA strand of the RNA:DNA duplex.

[0104] In certain embodiments, the antisense oligonucleotide is a uniformly sugar-modified oligonucleotide. The antisense oligonucleotide may contain a gapmer motif. In a gapmer, an inner region having a plurality of nucleotides that assist in cleavage by RNase H is positioned between outer regions having a plurality of nucleotides that are chemically different from the nucleosides of the inner region. In the case of an antisense oligonucleotide having a gapmer motif, the gap segment generally serves as a substrate for endonuclease cleavage, while the wing segment contains modified nucleosides. In certain embodiments, the regions of the gapmer are distinguished by the type of sugar moiety comprising each discrete region. Types of sugar moieties used to distinguish 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, inter alia, 2'-MOE and 2'-O-CH3), and bicyclic sugar-modified nucleosides. In certain embodiments, the wing may contain several modified sugar moieties, e.g., including 2'-MOE. In certain embodiments, the wing may contain several modified and unmodified sugar moieties. In certain embodiments, the wing may include various combinations of 2'-MOE nucleosides and 2'-deoxynucleosides.

[0105] The different regions may each contain a uniform sugar moiety, a variant, or an alternating sugar moiety. The wing-gap-wing motif is often described as "X-Y-Z", 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 contain a uniform sugar moiety, a variant sugar moiety, or an alternating sugar moiety. In certain embodiments, "X" and "Y" may contain one or more 2'-deoxynucleosides. "Y" may contain 2'-deoxynucleosides. As used herein, a gapmer described as "X-Y-Z" has a configuration such that the gap is positioned immediately adjacent to each of the 5'-wing and the 3'-wing. Thus, there are no intervening nucleotides between the 5'-wing and the gap, nor between the gap and the 3'-wing. Any of the antisense compounds described herein can have a gapmer motif. In certain embodiments, "X" and "Z" are the same, and in other embodiments, they are different. In certain embodiments, "Y" is a nucleoside between 8 and 15. 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, the 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] An antisense oligonucleotide targeting 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 include a narrowed motif. Antisense oligonucleotides with a narrowed gap targeting 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 nucleosides may include bicyclic sugars. The bicyclic sugar may include a 4'-(CH2)n-O-2' bridge where n is 1 or 2; and a 4' to 2' bridge selected from 4'-CH2-O-CH2-2'. The bicyclic sugar may include a 4'-CH(CH3)-O-2' bridge. The chemical modification may include an acyclic 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, for example, the alternative 5' splice site correlates with a TARDBP genomic position 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 position of the human TARDBP gene, and the alternative 3' splice site correlates with the TARDBP genomic position of chromosome 4:148,617,705, an antisense oligonucleotide comprising a gapmer motif targeting the TDP-43 mRNA sequence between the alternative 5' and 3' splice sites. In some embodiments, the siRNA includes a sequence targeting the TDP-43 mRNA sequence between the alternative 5' splice site and the alternative 3' splice site, wherein the alternative 5' splice site is within exon 6, for example, the alternative 5' splice site correlates with a TARDBP genomic position 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 position of the human TARDBP gene, and the alternative 3' splice site correlates with the TARDBP genomic position of chromosome 4:148,617,705.

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

[0109] The ASO or siRNA may also be covalently attached to one or more moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the resulting ASO or siRNA. Exemplary conjugating groups include cholesterol moieties and lipid moieties. Additional conjugating groups include carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes.

[0110] The ASO or siRNA may also be modified to have one or more stabilizing groups generally attached to one or both ends. Included in the stabilizing groups are cap structures. These terminal modifications can protect the ASO or siRNA having terminal nucleic acids from exonuclease degradation and can aid in intracellular delivery and / or localization. The cap can be present at the 5'-end (5' cap) or 3'-end (3' cap), or at both ends. Cap structures are well known and include, for example, inverted deoxyabasic 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 the desired effect. For example, the ASO can be from 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 can 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 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 consist 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 a specific example, the ASO can be about 15 to about 25 linked nucleosides.

[0112] An ASO or siRNA can be complementary to a target nucleic acid (e.g., a TDP-43 pre-mRNA, e.g., an mRNA sequence encoding PLD) and / or can specifically hybridize. When a sufficient number of nucleobases of the ASO can hydrogen bond to the corresponding nucleobases of the target nucleic acid, resulting in the desired effect, the ASO and the target nucleic acid are complementary to each other. Specifically hybridizable refers to an ASO that has complementarity between the ASO and the target nucleic acid to such an extent that it causes the 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 an equal-length portion of the TDP-43 pre-mRNA. Alternatively, the ASO can be about 100% complementary to an equal-length portion of the TDP-43 pre-mRNA. The percent complementarity of the ASO to the target nucleic acid can be determined using conventional methods. For example, an ASO that specifically hybridizes because 18 out of 20 nucleobases of the ASO are complementary to the target region exhibits 90 percent complementarity. The percent complementarity of the ASO to a region of the target nucleic acid can be routinely determined using the BLAST program (Basic Local Alignment 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). The percent homology, percent sequence identity, or percent complementarity can be determined using, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix®, Genetics Computer Group, University Research Park, Madison Wis.) with the default settings, using the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489).

[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 continue to specifically hybridize with the target nucleic acid. Further, the ASO or siRNA may hybridize over one or more segments of the TDP-43 pre-mRNA such that intervening or flanking segments are not involved in the hybridization event (e.g., loop structures, mismatches, or hairpin structures). The position of the non-complementary nucleobases may be at the 5' or 3' end of the ASO or siRNA. Alternatively, the non-complementary nucleobase(s) may be at an internal position of the ASO or siRNA. If two or more non-complementary nucleobases are present, they may be contiguous (i.e., linked) or may be discontinuous.

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

[0116] As shown herein, cells continue to survive even though they express only a mutant TDP-43 polypeptide lacking functional PLD. Also described herein is a clustered regularly interspaced short palindromic repeat (CRISPR) / CRISPR-associated (Cas) system, or one or more components of a CRISPR / Cas system, which can be used to delete the protein-like domain (or a portion thereof) of the endogenous TARDBP locus as described herein from a cell, such as 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, such as 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, such as a targeter RNA (e.g., a CRISPR RNA (crRNA) and an activator RNA (e.g., a 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 that are involved in the expression of Cas genes or induce 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 V-A or subtype V-B). The sequence encoding the prion-like domain (or a part thereof) of TDP-43 at 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 nucleic acids.

[0118] The CRISPR / Cas system described herein may include one or more gRNAs targeting a Cas protein (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 a gRNA recognition sequence. The CRISPR / Cas system as described herein may further include at least one expression construct containing 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 positions determined by both (i) the complementarity of base pairs between the gRNA and the target DNA and (ii) a short motif called the protospacer adjacent motif (PAM) in the target DNA. 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 the immediate 3'-side of 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 is any DNA nucleotide and is the immediate 5'-side of the guide RNA recognition sequence of the complementary strand of the target DNA. In some such cases, N1 and N2 can be complementary and the N1-N2 base pair can be any base pair (e.g., N1 = C and N2 = G; N1 = G and N2 = C; N1 = A and N2 = T; or N1 = T and N2 = A). In 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 can be provided in the form of either two molecules (separate crRNA and tracrRNA) or one molecule (sgRNA) of RNA, and optionally in the form of a complex with a Cas protein. The gRNA can also be provided in the form of DNA encoding the gRNA. In some embodiments, the DNA encoding the gRNA can encode a single RNA molecule (sgRNA) or separate RNA molecules (e.g., separate crRNA and tracrRNA) (wherein the separate RNA molecules may be provided as one DNA molecule or as separate DNA molecules encoding 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 crRNA and / or tracrRNA.

[0122] The targeted genetic manipulation can be generated by contacting the cell with a Cas protein and one or more guide RNAs that hybridize to one or more guide RNA recognition sequences within the target genomic locus. At least one of the one or more guide RNAs can form a complex with the Cas protein and direct it 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., if the Cas protein is a nickase). The resulting end sequences generated by the double-stranded break or single-stranded break can then undergo recombination.

[0123] C. Methods for Introducing Oligonucleotides

[0124] To enable the introduction of oligonucleotides into cells, various methods and compositions are provided herein. Methods for introducing oligonucleotides into various cell types are known and include, for example, stable transfection methods, transient transfection methods, and virus-mediated methods.

[0125] Transfection protocols, as well as protocols 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. U.S.A. 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 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 gene guns, or magnet - assisted transfection (Bertram, (2006), Current Pharmaceutical Biotechnology, 7, 277 - 28). Virus - based methods can also be used for transfection.

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

[0127] The introduction of oligonucleotides into cells can also be achieved by microinjection. In zygotes (i.e., embryos at the 1-cell stage), microinjection can be performed into the maternal and / or paternal pronuclei or cytoplasm. When microinjection is performed into only one pronucleus, the paternal pronucleus is preferred because of its larger size. Methods for performing microinjection are well known. See, for example, Nagy et al. (Nagy A, Gertsenstein M, Vintersten K, Behringer R., 2003, Manipulating the Mouse Embryo, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); 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 oligonucleotides 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. As specific examples, oligonucleotides can be introduced into cells or non-human animals using carriers such as poly(lactic acid) (PLA) microspheres, poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres, liposomes, micelles, reverse micelles, lipid coacervates, or lipid nanotubes.

[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. Viruses can infect dividing cells, non-dividing cells, or both dividing and non-dividing cells. Viruses can integrate into the host genome or alternatively not integrate into the host genome. Such viruses can also be engineered to have attenuated immunity. Viruses can be replication-competent or replication-defective (e.g., defective in one or more genes required for virion replication and / or additional rounds of packaging). Viruses can cause transient expression, long-term expression (e.g., at least 1 week, 2 weeks, 1 month, 2 months, or 3 months), or persistent expression. Exemplary virus titers (e.g., AAV titer) include 10 12 、10 13 、10 14 、10 15 、and 10 16 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 for the synthesis of complementary DNA strands. When constructing an AAV delivery 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) are involved in AAV replication. For example, transfection of HEK293 cells with a delivery plasmid, Rep / Cap, and a helper plasmid containing the adenoviral gene E1+ can produce infectious AAV particles. Alternatively, Rep, Cap, and the adenoviral helper genes can be combined into a single plasmid. Similar packaging cells and methods can also be used for other viruses such as retroviruses.

[0131] Multiple serotypes of AAV have been identified. These serotypes differ in the types of cells they infect (i.e., their tropism), allowing for preferential transduction of specific 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 epithelial 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, particularly AAV8.

[0132] Tropism can be further refined by pseudotyping, which is the mixing of genomes from different viral serotypes with the capsid. For example, AAV2 / 5 refers to a virus containing a serotype 2 genome packaged in a serotype 5 capsid. The use of pseudotyped viruses can improve transduction efficiency and, similarly, change tropism. Hybrid capsids derived from different serotypes can also be used to change the tropism of the virus. For example, AAV-DJ contains a hybrid capsid of 8 serotypes and exhibits high infectivity across a wide range of cell types in vivo. AAV-DJ8 is another example that displays the characteristics of AAV-DJ but with enhanced uptake into the brain. AAV serotypes can also be modified via mutations. Examples of AAV2 mutation modifications include Y444F, Y500F, Y730F, and S662V. Examples of AAV3 mutation modifications include Y705F, Y731F, and T492V. Examples of AAV6 mutation 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 the expression of the transgene, self-complementary AAV (scAAV) variants can be used. Since AAV relies on the cell's DNA replication machinery to synthesize the complementary strand of the AAV single-stranded DNA genome, the expression of the transgene may be delayed. To address this delay, scAAV containing complementary sequences that can spontaneously anneal upon infection can be used, 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 delivery mediated by lipid nanoparticles (LNPs). Lipid formulations can improve cellular uptake while protecting biomolecules from degradation. Lipid nanoparticles are particles containing multiple lipid molecules physically bound to each other by intermolecular forces. These include microspheres (including monolayer and multilayer vesicles, such as 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 prolong the time the nanoparticles can exist in the body. Examples of suitable cationic lipids, neutral lipids, anionic lipids, helper lipids, and stealth lipids can be found in WO2016 / 010840A1, which is hereby incorporated by reference in its entirety for all purposes.

