Anti-aggregation variants of tdp-43

Abstract

Provided herein are TDP-43 variants in which the prion-like domain (PLD) of the TDP-43 variant is mutated to aromatic amino acids having more aromatic amino acids and / or more uniformly spaced aromatic amino acids than PLD from the wild-type TDP-43, nucleic acids encoding such TDP-43 variants, and methods of using such TDP-43 variants, e.g., methods of treating TDP-43 proteinopathies.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Application No. 63 / 370,527, filed on August 5, 2022, which is incorporated herein by reference in its entirety for all purposes.

[0003] References to sequence listings submitted as text files via EFS WEB

[0004] The sequence listing written into file 599567SEQLIST.xml is 122 kilobytes, created on August 4, 2023, and is hereby incorporated by reference. Background Art

[0005] TAR DNA binding protein 43 (TDP-43) is a ubiquitous protein encoded by the highly conserved TARDBP gene. TDP-43 is a major nuclear RNA binding protein similar to members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family, which is developmentally regulated and essential for early embryonic development. TDP-43 binds thousands of RNAs and has a strong preference for UG-rich sequences. TDP-43 automatically regulates its synthesis by promoting optional cleavage events at the 3′ end of pre-mRNA. Although the entire 3D structure of TDP-43 has not been resolved, several structural features of TDP-43 protein have been identified. These include a nuclear localization signal (NLS), two RNA recognition motifs (RRM1 and RRM2), a putative nuclear extracellular transfer signal (NES), and a large domain in the carboxyl terminal half of the protein, which has been described as a low complexity, weakly ordered or prion-like domain (PLD). TDP-43 is a disease signature protein associated with several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), with 97% of ALS cases showing autopsy pathology of cytoplasmic TDP-43 aggregates. These aggregates are ubiquitinated, hyperphosphorylated, and truncated. In addition, mutations in TDP-43 are associated with ALS. Among these rare TDP-43 mutations, most mutations are present in its prion-like domain. The correlation between the physiological function of TDP 43 and these diseases remains unknown. Therefore, it is essential to understand the normal physiological role of TDP-43.

[0006] The mechanisms by which RNA binding proteins such as TDP-43 trigger neurodegeneration are not fully understood. TDP-43 is ubiquitously expressed and is thought to be involved in multiple levels of RNA metabolism, including transcription, splicing, transport, and translation. Although TDP-43 plays a key role in maintaining cellular life, the structures and functional domains by which it maintains these functions are not yet clear. Summary of the invention

[0007] Provided herein are TDP-43 variants in which the prion-like domain (PLD) of the TDP-43 variant is mutated to have more aromatic amino acids and / or more evenly spaced aromatic amino acids than the PLD from wild-type TDP-43, nucleic acids encoding such TDP-43 variants, cells and animals comprising such variants or nucleic acids, methods of making such cells and animals, and methods of using such TDP-43 variants, such as, for example, methods of treating TDP-43 proteinopathies (such as amyotrophic lateral sclerosis (ALS)).

[0008] In one aspect, TAR DNA binding protein 43 (TDP-43) variants are provided, wherein the prion-like domain (PLD) of the TDP-43 variant is mutated to have more aromatic amino acids and / or more evenly spaced aromatic amino acids than the PLD from wild-type TDP-43. In some such TDP-43 variants, the PLD is mutated to have more aromatic amino acids. In some such TDP-43 variants, the PLD is mutated to have more aromatic amino acids than the PLD from wild-type TDP-43. In some such TDP-43 variants, the PLD is mutated to have more aromatic amino acids and / or more evenly spaced aromatic amino acids than the PLD from wild-type TDP-43.

[0009] In some such TDP-43 variants, the PLD in the TDP-43 variant is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the sequence shown in SEQ ID NO: 22. In some such TDP-43 variants, the PLD in the TDP-43 variant is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the sequence shown in SEQ ID NO: 63. In some such TDP-43 variants, the PLD in the TDP-43 variant comprises, consists essentially of, or consists of the sequence shown in SEQ ID NO: 22. In some such TDP-43 variants, the PLD in the TDP-43 variant comprises, consists essentially of, or consists of the sequence shown in SEQ ID NO: 63. In some such TDP-43 variants, the TDP-43 variant is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the sequence shown in SEQ ID NO: 23. In some such TDP-43 variants, the TDP-43 variant is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the sequence shown in SEQ ID NO: 64. In some such TDP-43 variants, the TDP-43 variant comprises, consists essentially of, or consists of the sequence shown in SEQ ID NO: 23. In some such TDP-43 variants, the TDP-43 variant comprises, consists essentially of, or consists of the sequence shown in SEQ ID NO: 64.