[0135] Administration in vivo can be by any suitable route including, for example, parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. Systemic administration modes include, for example, oral and parenteral routes. Examples of parenteral routes include intravenous, intra-arterial, intraosseous, intramuscular, intradermal, subcutaneous, intranasal, and intraperitoneal routes. A specific example is intravenous injection. Intranasal injection and intravitreal injection are other specific examples. Topical administration modes include, for example, intrathecal, intraventricular, intrasubstantial (e.g., local intrasubstantial 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, tonsil, prefrontal cortex, thalamus, cerebellum, medulla, hypothalamus, tegmentum, substantia nigra), intraocular, intraorbital, subconjunctival, intravitreal, subretinal, and transscleral routes. A significantly smaller amount of the component may exert an effect when administered locally (e.g., intrasubstantially or intravitreally) compared to when administered systemically (e.g., intravenously). The topical administration mode may also reduce or eliminate the occurrence of potentially toxic side effects that may occur when a therapeutically effective amount of the component 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 for nucleic acid transfection. When a cationic lipid is mixed with a negatively charged nucleic acid, a complex can be obtained that can release the active nucleic acid into the cytoplasm of the cell by passing through the cell membrane. It is also possible to introduce reagents (e.g., antisense oligonucleotides) into cells by electroporation. This method is very effective and useful for cell lines that cannot be easily transfected by lipids.

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

[0138] In some methods, administration is by means such that the reagent being introduced 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 (which is hereby incorporated by reference in its entirety for all purposes).

[0139] For reagents (e.g., antisense oligonucleotides) to reach the nervous system, they must first pass through the blood-brain barrier or the blood-spinal cord barrier, which are vascular barriers. One mechanism that can be used to pass through 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 depending on the cell type and cargo. For example, systemically delivered antisense oligonucleotides tagged with arginine-rich CPPs can pass through the blood-brain barrier. Another delivery mechanism that can be used is exosomes, which are extracellular vesicles known to mediate cell-cell communication through the transfer of proteins and nucleic acids. For example, intravenous injection of exosomes transduced with a short viral peptide derived from the rabies virus glycoprotein (RVG) can pass through the blood-brain barrier and be delivered to the brain.

[0140] Techniques that bypass the blood-brain barrier through direct injection into cerebrospinal fluid can also be used. For example, a reagent (e.g., an antisense oligonucleotide) can be injected into the cerebral ventricle (ICV), and then the reagent (e.g., an antisense oligonucleotide) must pass through the ependymal cell layer that fills the ventricular system to substantially enter. Intrathecal (IT) delivery means the delivery of a reagent (e.g., an antisense oligonucleotide) to the subarachnoid space of the spinal cord. From here, the reagent (e.g., an antisense oligonucleotide) must pass through the pia mater to substantially enter. The reagent (e.g., an antisense oligonucleotide) can be delivered by ICT or IT via an exit catheter connected to an implanted reservoir. The drug is injected 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; those skilled in the art reading this disclosure will recognize various modifications that may be equivalent to such described embodiments or otherwise within the claims. Generally, terms will follow their meaning as understood in the art unless otherwise specified. References cited within the scope of this specification, or relevant portions thereof, are hereby incorporated herein by reference in their entirety.

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

[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 that includes "or" between one or more members of a group is satisfied if, as the context permits, 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 is otherwise clear from the context. The present invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The present 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. Further, it should be understood that the present invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc. from one or more of the listed claims are introduced into another claim that depends from the same basic claim (or, in the case of a related claim, another claim) unless otherwise indicated or unless it is apparent to one of ordinary skill in the art that a contradiction or inconsistency would result. When elements are presented as a list (e.g., in Markush group format or similar), it should be understood that each subgroup of the elements is also disclosed and that any element(s) can be removed from the group. Generally, when the present invention or an aspect of the present invention is referred to as including a particular element, feature, etc., it should be understood that embodiments of the present invention or aspects of the present invention consist of or consist essentially of such element, feature, etc. For the sake of brevity, such embodiments are not specifically described verbatim herein in every case. It should also be understood that any embodiment or aspect of the present invention can be explicitly excluded from the claims, regardless of whether a particular exclusion is recited in the specification.

[0144] "Control" includes the meaning understood in the art of "control" as the standard against which results are compared. Typically, a control is used to enhance the integrity in an experiment by separating such variables in order to draw conclusions about the variables. In some embodiments, the control is a reaction or assay that is performed concurrently with a test reaction or assay that provides a comparison standard. "Control" also includes "control animal". A "control animal" may have the modifications described herein, different modifications described herein, or no modifications (i.e., wild-type animals). In one experiment, a "test" (i.e., the variable being investigated) is applied. In a second experiment, for the "control", the variable being investigated is not applied. In some embodiments, the control is an existing control (i.e., a previously performed test or assay, or a previously known amount or result). In some embodiments, the control is, or includes, a record stored in printed or another manner. The control may be a positive control or a negative control.

[0145] "Determining", "measuring", "evaluating", "assessing", "assaying", and "analyzing" include any form of measurement and include determining whether an element is present. These terms include both quantitative and / or qualitative determinations. An assay may be relative or absolute. "Analyzing for presence" can be determining the amount of what is 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 modified forms thereof. They include single-stranded, double-stranded, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, unnatural, or derivatized nucleotide bases.

[0147] As used interchangeably herein, the terms "protein," "polypeptide," and "peptide" include polymeric forms of amino acids of any length, including both coded and non-coded amino acids and chemically or biochemically modified or derivatized amino acids. This term also includes modified polymers such as polypeptides having modified peptide backbones. The term "domain" refers to any portion of a protein or polypeptide that has 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 mutant, diseased, altered, etc.) state or situation. Wild-type genes and polypeptides are often present in multiple different forms (e.g., alleles).

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

[0150] The term "locus" refers to the specific location of a gene (or important sequence), DNA sequence, sequence encoding a polypeptide, or position on a chromosome in the genome of an organism. For example, the "TARDBP locus" may refer to the TARDBP gene, the DNA sequence of TARDBP, the specific location of the sequence encoding TARDBP2, or the TARDBP position on the chromosome in the genome of an organism where such a sequence is identified. The "TARDBP locus" may include regulatory elements of the TARDBP gene, such as, 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 encodes a product (e.g., an RNA product and / or a polypeptide product), including a coding region interrupted by non-coding introns such that the 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 insulator sequences, and matrix attachment regions. These sequences may be close to (e.g., within 10 kb of) or distant from the coding region of the gene, 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 various different forms located at the same position on a chromosome, or locus. A diploid organism has two alleles, each at the endogenous locus of a homologous chromosome. Each pair of alleles represents the genotype of a particular locus. The genotype is described as homozygous when there are two identical alleles at a particular locus and heterozygous when the two alleles are different.

[0153] "Operably linked" includes juxtapositions that enable the described components to function in their intended manner. A coding sequence "operably linked" to a control sequence is linked in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequence. "Operably linked" arrays include both expression control sequences contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. The term "expression control sequence" includes polynucleotide sequences necessary to influence the expression and processing of the coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing signals 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 a promoter, ribosome binding site, and transcription termination sequence, while in eukaryotes such control sequences usually include a promoter and transcription termination sequence. The term "control sequence" is intended to include components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences.

[0154] "Phenotype" includes traits, or classes or sets of traits, exhibited by a cell or an organism. In some embodiments, a particular phenotype may correlate with a particular allele or genotype. In some embodiments, the phenotype may be discrete; in some embodiments, the phenotype may be continuous. The phenotype may include cell viability or cell fitness. The phenotype may include the expression level, cellular localization and / or solubility / stability profile of a protein, e.g., a mutant TDP-43 polypeptide, and each of these phenotypes may be determined using well-known methods such as Western blot analysis, fluorescence in situ hybridization, qualitative RT-PCR, etc.

[0155] A "promoter" is a regulatory region of DNA that typically contains a TATA box that can direct RNA polymerase II to initiate RNA synthesis at an appropriate transcription start site of a particular polynucleotide sequence. The promoter may further include other regions that affect the rate of transcription initiation. The promoter sequences disclosed herein regulate the transcription of operably linked polynucleotides. The promoter can be active in one or more of the cell types disclosed herein (e.g., eukaryotic cells, non-human mammalian cells, human cells, rodent cells, pluripotent cells, 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 hereby 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 and is optionally embodied in a tangible medium. In some embodiments, reference may refer to a control. "Reference" also includes "reference cell". A "reference cell" may have the modifications described herein, different modifications as described herein, or no modifications (i.e., wild-type cells). Generally, as would be understood by one of ordinary skill in the art, the reference agent, cell, animal, cohort, individual, population, sample, sequence, or value is determined or characterized under conditions comparable to those utilized to determine or characterize the 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 one nucleotide), or a protein sequence that differs from a reference amino acid sequence (e.g., by one amino acid) but retains the biological function of the reference sequence. In some embodiments, the variant differs from the reference sequence due to the degeneracy of the genetic code and / or conservative codon / amino acid substitutions.

[0158] The terms "sequence identity" or "identity" in the context of the relationship between two polynucleotide sequences or polypeptide sequences refer to residues in the two sequences that are the same when the sequences are compared to maximize matches over a specified comparison window. When using percent sequence identity with respect to proteins, positions of non-identical residues often differ by conservative amino acid substitutions, where amino acid residues are substituted with other amino acid residues having similar chemical properties (e.g., charge or hydrophobicity), so as not to change the functional properties of the molecule. If the sequences differ by conservative substitutions, the percent sequence identity may be adjusted upward to correct 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 percent sequence identity. Thus, for example, if a score of 1 is given for identical amino acids and a score of zero for non-conservative substitutions, a score between zero and 1 is given for conservative substitutions. Scoring of conservative substitutions is calculated, for example, as implemented in the program PC / GENE (Intelligenetics, Mountain View, California).

[0159] "Percent sequence identity" includes the value (the number of residues that perfectly match) determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide sequence in the comparison window may include additions or deletions (i.e., gaps) as compared to a reference sequence (not including additions or deletions) for optimal alignment of the two sequences. The percent is calculated by determining the number of positions at which nucleic acid bases or amino acid residues match 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 percent sequence identity. Unless otherwise specified (e.g., the shorter sequence includes linked heterologous sequences), the comparison window is the full length of the shorter of the two sequences being compared.

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

[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 having similar size, charge, or polarity. Examples of conservative substitutions include the replacement of one nonpolar (hydrophobic) residue, such as isoleucine, valine, or leucine, with another nonpolar 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. Further, the replacement of a basic residue, such as lysine, arginine, or histidine, with another residue, or the replacement of one acidic residue, such as aspartic acid or glutamic acid, with another acidic residue 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 nonpolar (hydrophobic) amino acid residue, such as isoleucine, valine, leucine, alanine, or methionine, and / or the replacement of a nonpolar residue with a polar residue. A typical classification of amino acids is 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 the artificial environment. The term "in vivo" includes a natural environment (e.g., a cell or an organism or a living body) and processes or reactions that occur within the 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, wherein the mutant TDP-43 polypeptide lacks a functional structural domain found in the wild-type TDP-43 polypeptide, wherein the non-human animal or non-human animal cell expresses the mutant TDP-43 polypeptide, Optionally, the wild-type TDP-43 polypeptide comprises a sequence shown as SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, the non-human animal cell.

[0164] Embodiment 2. The non-human animal cell according to 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 according to Embodiment 1 or Embodiment 2, wherein the 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 according to any one of the preceding embodiments, wherein the mutated TARDBP gene is the mutated TARDBP gene of the non-human animal.

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

[0168] Embodiment 6. The mutant TDP-43 polypeptide is as follows: (a) a point mutation of an amino acid in the NLS, (b) a point mutation of an amino acid in the RRM1, (c) a point mutation of an amino acid in the RRM2, (d) deletion of at least a part of the nuclear export signal, and (e) deletion of at least a part of the prion-like domain, and the non-human animal cell according to any one of the preceding embodiments, lacking a functional structural domain due to one or more of the above.

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

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

[0171] Embodiment 9. The non-human animal cell according to 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 according to any one of the preceding embodiments, wherein the mutant TDP-43 polypeptide comprises F147L and F149L.

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

[0174] Embodiment 12. The non-human animal cell according to 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 according to any one of the preceding embodiments, wherein the mutated TARDBP gene encoding the mutant TDP-43 polypeptide replaces the endogenous TARDBP gene at the endogenous TARDBP locus.