[0010] In some such TDP-43 variants, a portion of the PLD from wild-type TDP-43 is replaced by at least a portion of the PLD from a different RNA binding protein in the variant TDP-43. In some such TDP-43 variants, the substituted portion of the PLD from wild-type TDP-43 is at least about 10, at least about 15, at least about 20, at least about 25, or at least about 28 amino acids. In some such TDP-43 variants, the substituted portion of the PLD from wild-type TDP-43 is about 10 to about 50, about 20 to about 40, or about 25 to about 35 amino acids. In some such TDP-43 variants, the substituted portion of the PLD is about 28 amino acids. In some such TDP-43 variants, the substituted portion of the PLD includes, consists essentially of, or consists of SEQ ID NO: 6, or the substituted portion of the PLD includes, consists essentially of, or consists of SEQ ID NO: 47. In some such TDP-43 variants, the different RNA binding protein is hnRNPA2B1 (e.g., human hnRNPA2B1 or mouse hnRNPA2B1). In some such TDP-43 variants, a portion of the PLD from hnRNPA2B1 is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the sequence shown in SEQ ID NO: 24. In some such TDP-43 variants, a portion of the PLD from hnRNPA2B1 is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the sequence shown in SEQ ID NO: 65. In some such TDP-43 variants, a portion of the PLD from hnRNPA2B1 comprises, consists essentially of, or consists of the sequence shown in SEQ ID NO: 24. In some such TDP-43 variants, a portion of the PLD from hnRNPA2B1 comprises, consists essentially of, or consists of the sequence set forth in SEQ ID NO: 65. In some such TDP-43 variants, the PLD in the TDP-43 variant is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the sequence set forth in SEQ ID NO: 25. In some such TDP-43 variants, the PLD in the TDP-43 variant is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the sequence set forth in SEQ ID NO: 66.In some such TDP-43 variants, the PLD in the TDP-43 variant comprises, consists essentially of, or consists of the sequence shown in SEQ ID NO: 25. In some such TDP-43 variants, the PLD in the TDP-43 variant comprises, consists essentially of, or consists of the sequence shown in SEQ ID NO: 66. In some such TDP-43 variants, the TDP-43 variant is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the sequence shown in SEQ ID NO: 26. In some such TDP-43 variants, the TDP-43 variant is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the sequence shown in SEQ ID NO: 67. In some such TDP-43 variants, the TDP-43 variant comprises, consists essentially of, or consists of the sequence set forth in SEQ ID NO: 26. In some such TDP-43 variants, the TDP-43 variant comprises, consists essentially of, or consists of the sequence set forth in SEQ ID NO: 67.

[0011] In some such TDP-43 variants, the PLD from wild-type TDP-43 is replaced by a PLD from a different RNA binding protein in the variant TDP-43. In some such TDP-43 variants, the different RNA binding protein is hnRNPA2B1 (e.g., human hnRNPA2B1 or mouse hnRNPA2B1). In some such TDP-43 variants, the PLD in the TDP-43 variant is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the sequence shown in SEQ ID NO: 27 or 13. In some such TDP-43 variants, the PLD in the TDP-43 variant is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the sequence shown in SEQ ID NO: 68 or 58. In some such TDP-43 variants, the PLD in the TDP-43 variant comprises, consists essentially of, or consists of the sequence shown in SEQ ID NO: 27 or 13. In some such TDP-43 variants, the PLD in the TDP-43 variant comprises, consists essentially of, or consists of the sequence shown in SEQ ID NO: 68 or 58. In some such TDP-43 variants, the TDP-43 variant is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the sequence shown in SEQ ID NO: 28. In some such TDP-43 variants, the TDP-43 variant is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the sequence shown in SEQ ID NO: 69. In some such TDP-43 variants, the TDP-43 variant comprises, consists essentially of, or consists of the sequence set forth in SEQ ID NO: 28. In some such TDP-43 variants, the TDP-43 variant comprises, consists essentially of, or consists of the sequence set forth in SEQ ID NO: 69.

[0012] In some such TDP-43 variants, the TDP-43 variant is less prone to aggregation than wild-type TDP-43. In some such TDP-43 variants, the TDP-43 variant retains the function of wild-type TDP-43 in splicing regulation. In some such TDP-43 variants, the TDP-43 variant is primarily nuclear and / or retains the subcellular distribution of wild-type TDP-43. In some such TDP-43 variants, the TDP-43 variant retains the function of wild-type TDP-43 during embryonic development. In some such TDP-43 variants, the TDP-43 variant is a human TDP-43 variant.

[0013] Some such TDP-43 variants are used to treat TDP-43 protein diseases in subjects, optionally wherein TDP-43 protein diseases are amyotrophic lateral sclerosis (ALS). Some such TDP-43 variants are used to prevent TDP-43 protein diseases in subjects, optionally wherein TDP-43 protein diseases are amyotrophic lateral sclerosis (ALS). Some such TDP-43 variants are used to manufacture medicines for treating TDP-43 protein diseases, optionally wherein TDP-43 protein diseases are amyotrophic lateral sclerosis (ALS). On the other hand, there is provided the use of any of the above-mentioned TDP-43 variants in the manufacture of medicines for preventing TDP-43 protein diseases, optionally wherein TDP-43 protein diseases are amyotrophic lateral sclerosis (ALS).