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

[0177] Embodiment 15. The non-human animal cell according to embodiment 13, wherein the non-human animal cell is homozygous for the mutated TARDBP gene encoding the 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 according to Embodiment 16, wherein the knockout mutation includes a conditional knockout mutation.

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

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

[0182] Embodiment 20. The non-human animal cell according to Embodiment 19, wherein the loxp sequence is adjacent to exon 3 of the TARDBP gene including the knockout mutation.

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

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

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

[0187] Embodiment 25. (i) The mRNA transcript level of the mutated TARDBP gene comparable to the mRNA transcript level of the wild-type TARDBP gene in control cells, (ii) An increase in the level of the mutant TDP-43 polypeptide as compared to the level of the wild-type TDP-43 polypeptide in control cells, (iii) A mutant TDP-43 polypeptide that is found at a higher concentration in the cytoplasm than in the nucleus of motor neurons, for example, (iv) A mutant TDP-43 polypeptide with increased insolubility compared to the wild-type TDP-43 polypeptide, (v) A cytoplasmic aggregate containing the mutant TDP-43 polypeptide, (vi) An increase in the splicing of cryptic exons, and / or (vii) A decrease in the level of the TDP-43 form that has undergone alternative splicing, The non-human animal cell according to any one of the preceding embodiments, comprising:

[0188] Embodiment 26. A non-human animal cell 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.

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

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

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

[0192] Embodiment 30. The 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 according to any one of the preceding embodiments, wherein the non-human animal cell is cultured in vitro.

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

[0195] Embodiment 33. A composition comprising the 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 non-human animal cell that expresses a mutant TDP-43 polypeptide, the method comprising manipulating the genome of the non-human animal or non-human animal cell to include 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 a sequence shown as SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5.

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

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

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

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

[0201] Embodiment 39. A method for identifying a therapeutic candidate 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 the composition according to embodiment 32 with a candidate drug; (b) evaluating the phenotype and / or TDP-43 biological activity of the non-human cell or tissue; (c) identifying the candidate drug that restores to the non-human cell or tissue a phenotype and / or TDP-43 biological activity comparable to the phenotype and / or TDP-43 biological activity of a control cell or tissue expressing 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; (c) evaluating the phenotype and / or TDP-43 biological activity of the genetically engineered ES cells, primitive ectoderm derived therefrom, motor neurons derived therefrom, or non-human animals derived therefrom.

[0203] The method according to embodiment 39 or 40, wherein the phenotype is evaluated by cell culture, fluorescence in situ hybridization, Western blot analysis, or a combination thereof.

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

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

[0206] The method according to any one of embodiments 39 to 43, wherein evaluating the biological activity of the mutant TDP-43 polypeptide comprises measuring splice products of a gene containing a cryptic exon regulated by TDP-43.

[0207] The method according to embodiment 44, wherein the gene containing a cryptic exon regulated by TDP-43 comprises Crem, Fyxd2, Clf1.

[0208] The method according to any one of embodiments 39 to 45, wherein evaluating the biological activity of the mutant TDP-43 polypeptide comprises measuring the level of TDP-43 that has undergone alternative splicing.

[0209] An antisense oligonucleotide comprising a gapmer motif that targets a TDP-43 mRNA sequence encoding the PLD of the TDP-43 polypeptide and / or comprising an untranslated sequence downstream of exon 6 and upstream of exon 7, Optionally, the TDP-mRNA comprises a sequence between a selected 5' splice site and a selected 3' splice site within exon 6, Optionally, the selective 5' splice site correlates with a TARDBP genomic position 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 position of the human TARDBP gene, and / or the selective 3' splice site correlates with the TARDBP genomic position of chromosome 4: 148,617,705, said antisense oligonucleotide.

[0210] Embodiment 48. An siRNA comprising a sequence encoding the PLD of the TDP-43 polypeptide and / or targeting a TDP-43 mRNA sequence comprising an untranslated sequence downstream of exon 6 and upstream of exon 7, Optionally, the TDP-mRNA sequence is between a selective 5' splice site within exon 6 and a downstream selective 3' splice site, Optionally, the selective 5' splice site correlates with a TARDBP genomic position 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 position of the human TARDBP gene, and / or the selective 3' splice site correlates with the TARDBP genomic position of chromosome 4: 148,617,705, said siRNA.

[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 a selective splice site that results in a selective mRNA encoding a truncated TDP-43 polypeptide lacking the PLD, Optionally, the selective splice site includes a selective 5' splice site within exon 6 and a downstream selective 3' splice site, Optionally, the selective 5' splice site correlates with a TARDBP genomic position 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 position of any of the human TARDBP genes, and / or the selective 3' splice site correlates with the TARDBP genomic position of chromosome 4: 148,617,705, said CRISPR / Cas system.

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

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

[0214] Embodiment 52. The non-human animal according to 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. The non-human animal according to Embodiment 51 or Embodiment 52, wherein the mutated TARDBP gene is the mutated TARDBP gene of the non-human animal.

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

[0217] Embodiment 55. The mutant TDP-43 polypeptide is as follows: (a) a point mutation of an amino acid in the NLS, (b) a point mutation of an amino acid in the RRM1, (c) a point mutation of an amino acid in the RRM2, (d) a deletion of at least a part of the nuclear export signal, and (e) a deletion of at least a part of the prion-like domain, and lacks a functional structural domain due to one or more of the above, the non-human animal according to any one of Embodiments 51 to 54.

[0218] Embodiment 56. (a) the point mutation of the amino acid in the NLS includes K82A K83A, R84A, K95A, K97A, K98A, or a combination thereof, (b) the point mutation in RRM1 includes F147L and / or F149L, (c) the point mutation in RRM2 includes F194L and / or F229L, (d) at least a part of the deletion of the nuclear export signal deletion includes a deletion of an amino acid at and between positions 239 and 250 of the wild-type TDP-43 polypeptide, and (e) at least a part of the deletion of the prion-like domain includes a deletion of an amino acid at and between positions 274 and 414 of the wild-type TDP-43 polypeptide, the non-human animal according to Embodiment 55.

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

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

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

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

[0223] Embodiment 61. The non-human animal according to 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. The non-human animal according to any one of Embodiments 51 to 61, wherein the mutated TARDBP gene encoding the mutant TDP-43 polypeptide replaces the endogenous TARDBP gene at the endogenous TARDBP locus.

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

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

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

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

[0229] Embodiment 67. The 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 according to Embodiment 67, wherein the loxp sequence is adjacent to exon 3 of the TARDBP gene containing a knockout mutation.

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

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

[0233] Embodiment 71. The 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) the mRNA transcript level of the mutated TARDBP gene comparable to the mRNA transcript level of the wild-type TARDBP gene in the control animal, (ii) an increase in the level of the mutant TDP-43 polypeptide compared to the level of the wild-type TDP-43 polypeptide in the control animal, (iii) for example, the mutant TDP-43 polypeptide found at a higher concentration in the cytoplasm than in the nucleus of motor neurons, (iv) the mutant TDP-43 polypeptide having increased insolubility compared to the wild-type TDP-43 polypeptide, (v) cytoplasmic aggregates containing the mutant TDP-43 polypeptide, (vi) an increase in the splicing of cryptic exons, (vii) A decrease in the level of TDP-43 forms that have undergone alternative splicing, (viii) Denervation of muscle tissue mainly composed of fast-twitch muscles such as the anterior tibialis muscle, and / or (ix) Normal innervation of muscle tissue mainly composed of slow-twitch muscles such as the intercostal muscles, The non-human animal according to any one of Embodiments 50 to 71, including.

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

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

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

[0238] Embodiment 76. The 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 a therapeutic candidate for the treatment of a disease, (a) Contacting a non-human animal according to any one of Embodiments 50 to 76 with a candidate drug, (b) Evaluating the phenotype and / or TDP-43 biological activity of the non-human animal, (c) Identifying the candidate drug that restores the phenotype and / or TDP-43 biological activity of the non-human. The method, including.

[0240] Embodiment 78. A mutant TDP-43 polypeptide, as follows: (a) A point mutation of an amino acid in the NLS, (b) A point mutation of an amino acid in RRM1, (c) A point mutation of an amino acid in RRM2, (d) A deletion of at least a part of the nuclear export signal, and (e) A deletion of at least a part of the prion-like domain, the mutant TDP-43 polypeptide comprising a sequence shown as SEQ ID NO: 1, 3, or 5 modified to include one or more of the foregoing.

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

[0242] Embodiment 80. The mutant TDP-43 polypeptide according to Embodiment 78 or Embodiment 79, including the K82A mutation, the K83A mutation, the R84A mutation, the K95A mutation, the K97A mutation, and / or the K98A mutation.

[0243] Embodiment 81. The mutant TDP-43 polypeptide according to any one of Embodiments 78 to 80, including a deletion of the prion-like domain with amino acids from position 274 to position 414 of the wild-type polypeptide.

[0244] Embodiment 82. The mutant TDP-43 polypeptide according to any one of Embodiments 78 to 81, wherein the mutant TDP-43 polypeptide includes the F147L mutation and / or the F149L mutation.

[0245] Embodiment 83. The 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 according to 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 the mutant TDP-43 polypeptide according to any one of Embodiments 78 to 84.

[0248] Embodiment 86. Further, 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 rodent cells. The nucleic acid according to Embodiment 85.

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

[0250] Embodiment 88. The nucleic acid according to Embodiment 86, wherein the nucleic acid undergoes homologous recombination at the endogenous mouse TARDBP locus, and the 5' and 3' homology arms are homologous to the mouse sequence 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 containing PLD while retaining a truncated TDP-43 mRNA encoding PLD-lacking TDP-43 in a cell, (i) An antisense oligonucleotide comprising a gapmer motif targeting a TDP-43 mRNA sequence encoding the PLD of the TDP-43 polypeptide and / or containing an untranslated sequence downstream of exon 6 and upstream of exon 7, (ii) An siRNA comprising a sequence targeting a TDP-43 mRNA sequence encoding the PLD of the TDP-43 polypeptide and / or containing an untranslated sequence downstream of exon 6 and upstream of exon 7, and / or (iii) A CRISPR / Cas system comprising a Cas9 protein and at least one of the gRNAs, 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, introducing the CRISPR / Cas system into the cell, the method comprising.

[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 CRISPR / Cas system is the CRISPR / Cas system of Embodiment 49, the method according to Embodiment 89.

[0253] Embodiment 91. The method according to Embodiment 89 or 90, wherein the cell is present in vivo. Brief Description of Sequences [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 Mutated TARDBP Gene Since TDP-43 is essential for viability, conditional knockout in the first endogenous TDP-43 allele and mutation in the other second endogenous TDP-43 allele are included so that wild-type TDP-43 from the first endogenous allele sustains the viability of ES cells until activation of the state, and after its activation, the effect of the mutant TDP-43 polypeptide expressed from the second allele may be confirmed. Embryonic stem (ES) cells may be generated.

[0256] To evaluate 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 encoding a mutant TDP-43 polypeptide that was altered or deleted in a way predicted to disable the function of one of five structural domains - the nuclear localization signal (NLS), RNA recognition motif 1 (RRM1), RNA recognition motif 2 (RRM2), putative nuclear export signal (E), or prion-like domain (PLD) - at the other TARDBP locus on the homologous chromosome. See Figure 3. The (1) phenotype and (2) biological activity of the mutant TDP-43 polypeptide of cells carrying the mutated TARDBP gene(s) internally and expressing a mutant TDP-43 polypeptide lacking a functional NLS, RRM1, RRM2, E, or PLD were analyzed as described in Examples 2 and 3, respectively.

[0257] The conditional alleles were designed based on previously published studies showing that deletion of exon 3 of TDP-43 does not produce a functional protein. Chiang et al. (2010), Proc. Natl. Acad. Sci. USA. 107:16320 - 324. Exon 3 of the endogenous mouse TARDBP gene was engineered to have loxP sites at each end. After Cre-mediated recombination, deletion of the genomic coordinates chr4:147995844 - 147996841 was achieved. ES cells containing exon 3 with loxP sites were further engineered with the mutated TARDBP gene as described herein. As a control, mouse ES cells were also generated that were engineered by conditional knockout mutation in one allele and deletion from the start codon to the stop codon of exon 2 (genomic coordinates chr4:147992370 - 147999471) in the other allele.