[0014] On the other hand, nucleic acids encoding any of the above-mentioned TDP-43 variants are provided. In some such nucleic acids, the nucleic acid is a messenger RNA. In some such nucleic acids, the nucleic acid comprises DNA, optionally wherein the DNA comprises complementary DNA (cDNA). In some such nucleic acids, the nucleic acid is in an expression construct, the expression construct comprising a promoter operably linked to the nucleic acid encoding the TDP-43 variant, optionally wherein the promoter is a neuron-specific promoter or a constitutive promoter. In some such nucleic acids, the promoter is a constitutive promoter, a tissue-specific promoter or an inducible promoter. In some such nucleic acids, the promoter is a neuron-specific promoter, optionally wherein the promoter is a synapsin-1 promoter, and optionally wherein the promoter is a human synapsin-1 promoter.

[0015] In some such nucleic acids, the nucleic acid is located in a vector. In some such nucleic acids, the vector is a viral vector. In some such nucleic acids, the viral vector is a lentiviral vector or an adeno-associated virus (AAV) vector. In some such nucleic acids, the vector is an AAV vector, optionally wherein the AAV vector is an AAV-PHP.eB vector.

[0016] In some such nucleic acids, the nucleic acid is codon-optimized for expression in human cells.

[0017] In another aspect, cells are provided comprising any of the above TDP-43 variants or comprising any of the above nucleic acids encoding TDP-43 variants. In some such cells, the TDP-43 variant is expressed. In some such cells, the cell is a mammalian cell. In some such cells, the mammalian cell is a human cell, a rodent cell, a mouse cell, or a rat cell. In some such cells, the cell is a human cell. In some such cells, the cell is a neuron, a glial cell, or a muscle cell. In some such cells, the cell is located in a subject. In some such cells, the cell is a neuron in the subject's brain.

[0018] In some such cells, endogenous TDP-43 is not expressed in cells. In some such cells, endogenous TARDBP locus includes a mutation that prevents endogenous TDP-43 from being expressed in cells. In some such cells, cells also include a preparation that reduces or eliminates endogenous TDP-43 expression in cells. In some such cells, preparations include antisense oligonucleotides or RNAi preparations targeting endogenous TARDBP messenger RNA or nucleic acids encoding the antisense oligonucleotides or RNAi preparations. In some such cells, preparations include a nuclease preparation targeting endogenous TARDBP locus or one or more nucleic acids encoding a nuclease preparation. In some such cells, nuclease preparations are zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) or clustered regularly spaced short palindromic repeats (CRISPR) related (Cas) proteins and guide RNAs. In some such cells, nuclease preparations are Cas proteins and guide RNAs, and optionally wherein Cas proteins are Cas9 proteins.

[0019] In some such cells, the cell has a genetically modified endogenous TARDBP locus, wherein the nucleic acid is integrated into the endogenous TARDBP locus. In some such cells, the cell is heterozygous for the integrated nucleic acid. In some such cells, the cell is homozygous for the integrated nucleic acid. In some such cells, the nucleic acid is operably connected to the endogenous TARDBP promoter. In some such cells, the TDP-43 variant is expressed from the endogenous TARDBP locus. In some such cells, the TDP-43 variant replaces the expression of endogenous TDP-43.

[0020] In some such cells, the cells have reduced TDP-43 aggregation compared to control cells that do not contain the TDP-43 variant or nucleic acid.

[0021] On the other hand, non-human animals comprising any of the above-mentioned TDP-43 variants or nucleic acids encoding any of the above-mentioned TDP-43 variants are provided. In some such non-human animals, the TDP-43 variants are expressed. In some such non-human animals, the non-human animals are mammals. In some such non-human animals, the non-human animals are rodents, mice or rats. In some such non-human animals, the non-human animals are mice. In some such non-human animals, the TDP-43 variants or nucleic acids are present in neurons, glial cells or muscle cells of the non-human animals. In some such non-human animals, the TDP-43 variants or nucleic acids are present in neurons. In some such non-human animals, neurons are present in the brain of the non-human animals.

[0022] In some such non-human animals, endogenous TDP-43 is not expressed in non-human animals. In some such non-human animals, endogenous TARDBP locus includes a mutation that prevents endogenous TDP-43 from being expressed in non-human animals. In some such non-human animals, non-human animals include a preparation that reduces or eliminates endogenous TDP-43 expression in non-human animals. In some such non-human animals, preparations include antisense oligonucleotides or RNAi preparations targeting endogenous TARDBP messenger RNA or nucleic acids encoding the antisense oligonucleotides or RNAi preparations. In some such non-human animals, preparations include a nuclease preparation targeting endogenous TARDBP locus or one or more nucleic acids encoding a nuclease preparation. In some such non-human animals, nuclease preparations are zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) or clustered regularly spaced short palindromic repeats (CRISPR) related (Cas) proteins and guide RNAs. In some such non-human animals, the nuclease preparation is a Cas protein and a guide RNA, optionally wherein the Cas protein is a Cas9 protein.

[0023] In some such non-human animals, the non-human animal has a genetically modified endogenous TARDBP locus, wherein the nucleic acid is integrated in the endogenous TARDBP locus. In some such non-human animals, the non-human animal is heterozygous for the nucleic acid integrated. In some such non-human animals, the non-human animal is homozygous for the nucleic acid integrated. In some such non-human animals, the nucleic acid is operably connected to the endogenous TARDBP promoter. In some such non-human animals, the TDP-43 variant is expressed from the endogenous TARDBP locus. In some such non-human animals, the TDP-43 variant replaces the expression of endogenous TDP-43.