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

[0259] Specifically, 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 a functional RRM1 or RRM2 survived. Figures 4 and 5.

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

[0261] Mutant TDP-43 polypeptides lacking a functional NLS or PLD showed increased solubility of mutant TDP-44. FIG. 9A. The solubility of mutant TDP-43 polypeptides lacking a functional E or RRM1 did not change compared to wild-type TDP-43 polypeptides. FIG. 9A. There was no difference in mRNA expression levels for any of the mutant TDP-43 polypeptides, but an increase in protein levels was seen for mutant TDP-43 polypeptides lacking a functional NLS, PLD, or RRM1. FIG. 9B. Since the mRNA expression levels of these mutant TDP-43 polypeptides were comparable to the expression level of wild-type TDP-43, the increase in protein levels may have been due to improved stability of the mutant TDP-43 polypeptides. FIG. 9C.

[0262] The materials and methods used to analyze the phenotypes 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 cells to support the viability of embryonic stem (ES) cells and motor neurons (ESMN) derived therefrom 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 trypsin treatment, the ES medium was changed to 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 further cultured for 4 days in ADFNK supplemented with retinoic acid (RA), smoothened agonist, and purmorphamine to obtain motor neurons (ESMN) resembling limbs. 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 of GDNF, BDNF, CNTF) to mature. Conditional knockout alleles were activated using Cre recombinase delivered via electroporation at the ES cell stage (Figures 4, 6 - 9) or 7 days after plating (Figure 5).

[0265] Intracellular localization of mutant TDP-43 polypeptide

[0266] The intracellular localization of TDP-43 variants was analyzed using an antibody that recognizes the N-terminus of the TDP-43 polypeptide (α-TDP-43N-term) and an antibody that recognizes 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 MNs derived from ES cells on ice for 10 minutes 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). Next, the cells were passed through a 27-gauge syringe five times. After centrifugation at 4000 rpm for 5 minutes at 4°C, the protein supernatant containing the soluble cytoplasmic extract was recovered. 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 protease and phosphatase. An equal volume of 2× SDS sample buffer was added to each fraction, and the samples were heated to 90°C. Next, equal volumes of each fraction were loaded onto a 14% SDS gel and electrophoresed at 225 V for 50 minutes, followed by Western blotting for TDP-43 using the α-TDP-43-N-term antibody or the α-TDP-43-C-term antibody, the latter of which does not recognize the PLD deletion variant. Densitometry was performed using ImageJ. Figure 6B. The cytoplasmic / nuclear TDP-43 ratio was plotted and statistically analyzed using GraphPad for Prism. Figure 6B; Figure 9B, right panel.

[0267] Fluorescence in situ hybridization (FISH)

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

[0269] Solubility of mutant TDP-43 polypeptide

[0270] This protocol was adopted from Jo et al. (2014), Nature Communications, 5:3496. 500 μl of ice-cold soluble buffer (0.1 M MES (pH 7), 1 mM EDTA, 0.5 mM MgSO4, 1 M sucrose) containing 50 mM N-ethylmaleimide (NEM), 1 mM NaF, 1 mM Na3VO4, 1 mM PMSF, and 10 μg / mL each of aprotinin, leupeptin, and pepstatin. Cells were passed through a 21-gauge needle 3 - 5 times and then through a 23-gauge needle 3 - 5 times. Next, an equal volume of homogenate was recovered from each sample, 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 μl of RAB buffer (100 mM MES (pH 6.8), 10% sucrose, 2 mM EGTA, 0.5 mM MgSO4, 500 mM NaCl, 1 mM MgCl2, 10 mM NaH2PO4, 20 mM NaF) containing 1% N-lauroyl sarcosine (Sarkosyl) and protease inhibitors (1 mM PMSF, 50 mM NEM, and 10 μg / mL each of aprotinin, leupeptin, pepstatin), vortexed for 1 minute at RT, and incubated overnight at 4°C with inversion rotation. Next, the samples were centrifuged at 200,000×g for 30 minutes at 12°C, and the supernatant was recovered as the sarcosyl-soluble fraction. The pellet was resuspended in 700 μl of RAB buffer, passed through a 26-gauge needle 3 - 5 times to completely disperse the pellet, and the sarcosyl-insoluble fraction was prepared. Next, equal volumes of the sarcosyl-soluble and insoluble fractions were aliquoted, and an equal volume of 2× SDS sample buffer was added to each. The samples were heated to 90°C. Next, equal volumes of each fraction were loaded onto a 14% SDS gel, electrophoresed at 225 V for 50 minutes, and then Western blotting for TDP-43 was performed. Densitometry was performed using ImageJ. Figure 9A. The ratio of soluble:insoluble TDP-43 was plotted and statistically analyzed using GraphPad for Prism. Figure 9A.

[0271] Expression level of mutant TDP-43 polypeptide

[0272] The expression level of the TDP-43 mutant was analyzed by Western blot analysis as described herein. The messenger RNA levels in this example were performed by quantitative polymerase chain reaction.

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

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

[0275] 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). The qRT-PCR reaction contained 2 μL of RNA and an 8 μL mixture containing RT-PCR master mix, ROX dye, RT mix, and a mix of 20× gene-specific primers and probes to bring the final volume to 10 μL.

[0276] Unless otherwise specified, the final concentrations of the primers and probes were 0.5 μM and 0.25 μM, respectively. qRT-PCR was performed on a ViiA™ 7 real-time PCR detection system (ThermoFisher). The PCR reaction was carried out in optical 384-well plates in four sets with a RT step at 45°C for 10 minutes followed by 95°C for 10 minutes and two-step cycling of 95°C for 5 seconds and 60°C for 30 seconds for 45 cycles. The sequences of the primers and probes used in the assay (pan assay) are provided in Table 2 below. Table 2

Table 2

[0277] Solubility of mutant TDP-43 polypeptide

[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. On day 4, the embryoid bodies were treated with cycloheximide (100 μg / mL) to block de novo 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 using TDP-43 antibody and GAPDH antibody. Figure 9C. Example 3: Analysis of TDP-43 biological activity by TDP-43 mutants

[0279] Cryptic exons often have TDP-43 binding sites rich in GU, and TDP-43 has been shown to suppress the recognition of cryptic exons, thereby promoting normal splicing. When TDP-43 is lost, the normal mRNA and protein levels of regulated genes are lost. TDP-43 also binds to the 3' end of its own transcripts as 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 evaluating the ability of mutant TDP-43 polypeptides to continue to suppress the splicing of cryptic exons and / or by examining their participation in the autoregulatory loop.

[0280] Heterozygous ESMNs for the wild-type TARDBP gene or a mutated TARDBP gene encoding a mutant TDP-43 polypeptide lacking functional RRM1, NLS, or PLD were analyzed for the expression products of three genes, Crem, Fyxd2, and Clf1, containing a cryptic exon whose splicing is known to be suppressed by wild-type TDP-43. Figure 10. The products of Crem, Fyxd2, and Clf1 that underwent normal splicing were seen in all ESMNs expressing a mutant TDP-43 polypeptide lacking functional RRM1, NLS, or PLD, and the normal splice products were seen in amounts equivalent to those in ESMNs expressing wild-type TDP-43 polypeptide. Figure 10 However, the splicing of the cryptic exon was increased in ESMNs expressing a 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 a mutant TDP-43 polypeptide lacking functional RRM1, NLS, or PLD cannot suppress the splicing of the cryptic exons of the Crem, Fyxd2, and Clf1 genes. Figure 10.

[0281] Heterozygous ESMNs for a mutated TARDBP gene encoding a wild-type TARDBP gene or a mutant TDP-43 polypeptide lacking a functional NLS, RRM1, RRM2, E, or PLD were analyzed for the levels of alternatively spliced TDP-43 mRNA. FIG. 11B. ESMNs expressing a mutant TDP-43 polypeptide lacking a functional NLS, RRM1, E, or PLD showed a decrease in the levels of alternatively spliced TDP-43 mRNA compared to control ESMNs expressing wild-type TDP-43 polypeptide. FIG. 11B. ESMNs expressing a mutant TDP-43 polypeptide lacking a functional E showed comparable levels of alternatively spliced TDP-43 mRNA. FIG. 11B. This data, combined with the data provided in Example 2 showing that ESMNs expressing TDP-43 mutants lacking a functional NLS or PLD exhibit an ALS phenotype (FIG. 5), suggests that strategies to reduce the levels of normally spliced TDP-43 mRNA while preserving alternatively spliced TDP-43 mRNA may be a therapeutic approach 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 was extracted from each sample and reverse transcribed using primers adjacent to the splicing region and probes to detect regions of the loci of the genes being investigated (Crem, Fxyd2, Clf1, TDP-43). Detectable regions for the genes Crem, Fxyd2, and Clf1 investigated included regions spanning the junctions of the normal exons and mouse sequences of cryptic exons of each gene investigated. Detectable regions for the TDP-43 region investigated included regions spanning alternative splicing regions. qPCR for DROSHA was performed using the probes and primers of an easily available kit.

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

[0286] Unless otherwise specified, the final concentrations of the primers and probes were 0.5 μM and 0.25 μM, respectively. qRT-PCR was performed on a ViiA™ 7 real-time PCR detection system (ThermoFisher). The PCR reaction was carried out in optical 384-well plates in four sets with an RT step at 45 °C for 10 minutes followed by 95 °C for 10 minutes and a two-step cycling of 50 cycles at 95 °C for 5 seconds and 60 °C for 30 seconds.

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

[0288] qRT-PCR to evaluate productive Fyxd2 splicing from exon 3 to exon 4 of Fyxd2 was performed with primers containing the nucleotide sequences shown as SEQ ID NO: 23 and SEQ ID NO: 24, and primers containing the nucleotide sequence shown as SEQ ID NO: 25. Splicing of Fyxd2 from exon 3 to the cryptic exon was evaluated using primers containing the nucleotide sequences shown as SEQ ID NO: 26 and SEQ ID NO: 27, and primers containing the nucleotide sequence shown as SEQ ID NO: 28. Splicing of Fyxd2 from the cryptic exon to exon 4 was evaluated using primers containing the nucleotide sequences shown as SEQ ID NO: 29 and SEQ ID NO: 30, and primers containing the nucleotide sequence shown as SEQ ID NO: 31.

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

[0290] TDP-43 mRNA that underwent alternative splicing lacking the sequence encoding the PLD domain was evaluated using primers comprising the nucleotide sequences shown as SEQ ID NO: 41 and SEQ ID NO: 42, and primers comprising 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) of 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 Protein Deletion of TDP-43 results in embryonic lethality, but embryonic stem cells expressing only the mutant ΔNLS TDP-43 gene or the mutant ΔPLD TDP-43 gene from the endogenous TARDBP locus are viable and may differentiate into motor neurons in vitro. This data increases the likelihood that embryonic stem cells expressing mutant TDP-43 polypeptides lacking functional structural domains from the endogenous TARDBP locus may be viable and may also be 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, that express mutant TDP-43 proteins lacking functional structural domains, in order to investigate the role of the TDP-43 structural domain in normal and pathological biological processes.

[0293] To generate embryos or animals that express mutant TDP-43 proteins lacking functional NLS or PLD domains, the VelociMouse® method (Dechiara, T.M., (2009), Methods Mol. Biol. 530:311-324; Poueymirou et al. (2007), Nat. Biotechnol. 25:91-99) was used, where (i) at the endogenous TARDBP locus, exon 3 with loxP introduced conditionally (loxP-Ex3-loxP), a null allele (-) upon Cre-mediated deletion of exon 3 with loxP introduced, a knockout mutation of the NLS (ΔNLS), a deletion of the prion-like domain (ΔPLD), or the wild-type TARDBP gene (WT) was included (see Fig. 3A), and (ii) at the other TARDBP locus on the homologous chromosome, the targeted ES cells containing the wild-type (WT) TARDBP gene or a null allele (-) upon Cre-mediated deletion of exon 3 with loxP introduced were injected into non-compact 8-cell stage Swiss Webster embryos. The survival rate of the embryos after fertilization was examined and the ability to produce live-born F0 generation mice was evaluated.

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

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

[0296] Example 4: Phenotypic analysis of mice expressing mutant TDP-43 polypeptides lacking functional structural domains The phenotypes of the animals generated in Example 3 were analyzed by evaluating the localization, phosphorylation status, and solubility of the TDP-43 polypeptide in spinal cord tissue or motor neurons isolated from the animals. Additionally, denervation or reinnervation of the animals' muscles was determined.

[0297] The cytoplasmic and nuclear fractions of motor neurons derived from the spinal cord of 16-week-old mice were evaluated by Western blot analysis using the following: (1) antibodies that bind to wild-type TDP-43, ΔNLS TDP-43, and ΔPLD TDP-43 because they recognize the N-terminus of the wild-type TDP-43 protein; (2) antibodies that bind to wild-type TDP-43 and ΔNLS TDP-43 but not to ΔPLD TDP-4 because they recognize the C-terminus of the wild-type TDP-43 protein; or (3) antibodies that recognize phosphorylated TDP-43.

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

[0299] A subset of motor neurons in 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 showed extensive cytoplasmic TDP-43 aggregation. Figure 14. Cytoplasmic aggregation was detected less frequently in motor neurons of mice expressing the ΔPLD mutant protein compared to mice expressing mutant TDP-43 polypeptides lacking a functional NLS. Figure 14.

[0300] Since denervation is one of the first pathological features that appears in ALS, muscles mainly containing fast muscle fibers (tibialis anterior muscle) or slow muscle fibers (intercostal muscles) were analyzed for denervation. The mislocalization of TDP-43 resulted in partial denervation of the endplates (*) and denervation (arrows) of muscles mainly containing fast muscle fibers but not slow muscle fibers. Figures 15A - 15B.

[0301] The data presented herein suggest that the animals described herein may be valuable disease models of ALS. In typical ALS patients, distal fast - fatigable (FF) motor units are affected earliest, and neurogenic changes in muscles can be observed before motor neurons are lost. Similarly, in the most widely used "ALS" model, SOD1 G93A mice, denervation of skeletal muscle with early and preferential involvement of FF motor units precedes motor neuron loss. In contrast, proximal muscles mainly innervated by slow muscle fibers, such as the intercostal muscles and diaphragm, 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 phenotypes of mice expressing both (a) mutant TDP-43 polypeptides lacking a functional NLS or PLD and (b) wild - type TDP-43 polypeptides are described below.

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

[0304] The intracellular localization of TDP-43 variants was analyzed using an antibody that recognizes the N-terminus of the TDP-43 polypeptide (α-TDP-43N-term) and an antibody that recognizes 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 tissue on ice for 10 minutes 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). Next, the cells were passed through a 27-gauge syringe five times. After centrifugation at 4000 rpm for 5 minutes 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 protease and phosphatase. An equal volume of 2× SDS sample buffer was added to each fraction, and the samples were heated to 90°C. Next, equal amounts of each fraction were loaded onto a 14% SDS gel and electrophoresed at 225 V for 50 minutes, followed by Western blotting of TDP-43 using an α-TDP-43-N-term antibody (Figure 13A), an α-TDP-43-C-term antibody (Figure 13A), or an α-phospho-TDP-43 antibody that detects phosphorylation of TDP-43 at amino acids 409 / 410 (Figure 13B) (Cosmo Bio USA; catalog number CAC-TIP-PTD-M01). Neither the α-TDP-43-C-term antibody nor the α-phospho-TDP-43 antibody recognizes the PLD deletion variant. Densitometry was performed using ImageJ (Figure 13A and 13B). The cytoplasmic / nuclear ratio of TDP-43 was plotted and statistically analyzed using GraphPad for Prism (Figure 13A, lower panel).

[0305] Fluorescence in situ hybridization (FISH)

[0306] The spinal cord was separated from the vertebral column, immersed and fixed overnight in 4% PFA (or for 1 hour in the case of FUS immunostaining), and washed with 1×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 floated. The floating 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 with primary antisera diluted in TBS-T with 5% normal donkey serum. The primary antibodies used were: ChAT (1:250) EMD Millpore Cat AB144P; TDP-43 (1:10,000) Proteintech 10782-2-AP and NeuN (1:500) EMD Millipore MAB377. After washing with TBS-T, the tissue sections were incubated for 4 hours at room temperature with species-specific secondary antibodies conjugated to Alexa 488, 555, 647 (1:1,000; Life Technologies, Carlsbad, CA, USA), Cy3 or Cy5 (dilution rate 1:500; Jackson Immunoresearch Labs, West Grove, PA, USA). After washing with TBS-T, the stained tissue sections were mounted on microscope slides with Flouromount G (Southern Biotech, Birmingham, AL, USA), and imaged at a magnification of 40× and 1.5× zoom using an LSM510 confocal microscope. (Figure 14)

[0307] Solubility of mutant TDP-43 polypeptide

[0308] This protocol was adopted from Jo et al. (2014), Nature Communications, 5:3496. 500 μl of ice-cold soluble buffer (0.1 M MES (pH 7), 1 mM EDTA, 0.5 mM MgSO4, 1 M sucrose) containing 50 mM N-ethylmaleimide (NEM), 1 mM NaF, 1 mM Na3VO4, 1 mM PMSF and 10 μg / ml each of aprotinin, leupeptin, and pepstatin. Cells of spinal cord tissue from 16-week-old mice were passed through a 21-gauge needle 3 - 5 times, followed by a 23-gauge needle 3 - 5 times for lysis. Next, an equal volume of homogenate was collected from each sample, 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 μl of RAB buffer (100 mM MES (pH 6.8), 10% sucrose, 2 mM EGTA, 0.5 mM MgSO4, 500 mM NaCl, 1 mM MgCl2, 10 mM NaH2PO4, 20 mM NaF) containing 1% N-lauroyl sarcosine (Sarkosyl) and protease inhibitors (1 mM PMSF, 50 mM NEM, and 10 μg / ml each of aprotinin, leupeptin, pepstatin), vortexed for 1 minute at RT, and incubated overnight at 4 °C with inversion rotation. Next, the samples were centrifuged at 200,000×g for 30 minutes at 12 °C, and the supernatant was recovered as the sarcosyl-soluble fraction. The pellet was resuspended in 700 μl of RAB buffer, passed through a 26-gauge needle 3 - 5 times to completely disperse the pellet, and a sarcosyl-insoluble fraction was created. Next, equal volumes of the sarcosyl-soluble and insoluble fractions were aliquoted, and an equal volume of 2× SDS sample buffer was added to each. The samples were heated to 90 °C. Next, equal volumes of each fraction were loaded onto a 14% SDS gel, electrophoresed at 225 V for 50 minutes, and then Western blotting for TDP-43 was performed. Densitometry was performed using ImageJ (Figure 13C). The ratio of soluble:insoluble TDP-43 was plotted and statistically analyzed using GraphPad for Prism. (Figure 13C).