[0024] In some such non-human animals, the non-human animal has reduced TDP-43 aggregation compared to a control non-human animal that does not contain the TDP-43 variant or nucleic acid.

[0025] In another aspect, a method for preparing any of the above-mentioned non-human animals is provided, the method comprising administering a TDP-43 variant or nucleic acid to the non-human animal. In another aspect, a method for preparing any of the above-mentioned non-human animals is provided, the method comprising: (I) (a) modifying the genome of a pluripotent non-human animal cell to include a genetically modified endogenous TARDBP locus; (b) identifying or selecting a genetically modified pluripotent non-human animal cell including a genetically modified endogenous TARDBP locus; (c) introducing the genetically modified pluripotent non-human animal cell into a non-human animal host embryo; and (d) gestating the non-human animal host embryo in a surrogate mother; or (II) (a) modifying the genome of a non-human animal one-cell stage embryo to include a genetically modified endogenous TARDBP locus; (b) selecting a genetically modified non-human animal one-cell stage embryo including a genetically modified endogenous TARDBP locus; and (c) gestating the genetically modified non-human animal one-cell stage embryo in a surrogate mother.

[0026] In another aspect, the following method is provided: the method comprises administering to a cell: (i) any of the above-mentioned TDP-43 variants; or (ii) any of the above-mentioned nucleic acids encoding TDP-43 variants. In some such methods, the TDP-43 variant is expressed. In some such methods, the cell is a mammalian cell. In some such methods, the mammalian cell is a human cell, a rodent cell, a mouse cell, or a rat cell. In some such methods, the cell is a human cell. In some such methods, the cell is a neuron, a glial cell, or a muscle cell. In some such methods, the cell is located in a subject. In some such methods, the cell is a neuron in the brain of the subject. In some such methods, the TDP-43 variant or nucleic acid is administered to the subject via intracerebroventricular injection, intracranial injection, or intrathecal injection.

[0027] In some such methods, the method includes applying to the cell a preparation that reduces or eliminates endogenous TDP-43 expression in the cell. In some such methods, the preparation includes antisense oligonucleotides or RNAi preparations targeting endogenous TARDBP messenger RNA or nucleic acids encoding the antisense oligonucleotides or RNAi preparations. In some such methods, the preparation includes a nuclease preparation targeting endogenous TARDBP locus or one or more nucleic acids encoding a nuclease preparation. In some such methods, the nuclease agent is zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) or clustered regularly spaced short palindromic repeats (CRISPR) related (Cas) protein and guide RNA. In some such methods, the nuclease preparation is Cas protein and guide RNA, and optionally wherein Cas protein is Cas9 protein.

[0028] In some such methods, the method comprises administering a nucleic acid encoding a TDP-43 variant, wherein the nuclease preparation cleaves an endogenous TARDBP locus, the nucleic acid encoding the TDP-43 variant is inserted into or recombined with the cleaved endogenous TARDBP locus, wherein the TDP-43 variant is expressed from the endogenous TARDBP locus. In some such methods, the TDP-43 variant replaces the expression of endogenous TDP-43.

[0029] In some such methods, endogenous TDP-43 in the cell is prone to aggregation, and wherein the method reduces TDP-43 aggregation in the cell. In some such methods, there is an aberrant splicing regulation of endogenous TDP-43 in the cell, and wherein the TDP-43 variant rescues the aberrant TDP-43 splicing regulation in the cell. In some such methods, there is an aberrant subcellular distribution of endogenous TDP-43 in the cell, and wherein the TDP-43 variant rescues the aberrant subcellular distribution of endogenous TDP-43 in the cell.

[0030] On the other hand, methods for treating TDP-43 proteinopathy in a subject are provided. Some such methods include administering to one or more cells in the subject: (i) any of the above-mentioned TDP-43 variants; or (ii) any of the above-mentioned nucleic acids encoding TDP-43 variants. In some such methods, the TDP-43 variants are expressed in one or more cells in the subject. On the other hand, methods for preventing TDP-43 proteinopathy in a subject are provided. Some such methods include administering to one or more cells in the subject: (i) any of the above-mentioned TDP-43 variants; or (ii) any of the above-mentioned nucleic acids encoding TDP-43 variants. In some such methods, the TDP-43 variants are expressed in one or more cells in the subject.

[0031] In some such methods, the TDP-43 proteinopathy is amyotrophic lateral sclerosis (ALS). In some such methods, the subject is a mammal. In some such methods, the subject is a human. In some such methods, one or more cells include neurons in the subject's brain. In some such methods, the TDP-43 variant or nucleic acid is administered to the subject via intraventricular injection, intracranial injection, or intrathecal injection.

[0032] In some such methods, the method also includes applying to one or more cells a preparation for reducing or eliminating endogenous TDP-43 expression in one or more cells. In some such methods, the preparation includes antisense oligonucleotides or RNAi preparations targeting endogenous TARDBP messenger RNA or nucleic acids encoding the antisense oligonucleotides or RNAi preparations. In some such methods, the preparation includes a nuclease preparation targeting endogenous TARDBP locus or one or more nucleic acids encoding a nuclease preparation. In some such methods, the nuclease agent is zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) or clustered regularly spaced short palindromic repeats (CRISPR) related (Cas) protein and guide RNA. In some such methods, the nuclease preparation is Cas protein and guide RNA, and optionally wherein Cas protein is Cas9 protein.

[0033] In some such methods, the method comprises administering a nucleic acid encoding a TDP-43 variant, wherein the nuclease preparation cleaves an endogenous TARDBP locus in one or more cells, and the nucleic acid encoding the TDP-43 variant is inserted into or recombined with the cleaved endogenous TARDBP locus, wherein the TDP-43 variant is expressed from the endogenous TARDBP locus and replaces expression of endogenous TDP-43.

[0034] In some such methods, endogenous TDP-43 in one or more cells is prone to aggregation, and wherein the method reduces TDP-43 aggregation in one or more cells. In some such methods, there is an abnormal splicing regulation of endogenous TDP-43 in one or more cells, and wherein the TDP-43 variant rescues the abnormal TDP-43 splicing regulation in one or more cells. In some such methods, there is an abnormal subcellular distribution of endogenous TDP-43 in one or more cells, and wherein the TDP-43 variant rescues the abnormal subcellular distribution of endogenous TDP-43 in one or more cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The amino acid composition of the prion-like domain (PLD) from TDP-43, hnRNPA1, and hnRNPA2B1 is shown. Aromatic residues are highlighted in orange. Biochemistry has shown that uniform spacing of aromatic residues throughout the PLD allows efficient liquid-liquid phase separation (LPPS) but prevents irreversible association that leads to aggregation. The PLD of TDP-43 has fewer and more unevenly spaced aromatic amino acids than other RNA binding proteins, suggesting that reorganizing the spacing of aromatic residues throughout the PLD may prevent aggregation.

[0036] Figure 2A The structure of TDP-43 is shown. Figure 2B To determine whether the PLD of another RNA-binding protein could replace the PLD of TDP-43, we replaced the PLD of TDP-43 with the PLD from the less aggregation-prone RNA-binding protein hnRNPA2B1.

[0037] Figure 3 Wild-type and PLDswap TDP-43 proteins are shown, which are detected at an expected size of about 43 kDa. Using an antibody that recognizes the N-terminus of TDP-43, mutant TDP-43 polypeptides lacking functional PLD are redistributed from the nucleus to the cytoplasm in ES cell-derived motor neurons. Similar to wild-type TDP-43, TDP-43PLDswap is mainly nuclear. As expected, antibodies that recognize the C-terminus of wild-type TDP-43 protein cannot detect TDP-43PLDswap protein (data not shown).

[0038] Figure 4 The lack of functional TDP-43 protein (TDP-43 - / - ) were nonviable and did not survive beyond E3.5. Similarly, embryos expressing only TDP-43 protein lacking a functional NLS ( ΔNLS / - ) or only expressing TDP-43 protein lacking functional PLD (TDP-43 ΔPLD / - Embryos expressing only the PLDswap protein were not viable, although they survived longer, suggesting that they could compensate for some of TDP-43's embryonic functions. Embryos expressing only the PLDswap protein were able to survive until birth, suggesting that this form can fully compensate for wild-type TDP-43 during embryogenesis.

[0039] Figure 5 RT-PCR analysis of the indicated TDP-43-dependent splicing events is shown. The specific splicing events monitored are shown in the mRNA schematic on the right, and the primer positions are indicated by black arrows. Cryptic exons are indicated by red boxes in the schematic. In MNs derived from ES cells, in which ΔNLS and ΔNES are the only forms of TDP-43 protein, there is a clear loss of function in splicing regulation. In mutants in which ΔPLD is the only form of TDP-43, this loss of function is less severe. In ES cell-derived MNs in which the only form of TDP-43 is the PLDswap chimeric protein, TDP-43 function is largely restored. The Adnp2 and Dnajc5 assays monitor aberrant increases in cryptic exons, while Poldip3 and Tsn monitor alternative exon skipping, and Sortilin1 monitors alternative exon increases.

[0040] Figure 6A-6BPLDswap neonatal survival is shown. Mice carrying the PLDswap protein as the sole form of TDP-43 after Cre-mediated ablation of the WT allele either ubiquitously (CAG-Cre) or restricted to neurons (SYN-Cre) ( Fig. 6A ), survived at similar levels compared to WT heterozygous control mice ( Figure 6B ). After Cre-mediated removal of the conditional WT allele across genotypes, TDP-43 ΔEx3 / ΔEx3 (CAG-Cre, n=18; SYN-Cre, n=12), TDP-43 ΔEx3 / WT (CAG-Cre, n=7; SYN-Cre, n=9), and TDP-43 ΔEx3 / PLDswap Survival curves of CAG-Cre, n=5; SYN-Cre, n=7. The median survival time is as follows: For CAG-Cre: TDP-43 ΔEx3 / ΔEx3 : 4 weeks; TDP-43 ΔEx3 / WT : 17.36 weeks; TDP-43 ΔEx3 / PLDswap : 29.43 weeks; for SYN-Cre: TDP-43 ΔEx3 / ΔEx3 : 3.93 weeks; TDP-43 ΔEx3 / WT : 52 weeks; TDP-43 ΔEx3 / PLDswap : 37 weeks.