[0309] Denervation test

[0310] For muscle analysis, the tibialis anterior (TA) and intercostal muscles were dissected, immersed in 4% PFA and fixed after 2 hours, and washed with 1× phosphate buffered saline, pH 7.4 (PBS). Next, the muscles were equilibrated in a sucrose gradient (10%-20%-30% sucrose in 0.1 M phosphate buffer, pH 7.4), embedded in O.C.T. compound (Sakura, Torrance, CA), and frozen at -20 °C. Serial sections (30 μm thick) were cut using a cryomicrotome (Leica CM 3050S). Frozen muscle sections (30 μm) were stained with an antibody against synaptophysin (invitrogen) to identify presynaptic terminals, and postsynaptic acetylcholine receptors were detected with Alexa488-conjugated α-BTX (invitrogen). Images were acquired using a Zeiss Pascal LSM510 confocal microscope with ×10 and ×40 objective lenses. The NMJ nerve innervation percentage (%) was determined by dividing the total number of overlapping areas (total number of innervated endplates) between the VAChT signal and the α-BTX signal by the number of areas of the 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, 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 found in the 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 a sequence shown as SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, the non-human animal cell. (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) The non-human animal cell according to item 1 or item 2, wherein the mutated TARDBP gene is the mutated TARDBP gene of the non-human animal. (Item 4) The non-human animal cell according to any one of items 1 to 2, wherein the mutated TARDBP gene is the mutated human TARDBP gene. (Item 5) The mutant TDP-43 polypeptide is as follows: (a) A point mutation of an amino acid in the NLS, (b) A point mutation of an amino acid in the RRM1, (c) A point mutation of an amino acid in the RRM2, (d) Deletion of at least a part of the nuclear export signal, and (e) Deletion of at least a part of the prion-like domain, resulting in the lack of a functional structural domain, the non-human animal cell according to any one of the preceding items. (Item 6) (a) The point mutation of the amino acid in the NLS includes K82A K83A, R84A, K95A, K97A, K98A, or a combination thereof, (b) The point mutation in the RRM1 includes F147L and / or F149L, (c) The point mutation in the RRM2 includes F194L and / or F229L, (d) At least a part of the deletion of the nuclear export signal deletion includes a deletion of an amino acid at and between positions 239 and 250 of the wild-type TDP-43 polypeptide, and (e) At least a part of the deletion of the prion-like domain deletion includes a deletion of an amino acid at and between positions 274 and 414 of the wild-type TDP-43 polypeptide, the non-human animal cell according to item 5. (Item 7) The non-human animal cell according to any one of the preceding items, wherein the mutant TDP-43 polypeptide includes K82A K83A, R84A, K95A, K97A, and K98A. (Item 8) The non-human animal cell according to 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) 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) 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) The non-human animal cell according to 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) The non-human animal cell according to any one of the preceding items, wherein the mutated TARDBP gene encoding the mutant TDP-43 polypeptide replaces the endogenous TARDBP gene at the endogenous TARDBP locus. (Item 13) The non-human animal cell according to Item 13, wherein the non-human animal cell is heterozygous for the mutated TARDBP gene encoding the mutant TDP-43 polypeptide. (Item 14) The non-human animal cell according to Item 13, wherein the non-human animal cell is homozygous for the mutated TARDBP gene encoding the mutant TDP-43 polypeptide. (Item 15) 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) The non-human animal cell according to Item 16, wherein the knockout mutation comprises a conditional knockout mutation. (Item 17) The non-human animal cell according to Item 15 or Item 16, wherein the knockout mutation comprises a site-specific recombination recognition sequence. (Item 18) The non-human animal cell according to any one of items 15 to 17, wherein the knockout mutation contains a loxp sequence. (Item 19) The non-human animal cell according to item 18, wherein the loxp sequence is adjacent to exon 3 of the TARDBP gene containing the knockout mutation. (Item 20) The non-human animal cell according to item 16, wherein the knockout mutation contains 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) substitution by the mutated TARDBP gene encoding a mutant TDP-43 polypeptide at the endogenous TARDBP locus on one chromosome, and (ii) either the TARDBP gene containing the knockout mutation or the wild-type TARDBP gene at the endogenous TARDBP locus on the other homologous chromosome, the non-human animal cell according to any one of items 15 to 20. (Item 22) The non-human animal cell according to any one of the preceding items, wherein the non-human animal cell does not express the wild-type TDP-43 polypeptide. (Item 23) The non-human animal cell according to any one of items 1 to 21, wherein the non-human animal cell expresses the wild-type TDP-43 polypeptide. (Item 24) (i) the mRNA transcript level of the mutated TARDBP gene comparable to the mRNA transcript level of the wild-type TARDBP gene in the control cell, (ii) an increase in the level of the mutant TDP-43 polypeptide compared to the level of the wild-type TDP-43 polypeptide in the control cell, (iii) for example, a mutant TDP-43 polypeptide found at a higher concentration in the cytoplasm than in the nucleus of motor neurons, (iv) a mutant TDP-43 polypeptide with increased insolubility compared to the wild-type TDP-43 polypeptide, (v) cytoplasmic aggregates containing the mutant TDP-43 polypeptide, (vi) increased splicing of cryptic exons, and / or (vii) decreased levels of TDP-43 forms that have undergone alternative splicing, a non-human animal cell according to any one of the preceding items. (Item 25) (i) a conditional knockout mutation of the TARDBP gene at the endogenous TARDBP locus on one chromosome, and (ii) deletion of the entire TARDBP coding sequence at the endogenous TARDBP locus on the other homologous chromosome, a non-human animal cell comprising the same. (Item 26) The non-human animal cell according to any one of the preceding items, wherein the cell is an embryonic stem (ES) cell, a primitive ectoderm cell, or a motor neuron derived from a motor neuron (ESMN). (Item 27) The non-human animal cell according to any one of the preceding items, wherein the non-human animal cell is a rodent cell. (Item 28) 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) 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) The non-human animal cell according to any one of the preceding items, wherein the non-human animal cell is cultured in vitro. (Item 31) A non-human animal tissue comprising the non-human animal cell according to any one of the preceding items. (Item 32) A composition comprising the 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 non-human animal cell that expresses a mutant TDP-43 polypeptide, the method comprising manipulating the genome of the non-human animal or non-human animal cell to include 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 a sequence shown as SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5. (Item 34) The method according to item 33, wherein the manipulation comprises replacing the endogenous TARDBP gene with the mutated TARDBP gene encoding the mutant TDP-43 polypeptide. (Item 35) The method according to item 33 or item 34, wherein the manipulation further comprises replacing the endogenous TARDBP gene with a TARDBP gene comprising a knockout mutation. (Item 36) The method according to item 35, wherein the knockout mutation comprises a conditional knockout mutation. (Item 37) The method according to item 36, further comprising culturing the cells under conditions that exclude expression of the TARDBP gene comprising the knockout mutation. (Item 38) A method for identifying a therapeutic candidate for the treatment of a disease, (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 agent; (b) evaluating the phenotype and / or TDP-43 biological activity of the non-human cell or tissue; (c) identifying the candidate agent that restores to the non-human cell or tissue a phenotype and / or TDP-43 biological activity comparable to the phenotype and / or TDP-43 biological activity of a control cell or tissue expressing wild-type TDP-43. (Item 39) A method for evaluating the biological function of the 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; (c) evaluating the phenotype and / or TDP-43 bioactivity of the genetically engineered ES cells, the primitive ectoderm derived therefrom, the motor neurons derived therefrom, or the non-human animal derived therefrom. The method as described above. (Item 40) The method according to item 38 or item 39, wherein the phenotype is evaluated by cell culture, fluorescence in situ hybridization, Western blot analysis, or a combination thereof. (Item 41) The method according to any one of items 38 to 40, wherein evaluating the phenotype includes 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. (Item 42) The method according to any one of items 38 to 41, wherein evaluating the phenotype includes determining the cellular location of the mutant TDP-43 polypeptide. (Item 43) The method according to any one of items 38 to 42, wherein evaluating the bioactivity of the mutant TDP-43 polypeptide includes measuring the splice products of a gene containing a cryptic exon regulated by TDP-43. (Item 44) The method according to item 43, wherein the gene containing a cryptic exon regulated by TDP-43 includes Crem, Fyxd2, and Clf1. (Item 45) The method according to any one of Items 38 to 44, wherein evaluating the biological activity of the mutant TDP-43 polypeptide includes measuring the level of TDP-43 that has undergone alternative splicing. (Item 46) An antisense oligonucleotide comprising a gapmer motif targeting a TDP-43 mRNA sequence encoding the PLD of the TDP-43 polypeptide and / or comprising an untranslated sequence downstream of exon 6 and upstream of exon 7. (Item 47) An siRNA comprising a sequence targeting a TDP-43 mRNA sequence encoding the PLD of the TDP-43 polypeptide and / or comprising an 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 cells according to Item 2. (Item 50) A non-human animal comprising a mutated TARDBP gene encoding a mutant TDP-43 polypeptide, 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 found in the wild-type TDP-43 polypeptide, and 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, the non-human animal. (Item 51) The non-human animal according to item 50, wherein the mutated TARDBP gene is the mutated TARDBP gene of the non-human animal. (Item 52) The non-human animal according to item 50 or item 51, wherein the mutated TARDBP gene is the mutated human TARDBP gene. (Item 53) The mutant TDP-43 polypeptide is as follows: (a) a point mutation of an amino acid in the NLS, (b) a point mutation of an amino acid in the RRM1, (c) a point mutation of an amino acid in the RRM2, (d) deletion of at least a part of the nuclear export signal, and (e) deletion of at least a part of the prion-like domain, the non-human animal according to any one of items 50 to 52, lacking a functional structural domain due to one or more of the above. (Item 54) (a) the point mutation of the amino acid in the NLS includes K82A K83A, R84A, K95A, K97A, K98A, or a combination thereof, (b) the point mutation in the RRM1 includes F147L and / or F149L, (c) the point mutation in the RRM2 includes F194L and / or F229L, (d) at least a part of the deletion of the nuclear export signal deletion includes a deletion of an amino acid at and between positions 239 and 250 of the wild-type TDP-43 polypeptide, and (e) at least a part of the deletion of the prion-like domain deletion includes a deletion of an amino acid at and between positions 274 and 414 of the wild-type TDP-43 polypeptide, the non-human animal according to item 53. (Item 55) The non-human animal according to any one of items 50 to 54, wherein the mutant TDP-43 polypeptide includes K82A K83A, R84A, K95A, K97A, and K98A. (Item 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 in the amino acids at positions 274 to 414 of the wild-type polypeptide. (Item 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) 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) The non-human animal according to any one of items 50 to 58, wherein the mutant TDP-43 polypeptide lacks the nuclear export signal in the amino acids at positions 239 to 250. (Item 60) The non-human animal according to any one of items 50 to 59, wherein the mutated TARDBP gene encoding the mutant TDP-43 polypeptide replaces the endogenous TARDBP gene at the endogenous TARDBP locus. (Item 61) The non-human animal according to item 60, wherein the non-human animal is heterozygous for the mutated TARDBP gene encoding the mutant TDP-43 polypeptide. (Item 62) The non-human animal according to any one of items 50 to 61, wherein the non-human animal further comprises a TARDBP gene containing a knockout mutation. (Item 63) The non-human animal according to item 62, wherein the knockout mutation comprises a conditional knockout mutation. (Item 64) The non-human animal according to item 62 or item 63, wherein the knockout mutation comprises a site-specific recombination recognition sequence. (Item 65) The non-human animal according to any one of items 62 to 64, wherein the knockout mutation comprises a loxp sequence. (Item 66) The non-human animal according to item 65, wherein the loxp array is adjacent to exon 3 of the TARDBP gene containing a knockout mutation. (Item 67) The non-human animal according to item 62, wherein the knockout mutation includes 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) substitution of the endogenous TARDBP gene at the endogenous TARDBP locus on one chromosome with the mutated TARDBP gene encoding a mutant TDP-43 polypeptide, and (ii) either the TARDBP gene containing the knockout mutation or the wild-type TARDBP gene at the endogenous TARDBP locus on the other homologous chromosome, the non-human animal according to any one of items 62 to 67. (Item 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) the mRNA transcript level of the mutated TARDBP gene 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 compared to the level of the wild-type TDP-43 polypeptide in a control animal, (iii) for example, a mutant TDP-43 polypeptide found at a higher concentration in the cytoplasm than in the nucleus of motor neurons, (iv) a mutant TDP-43 polypeptide with increased insolubility compared to the wild-type TDP-43 polypeptide, (v) cytoplasmic aggregates containing the mutant TDP-43 polypeptide, (vi) an increase in the splicing of cryptic exons, (vii) a decrease in the level of the TDP-43 form undergoing alternative splicing, (viii) denervation of muscle tissue mainly composed of fast-twitch muscles such as the anterior tibial muscle, and / or A non-human animal according to any one of items 49 to 69, including (ix) normal innervation of muscle tissue mainly composed of slow 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) A non-human animal according to any one of items 49 to 71, wherein the non-human animal is a rodent. (Item 73) A non-human animal according to any one of items 49 to 72, wherein the non-human animal is a rat. (Item 74) A non-human animal according to any one of items 49 to 72, wherein the non-human animal is a mouse. (Item 75) A method for identifying a therapeutic candidate for treating a disease, comprising: (a) contacting a non-human animal according to any one of items 49 to 74 with a candidate drug; (b) evaluating the phenotype and / or TDP-43 biological activity of the non-human animal; (c) identifying the candidate drug that restores the phenotype and / or TDP-43 biological activity of the non-human. (Item 76) A mutant TDP-43 polypeptide, comprising: (a) a point mutation of an amino acid in the NLS; (b) a point mutation of an amino acid in RRM1; (c) a point mutation of an amino acid in RRM2; (d) a deletion of at least a part of the nuclear export signal, and (e) a deletion of at least a part of the prion-like domain, and comprising a sequence shown as SEQ ID NO: 1, 3, or 5 modified to include one or more of the above. (Item 77) (a) the point mutation of the amino acid in the NLS includes K82A, K83A, R84A, K95A, K97A, K98A, or a combination thereof, (b) the point mutation in RRM1 includes F147L and / or F149L, (c) the point mutation in RRM2 includes F194L and / or F229L, (d) at least a part of the deletion of the nuclear export signal includes a deletion of amino acids at and between positions 239 and 250 of the wild-type TDP-43 polypeptide, and (e) at least a part of the deletion of the prion-like domain includes a deletion of amino acids at and between positions 274 and 414 of the wild-type TDP-43 polypeptide, the mutant TDP-43 polypeptide according to item 76. (Item 78) The mutant TDP-43 polypeptide according to item 76 or item 77, including the K82A mutation, K83A mutation, R84A mutation, K95A mutation, K97A mutation, and / or K98A mutation. (Item 79) The mutant TDP-43 polypeptide according to any one of items 76 to 78, including a deletion of the prion-like domain with amino acids from position 274 to 414 of the wild-type polypeptide. (Item 80) The mutant TDP-43 polypeptide according to any one of items 76 to 79, wherein the mutant TDP-43 polypeptide includes the F147L mutation and / or the F149L mutation. (Item 81) The mutant TDP-43 polypeptide according to any one of items 76 to 80, wherein the mutant TDP-43 polypeptide includes the F194L mutation and / or the F229L mutation. (Item 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 with amino acids from position 239 to 250. (Item 83) A nucleic acid comprising a nucleic acid sequence encoding a mutant TDP-43 polypeptide according to any one of items 76 to 82. (Item 84) Furthermore, 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 rodent cells, the nucleic acid according to item 83. (Item 85) The nucleic acid undergoes homologous recombination at the endogenous rat TARDBP locus, and the 5' and 3' homology arms are homologous to the rat sequence such that the nucleic acid sequence encoding the mutant TDP-43 polypeptide replaces the endogenous TARDBP coding sequence, the nucleic acid according to item 84. (Item 86) The nucleic acid undergoes homologous recombination at the endogenous mouse TARDBP locus, and the 5' and 3' homology arms are homologous to the mouse sequence such that the nucleic acid sequence encoding the mutant TDP-43 polypeptide replaces the endogenous TARDBP coding sequence, the nucleic acid according to item 84. (Item 87) While preserving the alternative TDP-43 mRNA encoding truncated TDP-43 lacking PLD in cells, TDP-43 mRNA encoding a TDP-43 polypeptide containing PLD is selectively decreased, a method comprising (i) an antisense oligonucleotide comprising a gapmer motif targeting a TDP-43 mRNA sequence encoding the PLD of the TDP-43 polypeptide and / or comprising an untranslated sequence downstream of exon 6 and upstream of exon 7, (ii) an siRNA comprising a sequence targeting a TDP-43 mRNA sequence encoding the PLD of the TDP-43 polypeptide and / or comprising an 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 of the gRNAs, wherein the gRNA recognizes a sequence at or near a sequence encoding an alternative splicing site that results in an alternative mRNA encoding a truncated TDP-43 polypeptide lacking PLD. (Item 88) Any embodiment or any applicable item category characterized by a product, process, or use, including any mutant TDP-43-expressing non-human animals, 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 produced, 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, as first described, disclosed, or illustrated in this patent application. 