[0041] Figure 7 PLDswap nascent RT-PCR analysis showing TDP-43-dependent splicing. RT-PCR analysis showing the indicated TDP-43-dependent splicing events. The specific splicing events monitored are shown in the mRNA schematic on the right, and the primer positions are indicated by black arrows. Adnp2 is monitoring the abnormal increase of cryptic exons, while Tsn is monitoring variable exon skipping. The cryptic exons in Adnp2 are shown as boxes between exon 2 and exon 3 in the schematic. The loss of TDP-43 (ΔEx3 / ΔEx3) triggers mis-splicing of both Adnp2 and Tsn, both of which tend to rescue when PLDswap is the only form of TDP-43.

[0042] definition

[0043] The terms "protein", "polypeptide", and "peptide" used interchangeably herein include amino acids of any length in polymeric form, including coded amino acids and non-coded amino acids as well as chemically or biochemically modified amino acids or chemically or biochemically derivatized amino acids. These terms also include polymers that have been modified, such as polypeptides with modified peptide backbones. The term "domain" refers to any portion of a protein or polypeptide that has a specific function or structure.

[0044] The terms "nucleic acid" and "polynucleotide", which are used interchangeably herein, include polymeric forms of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, or analogs or modified forms thereof. These terms include single-stranded, double-stranded, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers containing purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural, or derived nucleotide bases.

[0045] The term "expression vector" or "expression construct" or "expression cassette" refers to a recombinant nucleic acid containing a desired coding sequence operably linked to appropriate nucleic acid sequences necessary for expression of the operably linked coding sequence in a particular host cell or organism. The nucleic acid sequences necessary for expression in prokaryotes typically include a promoter, an operator (optional), and a ribosome binding site, as well as other sequences. It is well known that eukaryotic cells utilize promoters, enhancers, and termination and polyadenylation signals, but some elements may be deleted and others added without sacrificing the necessary expression.

[0046] The term "viral vector" refers to a recombinant nucleic acid that contains at least one element of viral origin and contains elements sufficient or allowing packaging into viral vector particles. The vector and / or particle can be used for the purpose of transferring DNA, RNA or other nucleic acids into cells in vitro or in vivo. Various forms of viral vectors are known.

[0047] The term "isolated" with respect to proteins, nucleic acids and cells includes proteins, nucleic acids and cells that are relatively purified relative to other cellular or biological components that may normally be present in situ, up to and including substantially pure preparations of proteins, nucleic acids or cells. The term "isolated" may include proteins and nucleic acids that do not have naturally occurring counterparts, or that have been chemically synthesized and are therefore substantially uncontaminated by other proteins or nucleic acids. The term "isolated" may include proteins, nucleic acids or cells that have been separated or purified from most other cellular components or biological components (e.g., but not limited to other cellular proteins, nucleic acids or cellular or extracellular components) that naturally accompany the protein, nucleic acid or cell.

[0048] The term "wild-type" includes an entity having structure and / or activity as found in a normal (as compared to a mutation, disease, alteration, etc.) state or condition. Wild-type genes and polypeptides typically exist in multiple different forms (eg, alleles).

[0049] The term "endogenous sequence" refers to a nucleic acid sequence naturally present in a cell or animal. For example, an endogenous TARDBP sequence of an animal refers to a native TARDBP sequence naturally present at the TARDBP locus of the animal.

[0050] "Exogenous" molecules or sequences include molecules or sequences that are not usually present in the cell in this form, or molecules or sequences introduced into the cell from an external source. Normal existence includes the presence of a specific developmental stage and environmental conditions of the cell. For example, an exogenous molecule or sequence may include a mutant form of a corresponding endogenous sequence in the cell (such as a humanized form of an endogenous sequence), or may include a sequence that corresponds to an endogenous sequence in the cell but is in a different form (i.e., not in a chromosome). In contrast, an endogenous molecule or sequence includes a molecule or sequence that is usually present in the form in a specific cell at a specific developmental stage under specific environmental conditions.

[0051] When used in the context of a nucleic acid or protein, the term "heterologous" indicates that the nucleic acid or protein comprises at least two segments that do not naturally occur together in the same molecule. For example, when used with respect to a nucleic acid segment or a protein segment, the term "heterologous" indicates that the nucleic acid or protein comprises two or more subsequences that are not found in the same relationship (e.g., linked together) to each other in nature. As an example, a "heterologous" region of a nucleic acid vector is a nucleic acid segment that is not found in nature in association with another nucleic acid molecule or attached to it. For example, a heterologous region of a nucleic acid vector may comprise a coding sequence that is flanked by a heterologous promoter that is not found in nature in association with the coding sequence. Similarly, a "heterologous" region of a protein is an amino acid segment that is not found in nature in association with another peptide molecule (e.g., a fusion protein or a protein with a tag) or attached to it. Similarly, a nucleic acid or protein may comprise a heterologous marker or a heterologous secretion or localization sequence.