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, 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 found in the 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 a sequence shown as SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, the non-human animal cell. (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 the 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 as follows: (a) a point mutation of an amino acid in the NLS, (b) a point mutation of an amino acid in the RRM1, (c) a point mutation of an amino acid in the RRM2, (d) a deletion of at least a part of the nuclear export signal, and (e) a deletion of at least a part of the prion-like domain, resulting in a lack of a functional structural domain, the non-human animal cell according to any one of the preceding Items A. (Item A6) (a) the point mutation of the amino acid in the NLS includes K82A K83A, R84A, K95A, K97A, K98A, or a combination thereof, (b) the point mutation in the RRM1 includes F147L and / or F149L, (c) the point mutation in RRM2 includes F194L and / or F229L, (d) at least a part of the deletion of the nuclear export signal includes a deletion of amino acids at and between positions 239 and 250 of the wild-type TDP-43 polypeptide, and (e) at least a part of the deletion of the prion-like domain includes a deletion of amino acids at and between positions 274 and 414 of the wild-type TDP-43 polypeptide, the non-human animal cell according to item A5. (Item A7) The non-human animal cell according to any one of the preceding items A, wherein the mutant TDP-43 polypeptide includes K82A K83A, R84A, K95A, K97A, and K98A. (Item A8) The non-human animal cell according to any one of the preceding items A, wherein the mutant TDP-43 polypeptide lacks the prion-like domain with amino acids from position 274 to position 414 of the wild-type polypeptide. (Item A9) The non-human animal cell according to any one of the preceding items A, wherein the mutant TDP-43 polypeptide includes F147L and F149L. (Item A10) The non-human animal cell according to any one of the preceding items A, wherein the mutant TDP-43 polypeptide includes F194L and F229L. (Item A11) The non-human animal cell according to any one of the preceding items A, wherein the mutant TDP-43 polypeptide lacks the nuclear export signal with amino acids from position 239 to position 250. (Item A12) The non-human animal cell according to any one of the preceding items A, wherein the mutated TARDBP gene encoding the 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 the 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 the 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 includes a TARDBP gene containing a knockout mutation. (Item A16) The non-human animal cell according to item A16, wherein the knockout mutation includes a conditional knockout mutation. (Item A17) The non-human animal cell according to item A15 or item A16, wherein the knockout mutation contains 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 contains a loxp sequence. (Item A19) The non-human animal cell according to item A18, wherein the loxp sequence is adjacent to exon 3 of the TARDBP gene containing the knockout mutation. (Item A20) The non-human animal cell according to item A16, wherein the knockout mutation contains 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) substitution by the mutated TARDBP gene encoding a mutant TDP-43 polypeptide at the endogenous TARDBP locus on one chromosome, and (ii) contains either the TARDBP gene containing the knockout mutation or the wild-type TARDBP gene at the endogenous TARDBP locus on the other homologous chromosome. The non-human animal cell according to any one of items A15 to A20. (Item A22) The non-human animal cell according to any one of the preceding items A, wherein the non-human animal cell does not express the 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 the wild-type TDP-43 polypeptide. (Item A24) (i) The mRNA transcript level of the mutated TARDBP gene comparable to the mRNA transcript level of the wild-type TARDBP gene in control cells, (ii) an increase in the level of the mutant TDP-43 polypeptide compared to the level of the wild-type TDP-43 polypeptide in control cells, (iii) for example, a mutant TDP-43 polypeptide found at a higher concentration in the cytoplasm than in the nucleus of motor neurons, (iv) a mutant TDP-43 polypeptide with increased insolubility compared to the wild-type TDP-43 polypeptide, (v) cytoplasmic aggregates containing the mutant TDP-43 polypeptide, (vi) an increase in the splicing of cryptic exons, and / or (vii) a decrease in the level of the TDP-43 form undergoing alternative splicing. The non-human animal cell according to any one of the preceding items A. (Item A25) (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, in a non-human animal cell. (Item A26) The non-human animal cell according to any one of the preceding items A, wherein the cell is an embryonic stem (ES) cell, a primitive ectoderm cell, or a motor neuron derived from embryonic stem cells (ESMN). (Item A27) The non-human animal cell according to any one of the preceding items A, wherein the non-human animal cell is a rodent cell. (Item A28) The non-human animal cell according to any one of the preceding items 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) The non-human animal cell according to any one of the preceding items A, wherein the non-human animal cell is cultured in vitro. (Item A31) A non-human animal tissue comprising the non-human animal cell according to any one of the preceding items A. (Item A32) A composition comprising the non-human animal cell or tissue according to any one of the preceding items A. (Item A33) A method for producing a non-human animal or non-human animal cell that expresses a mutant TDP-43 polypeptide, the method comprising manipulating the genome of the non-human animal or non-human animal cell to include 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 a sequence shown as SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5. (Item A34) The method according to item A33, wherein the manipulation comprises replacing the 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 manipulation further comprises replacing the endogenous TARDBP gene with a TARDBP gene comprising a knockout mutation. (Item A36) The method according to item 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 eliminate the expression of the TARDBP gene containing a knockout mutation. (Item A38) A method for identifying a therapeutic candidate 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) evaluating the phenotype and / or TDP-43 biological activity of the non-human cell or tissue; (c) identifying the candidate drug that restores to the non-human cell or tissue a phenotype and / or TDP-43 biological activity comparable to that of a control cell or tissue expressing wild-type TDP-43 polypeptide. (Item A39) 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; (c) evaluating the phenotype and / or TDP-43 biological activity of the genetically engineered ES cells, primitive ectoderm derived therefrom, motor neurons derived therefrom, or non-human animals derived therefrom. (Item A40) The method according to item A38 or item A39, wherein the phenotype is evaluated by cell culture, fluorescence in situ hybridization, Western blot analysis, or a combination thereof. (Item A41) The method according to any one of items A38 to A40, wherein evaluating the phenotype comprises measuring the viability of the genetically engineered ES cells, primitive ectoderm derived therefrom, motor neurons derived therefrom, or non-human animals derived therefrom. (Item A42) The method according to any one of items A38 to A41, wherein evaluating the phenotype comprises determining the intracellular location of the mutant TDP-43 polypeptide. (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 splice products of a gene containing a cryptic exon regulated by TDP-43. (Item A44) The method according to Item A43, wherein the gene containing a cryptic exon regulated by TDP-43 comprises 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 TDP-43 that has undergone alternative splicing. (Item A46) An antisense oligonucleotide comprising a gapmer motif targeting a TDP-43 mRNA sequence encoding the PLD of the TDP-43 polypeptide and / or comprising an untranslated sequence downstream of exon 6 and upstream of exon 7. (Item A47) An siRNA comprising a sequence targeting a TDP-43 mRNA sequence encoding the PLD of the TDP-43 polypeptide and / or comprising an 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 cells according to Item A2. (Item A50) A non-human animal comprising a mutated TARDBP gene encoding a mutant TDP-43 polypeptide, 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 found in the wild-type TDP-43 polypeptide, and optionally, the wild-type TDP-43 polypeptide comprises a 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 the mutated TARDBP gene is the mutated TARDBP gene of the non-human animal. (Item A52) The non-human animal according to item A50 or item A51, wherein the mutated TARDBP gene is the mutated human TARDBP gene. (Item A53) The mutant TDP-43 polypeptide is as follows: (a) a point mutation of an amino acid in the NLS, (b) a point mutation of an amino acid in the RRM1, (c) a point mutation of an amino acid in the RRM2, (d) a deletion of at least a part of the nuclear export signal, and (e) a deletion of at least a part of the prion-like domain, the non-human animal according to any one of items A50 to A52, lacking a functional structural domain due to one or more of the above. (Item A54) (a) the point mutation of the amino acid in the NLS includes K82A K83A, R84A, K95A, K97A, K98A, or a combination thereof, (b) the point mutation in RRM1 includes F147L and / or F149L, (c) the point mutation in RRM2 includes F194L and / or F229L, (d) at least a part of the deletion of the nuclear export signal deletion includes a deletion of an amino acid at and between positions 239 and 250 of the wild-type TDP-43 polypeptide, and (e) at least a part of the deletion of the prion-like domain deletion includes a deletion of an amino acid at and between positions 274 and 414 of the wild-type TDP-43 polypeptide, the non-human animal according to item A53. (Item A55) The non-human animal according to any one of items A50 to A54, wherein the mutant TDP-43 polypeptide includes K82A K83A, R84A, K95A, K97A, and K98A. (Item 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 with amino acids from position 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 includes F147L and F149L. (Item A58) The non-human animal according to any one of items A50 to A57, wherein the mutant TDP-43 polypeptide includes 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 with amino acids from position 239 to 250. (Item A60) The non-human animal according to any one of items A50 to A59, wherein the mutated TARDBP gene encoding the 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 the 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 containing a knockout mutation. (Item A63) The non-human animal according to 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 according to item A65, wherein the loxp sequence is adjacent to exon 3 of the TARDBP gene containing the knockout mutation. (Item A67) The non-human animal according to 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) substitution of the endogenous TARDBP gene at the endogenous TARDBP locus on one chromosome with the mutated TARDBP gene encoding the mutant TDP-43 polypeptide, and (ii) either the TARDBP gene containing the knockout mutation or the wild-type TARDBP gene at the endogenous TARDBP locus on the other homologous chromosome, The non-human animal according to any one of items A62 to A67. (Item A69) The non-human animal according to any one of items A49 to A68, wherein the non-human animal expresses the wild-type TDP-43 polypeptide. (Item A70) (i) The mRNA transcript level of the mutated TARDBP gene comparable to the mRNA transcript level of the wild-type TARDBP gene in the control animal, (ii) An increase in the level of the mutant TDP-43 polypeptide compared to the level of the wild-type TDP-43 polypeptide in the control animal, (iii) For example, a mutant TDP-43 polypeptide found at a high concentration in the cytoplasm rather than in the nucleus of motor neurons, (iv) A mutant TDP-43 polypeptide with increased insolubility compared to the wild-type TDP-43 polypeptide, (v) A cytoplasmic aggregate containing the mutant TDP-43 polypeptide, (vi) An increase in the splicing of cryptic exons, (vii) A decrease in the level of the TDP-43 form that has undergone alternative splicing, (viii) Denervation of muscle tissue mainly composed of fast-twitch muscles such as the anterior tibial muscle, and / or (ix) Normal innervation of muscle tissue mainly composed of slow-twitch muscles such as the intercostal muscles, The non-human animal according to any one of items A49 to A69, comprising (Item A71) (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, A non-human animal comprising (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) A method for identifying a therapeutic candidate for the treatment of a disease, comprising: (a) Contacting a non-human animal according to any one of items A49 to A74 with a candidate drug, (b) Evaluating the phenotype and / or TDP-43 biological activity of the non-human animal, (c) Identifying the candidate drug that restores the phenotype and / or TDP-43 biological activity of the non-human, The method comprising (Item A76) A mutant TDP-43 polypeptide, comprising: (a) A point mutation of an amino acid in the NLS, (b) A point mutation of an amino acid in RRM1, (c) A point mutation of an amino acid in RRM2, (d) A deletion of at least a part of the nuclear export signal, and (e) A deletion of at least a part of the prion-like domain, The mutant TDP-43 polypeptide comprising a sequence shown as SEQ ID NO: 1, 3, or 5 modified to include one or more of (Item A77) (a) the point mutation of the amino acid in the NLS includes K82A, K83A, R84A, K95A, K97A, K98A, or a combination thereof, (b) the point mutation in RRM1 includes F147L and / or F149L, (c) the point mutation in RRM2 includes F194L and / or F229L, (d) at least a part of the deletion of the nuclear export signal deletion includes a deletion of amino acids at and between positions 239 and 250 of the wild-type TDP-43 polypeptide, and (e) at least a part of the deletion of the prion-like domain includes a deletion of amino acids at and between positions 274 and 414 of the wild-type TDP-43 polypeptide, the mutant TDP-43 polypeptide according to item A76. (Item A78) The mutant TDP-43 polypeptide according to item A76 or item A77, which includes the K82A mutation, the K83A mutation, the R84A mutation, the K95A mutation, the K97A mutation, and / or the K98A mutation. (Item A79) The mutant TDP-43 polypeptide according to any one of items A76 to A78, which includes a deletion of the prion-like domain with amino acids from position 274 to position 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 includes the F147L mutation and / or the F149L mutation. (Item A81) The mutant TDP-43 polypeptide according to any one of items A76 to A80, wherein the mutant TDP-43 polypeptide includes the F194L mutation and / or the 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 with amino acids from position 239 to position 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 82. (Item A84) Furthermore, from 5' to 3': a 5' homology arm, the nucleic acid sequence encoding the mutant TDP-43 polypeptide, and a 3' homology arm, the nucleic acid according to item A83, wherein the nucleic acid undergoes homologous recombination in rodent cells. (Item A85) The nucleic acid according to item A84, wherein the nucleic acid undergoes homologous recombination at the endogenous rat TARDBP locus, and the 5' and 3' homology arms are homologous to the rat sequence such that the nucleic acid sequence encoding the mutant TDP-43 polypeptide replaces the endogenous TARDBP coding sequence. (Item A86) The nucleic acid according to item A84, wherein the nucleic acid undergoes homologous recombination at the endogenous mouse TARDBP locus, and the 5' and 3' homology arms are homologous to the mouse sequence such that the nucleic acid sequence encoding the mutant TDP-43 polypeptide replaces the endogenous TARDBP coding sequence. (Item A87) A method for selectively reducing TDP-43 mRNA encoding a TDP-43 polypeptide containing PLD while retaining alternative TDP-43 mRNA encoding truncated TDP-43 lacking PLD in a cell, comprising: (i) an antisense oligonucleotide containing a gapmer motif targeting a TDP-43 mRNA sequence encoding PLD of the TDP-43 polypeptide and / or containing an untranslated sequence downstream of exon 6 and upstream of exon 7; (ii) an siRNA containing a sequence targeting a TDP-43 mRNA sequence encoding PLD of the TDP-43 polypeptide and / or containing an 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 guide RNA (gRNA) that 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 PLD. (Item A88) (Item A88) Any embodiment or any applicable item category, e.g., a product, a process, or a use, characterized by a variant TDP-43-expressing non-human animal, a method of producing a non-human animal, a nucleic acid for use in the method of producing a non-human animal, a cell for use in the method of producing a non-human animal, the use of the non-human animal thus produced, and cells derived from the non-human animal and / or cells expressing a variant TDP-43 polypeptide, a variant TDP-43 polypeptide and a nucleic acid encoding the variant polypeptide, an antisense oligonucleotide, or siRNA, a CRISPR / Cas system, as first described, disclosed, or illustrated in this patent application.