[0052] "Codon optimization" utilizes the degeneracy of codons, as shown by the diversity of the three base pair codon combinations of the specified amino acid, and generally includes the process of modifying the nucleic acid sequence to enhance expression in a specific host cell by replacing at least one codon of the native sequence with a more frequent or most frequently used codon in the gene of the host cell while maintaining the native amino acid sequence. For example, compared with a naturally occurring nucleic acid sequence, the nucleic acid encoding TAR DNA binding protein 43 (TDP-43) protein can be modified to replace a codon with a higher frequency of use in a given prokaryotic or eukaryotic cell, the prokaryotic or eukaryotic cell including bacterial cells, yeast cells, human cells, non-human cells, mammalian cells, rodent cells, mouse cells, rat cells, hamster cells or any other host cell. Codon usage tables are readily available, such as at "codon usage databases". These tables can be modified in a variety of ways. See Nakamura et al., (2000) Nucleic Acids Research 28:292, which is incorporated herein by reference as a whole for all purposes. Computer algorithms are also available for codon optimization of a specific sequence for expression in a specific host (see, e.g., Gene Forge).

[0053] "Promoter" is a regulatory region of DNA, which generally comprises a TATA box capable of guiding RNA polymerase II to initiate RNA synthesis at an appropriate transcription start site of a specific polynucleotide sequence. The promoter may also comprise other regions that affect the transcription start rate. The promoter sequence disclosed herein regulates the transcription of an operably linked polynucleotide. The promoter may be active in one or more cell types in a cell type disclosed herein (e.g., eukaryotic cells, non-human mammalian cells, human cells, rodent cells, pluripotent cells, single-cell embryos, differentiated cells, or a combination thereof). The promoter may be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a time-limited promoter (e.g., a promoter regulated by development) or a spatially limited promoter (e.g., a cell-specific or tissue-specific promoter). Examples of promoters may be present in, for example, WO 2013 / 176772, which is incorporated herein by reference in its entirety for all purposes.

[0054] A constitutive promoter is a promoter that is active in all tissues or specific tissues at all developmental stages. Examples of constitutive promoters include human cytomegalovirus immediate early (hCMV) promoter, mouse cytomegalovirus immediate early (mCMV) promoter, human elongation factor 1α (hEF1α) promoter, mouse elongation factor 1α (mEF1α) promoter, mouse phosphoglycerate kinase (PGK) promoter, chicken β-actin hybrid (CAG or CBh) promoter, SV40 early promoter, and β2 tubulin promoter.

[0055] Examples of inducible promoters include, for example, chemically regulated promoters and physically regulated promoters. Chemically regulated promoters include, for example, alcohol regulated promoters (e.g., alcohol dehydrogenase (alcA) gene promoters), tetracycline regulated promoters (e.g., tetracycline responsive promoters, tetracycline operator sequences (tetO), tet-On promoters or tet-Off promoters), steroid regulated promoters (e.g., rat glucocorticoid receptor, estrogen receptor promoter or ecdysone receptor promoter) or metal regulated promoters (e.g., metalloprotein promoters). Physically regulated promoters include, for example, temperature regulated promoters (e.g., heat shock promoters) and light regulated promoters (e.g., light-inducible promoters or light-inhibitory promoters).

[0056] The tissue-specific promoter can be, for example, a neuron-specific promoter or a glial-specific promoter or a muscle-specific promoter.

[0057] Developmentally regulated promoters include, for example, promoters that are active only during embryonic development or only in adult cells.

[0058] "Operable linkage" or "operably linked" includes the juxtaposition of two or more components (e.g., a promoter and another sequence element) so that both components function normally and allow at least one component to mediate a function imposed on at least one other component. For example, a promoter can be operably linked to a coding sequence if it controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. Operable linkage can include such sequences that are adjacent to each other or act in trans (e.g., a regulatory sequence can act at a distance to control the transcription of a coding sequence).

[0059] The term "in vitro" includes artificial environments and processes or reactions that occur within artificial environments (e.g., test tubes or isolated cells or cell lines). The term "in vivo" includes natural environments (e.g., cells, organisms, or the body) and processes or reactions that occur within natural environments. The term "ex vivo" includes cells that have been removed from an individual's body and processes or reactions that occur within such cells.

[0060] A composition or method that “comprises” or “includes” one or more of the recited elements may include other elements not specifically recited. For example, a composition that “comprises” or “includes” a protein may contain the protein alone or in combination with other ingredients. The transition phrase “consisting essentially of” means that the scope of the claim should be interpreted to encompass the specified elements recited in the claim as well as those elements that do not materially affect the basic and novel characteristics of the claimed invention. Therefore, when used in the claims of the present invention, the term “consisting essentially of” should not be interpreted as being equivalent to “comprising.”

[0061] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur and that the description includes instances where said event or circumstance occurs and instances where it does not occur.

[0062] The designation of a numerical range includes all integers within or defining the range and all subranges defined by the integers within the range. For example, 5-10 nucleotides is understood to be 5, 6, 7, 8, 9 or 10 nucleotides, and 5%-10% is understood to include 5% and all possible values ​​up to 10%.