Claims

1. A rodent cell, comprising: (i) a mutated TARDBP gene on one chromosome at the endogenous TARDBP locus, which encodes a mutant TDP-43 polypeptide, wherein the mutated TARDBP gene encoding the mutant TDP-43 polypeptide replaces the endogenous TARDBP gene, 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 (NES), a prion-like domain (PLD), or a combination thereof found in the wild-type TDP-43 polypeptide, and the rodent cell expresses the mutant TDP-43 polypeptide, the mutated TARDBP gene, and (ii) a TARDBP gene on the other homologous chromosome at the endogenous TARDBP locus, which contains a conditional knockout mutation, wherein the TARDBP gene containing the conditional knockout mutation replaces the endogenous TARDBP gene, the TARDBP gene containing the conditional knockout mutation ; and the rodent cell is a rat cell or a mouse cell. A rodent cell.

2. The rodent cell according to claim 1, wherein the wild-type TDP-43 polypeptide comprises the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO:

5.

3. (a) the mutated TARDBP gene is the mutated TARDBP gene of the rodent cell, or (b) the mutated TARDBP gene is the mutated human TARDBP gene. The rodent cell according to claim 1 or claim 2.

4. The mutant TDP-43 polypeptide is as follows: (a) a point mutation of an amino acid in the NLS, (b) a point mutation of an amino acid in the RRM1, (c) a point mutation of an amino acid in the RRM2, (d) a deletion of at least a part of the NES, and (e) a deletion of at least a part of the PLD due to one or more of the above, resulting in the lack of a functional structural domain. The rodent cell according to any one of claims 1 to 3.

5. (a) the point mutation of the amino acid in the NLS comprises K82A, K83A, R84A, K95A, K97A, K98A, or a combination thereof with respect to the wild-type TDP-43 polypeptide, (b) the point mutation in the RRM1 includes F147L and / or F149L relative to the wild-type TDP-43 polypeptide, (c) the point mutation in the RRM2 includes F194L and / or F229L relative to the wild-type TDP-43 polypeptide, (d) at least a part of the deletion of the NES includes a deletion of amino acids at and between positions 239 and 250 of the wild-type TDP-43 polypeptide, and (e) at least a part of the deletion of the PLD includes a deletion of amino acids at and between positions 274 and 414 of the wild-type TDP-43 polypeptide. The rodent cell according to claim 4. **Claim 6** (a) the mutant TDP-43 polypeptide includes K82A, K83A, R84A, K95A, K97A, and K98A point mutations relative to the wild-type TDP-43 polypeptide, (b) the mutant TDP-43 polypeptide lacks the PLD with amino acids at and between positions 274 and 414 of the wild-type TDP-43 polypeptide, (c) the mutant TDP-43 polypeptide includes F147L and F149L point mutations relative to the wild-type TDP-43 polypeptide, (d) the mutant TDP-43 polypeptide includes F194L and F229L point mutations relative to the wild-type TDP-43 polypeptide, and / or (e) the mutant TDP-43 polypeptide lacks the NES with amino acids at and between positions 239 and 250 of the wild-type TDP-43 polypeptide. The rodent cell according to any one of claims 1 to 5. **Claim 7** The rodent cell according to any one of claims 1 to 6, wherein the conditional knockout mutation includes a site-specific recombination recognition sequence. **Claim 8** The rodent cell according to claim 7, wherein the conditional knockout mutation includes a loxp sequence. **Claim 9** The rodent cell according to claim 8, wherein the site-specific recombination recognition sequence includes a loxp sequence adjacent to exon 3 of the TARDBP gene. **Claim 10** The rodent cell according to any one of claims 1 to 9, wherein the rodent cell does not express the wild-type TDP-43 polypeptide. **Claim 11** (a) the mRNA transcript level of the mutated TARDBP gene comparable to the mRNA transcript level of the wild-type TARDBP gene in control rodent cells, (b) an increase in the level of the mutant TDP-43 polypeptide as compared to the level of the wild-type TDP-43 polypeptide in control rodent cells, (c) the mutant TDP-43 polypeptide found at a higher concentration in the cytoplasm than in the nucleus of the rodent cells, (d) the mutant TDP-43 polypeptide having increased insolubility as compared to the wild-type TDP-43 polypeptide, (e) cytoplasmic aggregates containing the mutant TDP-43 polypeptide, (f) an increase in the splicing of cryptic exons, and / or (g) a decrease in the level of TDP-43 forms that have undergone alternative splicing, The rodent cells according to any one of claims 1 to 10, comprising

12. The rodent cells according to any one of claims 1 to 11, wherein the rodent cells are embryonic stem (ES) cells, primitive ectoderm cells, or embryonic stem cell-derived motor neurons (ESMN).

13. The rodent cells according to any one of claims 1 to 12, wherein the rodent cells are mouse cells.

14. The rodent cells according to any one of claims 1 to 13, wherein the rodent cells are cultured in vitro.

15. A rodent tissue or composition comprising the rodent cells according to any one of claims 1 to 14, wherein the composition further comprises a medium.

16. A method for producing a rodent or rodent cell that expresses a mutant TDP-43 polypeptide, (a) a mutated TARDBP gene encoding the mutant TDP-43 polypeptide at the endogenous TARDBP locus on one chromosome, the mutated TARDBP gene encoding the mutant TDP-43 polypeptide replaces the endogenous TARDBP gene at the endogenous TARDBP locus, 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 (NES), a prion-like domain (PLD), or a combination thereof as compared to the wild-type TDP-43 polypeptide, the mutated TARDBP gene, and (b) a TARDBP gene containing a conditional knockout mutation at the endogenous TARDBP locus on the other homologous chromosome comprising manipulating the genome of the rodent or the rodent cell to include wherein the rodent is a rat or a mouse, or the rodent cell is a rat cell or a mouse cell, the method.

17. The method according to claim 16, wherein the wild-type TDP-43 polypeptide comprises the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, and / or the manipulating comprises replacing the endogenous TARDBP gene with the mutated TARDBP gene encoding the mutant TDP-43 polypeptide.

18. The method according to claim 16, wherein the manipulating comprises replacing the endogenous TARDBP gene with the TARDBP gene comprising the conditional knockout mutation, and / or culturing the rodent cells under conditions that eliminate the expression of the TARDBP gene comprising the conditional knockout mutation.

19. A method for identifying a therapeutic candidate agent for the treatment of a disease, comprising: (a) contacting the rodent cells according to any one of claims 1 to 14, or the rodent tissue or composition according to claim 15, with the therapeutic candidate agent; (b) evaluating the phenotype and / or TDP-43 biological activity of the rodent cells or rodent tissue; (c) identifying the therapeutic candidate agent that restores to the rodent cells or the rodent tissue a phenotype and / or TDP-43 biological activity comparable to that of a control rodent cell or rodent tissue expressing a wild-type TDP-43 polypeptide.

20. The method according to claim 19, wherein evaluating the phenotype and / or TDP-43 biological activity of the rodent cells or the rodent tissue is performed using cell culture, fluorescence in situ hybridization, Western blot analysis, or a combination thereof.

21. The method according to claim 19, wherein evaluating the phenotype of the rodent cells comprises (i) measuring the viability of genetically engineered ES cells, primitive ectoderm derived therefrom, motor neurons derived therefrom, or non-human animals derived therefrom, and / or (ii) determining the cellular location of the mutant TDP-43 polypeptide.

22. Evaluating the biological activity of the mutant TDP-43 polypeptide comprises (i) measuring the splice products of genes containing cryptic exons regulated by TDP-43, and / or (ii) measuring the levels of TDP-43 polypeptides that have undergone alternative splicing, the method according to claim 19.

23. A method for evaluating the biological function of a TDP-43 structural domain, comprising: (a) (i) A mutated TARDBP gene encoding a mutant TDP-43 polypeptide at the endogenous TARDBP locus on one chromosome, the mutated TARDBP gene encoding the mutant TDP-43 polypeptide replaces the endogenous TARDBP gene, the mutant TDP-43 polypeptide lacks a functional structural domain comprising a nuclear localization signal (NLS), RNA recognition motif 1 (RRM1), RNA recognition motif 2 (RRM2), putative nuclear export signal (NES), prion-like domain (PLD), or a combination thereof found in the wild-type TDP-43 polypeptide, the mutated TARDBP gene, and (ii) A TARDBP gene containing a conditional knockout mutation at the endogenous TARDBP locus on the other homologous chromosome, the TARDBP gene containing the conditional knockout mutation replaces the endogenous TARDBP gene, the TARDBP gene manipulating rodent embryonic stem (ES) cells to contain the above, wherein the rodent ES cells are rat ES cells or mouse ES cells, (b) evaluating the genetically engineered rodent ES cells, the primitive ectoderm derived therefrom, the motor neurons derived therefrom, or the phenotype and / or TDP-43 biological activity of the rodent derived therefrom The method as described above.

24. Following (a), the method further comprises differentiating the genetically engineered rodent ES cells in vitro and / or obtaining genetically engineered rodents from the genetically engineered rodent ES cells, the method according to claim 23.

25. The method according to claim 23, wherein evaluating the genetically engineered rodent ES cells, the primitive ectoderm derived therefrom, the motor neurons derived therefrom, or the phenotype and / or TDP-43 biological activity of the rodent derived therefrom is performed using cell culture, fluorescence in situ hybridization, Western blot analysis, or a combination thereof.

26. The method according to claim 23, wherein evaluating the genetically engineered rodent ES cells, the primitive ectoderm derived therefrom, the motor neurons derived therefrom, or the phenotype of the rodent derived therefrom comprises (i) measuring the viability of the genetically engineered rodent ES cells, the primitive ectoderm derived therefrom, the motor neurons derived therefrom, or the rodent derived therefrom, and / or (ii) determining the cellular location of the mutant TDP-43 polypeptide.

27. The method according to claim 23, wherein evaluating the biological activity of the mutant TDP-43 polypeptide comprises (i) measuring splice products of a gene comprising a cryptic exon regulated by TDP-43, and / or (ii) measuring the level of a TDP-43 polypeptide that has undergone alternative splicing.

28. The method according to claim 22 or claim 27, wherein the gene comprising a cryptic exon regulated by TDP-43 is selected from the group consisting of Crem, Fyxd2, Clf1, or a combination thereof.

Images