[0063] At least 17 nucleotides in a 20 nucleotide sequence are understood to include 17, 18, 19 or 20 nucleotides in the sequence provided, thereby providing an upper limit, even if an upper limit is not clearly provided as will be clearly understood. Similarly, at most 3 nucleotides will be understood to cover 0, 1, 2 or 3 nucleotides, thereby providing a lower limit, even if a lower limit is not clearly provided. When "at least", "at most" or other similar language modifies a number, it can be understood to modify each number in the series.

[0064] As used herein, "no more than" or "less than" is understood to mean the value adjacent to the phrase and any logically lower value or integer to zero that is reasonable from the context. For example, a duplex region of "no more than 2 nucleotide base pairs" has 2, 1, or 0 nucleotide base pairs. When "no more than" or "less than" is preceded by a series of numbers or ranges, it is understood that each number in the series or range is modified.

[0065] As used herein, it is understood that when the maximum amount of a value is represented by 100% (eg, 100% inhibition), the value is limited by the detection method. For example, 100% inhibition is understood to be inhibition to a level below the detection level of the assay.

[0066] Unless the context indicates otherwise, the term "about" encompasses values ​​of ±5% of the stated value. In certain embodiments, the term "about" should be understood to encompass variations or errors that are tolerated in the art, such as 2 standard deviations from the mean or the sensitivity of the method used to make the measurement, or a percentage of a value that is tolerated in the art, such as changes with age. When "about" is present before the first value in a series, it can be understood to modify each value in the series.

[0067] The term "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items as well as the lack of a combination when interpreted in the alternative ("or").

[0068] The term "or" refers to any one member of a particular list and also includes any combination of members of that list.

[0069] As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a protein" or "at least one protein" may include a plurality of proteins, including mixtures thereof.

[0070] Statistically significant means p≤0.05.

[0071] In the event of a conflict between a sequence in this application and a designated accession number or position in an accession number, the sequence in this application controls. DETAILED DESCRIPTION

[0072] I. Overview

[0073] Provided herein are TDP-43 variants in which the prion-like domain (PLD) of the TDP-43 variant is mutated to have more aromatic amino acids and / or more evenly spaced aromatic amino acids than the PLD from wild-type TDP-43, nucleic acids encoding such TDP-43 variants, and methods of using such TDP-43 variants, such as, for example, methods of treating TDP-43 protein diseases such as amyotrophic lateral sclerosis (ALS). ALS is a devastating neurodegenerative disease that affects motor neurons, leading to paralysis and eventual death. An almost ubiquitous pathological finding in autopsies of ALS patient tissue is the accumulation of TDP-43 (trans-responsive DNA binding protein 43 kDa) in cytoplasmic inclusion bodies. TDP-43 is a major nuclear RNA binding protein that is structurally similar to members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family, which is essential for the survival of all mammalian cells and the normal development and life of animals. The redistribution of TDP-43 from the nucleus to the cytoplasm and its accumulation in insoluble aggregates are two key diagnostic hallmarks of ALS disease. The biological functions of TDP-43 may not yet be fully elucidated, but there is evidence that this protein is involved in the regulation of pre-messenger RNA (pre-mRNA) splicing by preventing the use of cryptic exons in large introns and by affecting the alternative splicing of several pre-mRNAs. TDP-43 has also been proposed to have functions in the cytoplasm, possibly in RNA shuttling between the nucleus and the cytoplasm and in mRNA transport within neuronal axons.

[0074] Several structural features of the TDP-43 protein have been identified, including a nuclear localization signal (NLS), two RNA recognition motifs (RRM1 and RRM2), a putative nuclear extracellular transport signal (NES), and a large domain in the carboxyl-terminal half of the protein that has been described as a low-complexity, weakly ordered, or prion-like domain (PLD). Of the TDP-43 mutations associated with familial cases of ALS, the majority are found in the PLD.

[0075] Studies of TDP-43 domain mutants identified two mutants (ΔPLD and ΔNLS) that are viable as ES cell-derived motor neurons and cause mislocalization of TDP-43 protein from the nucleus to the cytoplasm, inducing cytoplasmic aggregation. Mice expressing only these mutants (ΔPLD / - and ΔNLS / -) are embryonic lethal. Mice heterozygous for ΔNLS (ΔNLS / +) and ΔPLD (ΔPLD / +) are viable and exhibit TDP-43 mislocalization, aggregation, insolubility, and phosphorylation.

[0076] In ΔPLD / + mice, the mutant (~30kDa) protein can be distinguished from the native (~43kDa) WT protein. PTDP-43ΔPLD protein is almost exclusively present in the cytoplasmic fraction, but some wild-type TDP-43 is also present in the cytoplasmic fraction. The wild-type protein, but not the ΔPLD protein, is phosphorylated and present in the detergent-insoluble fraction. These results suggest that the presence of mutant TDP-43 protein affects the behavior of the wild-type protein. In other experiments, ΔNLS / + mice were found to have progressive motor system degeneration. Mice heterozygous for ΔPLD exhibit degeneration, but unlike ΔNLS, this degeneration does not progress between the 4th and 6th months. TDP-43 without PLD can keep adult motor neurons alive, and less aggregation is seen in cells with ΔPLD as the only form of TDP-43. This suggests that the PLD of TDP-43 mediates aggregate formation, suggesting that TDP-43 with PLD removed or recombined may provide new therapeutic approaches. Biochemistry has shown that uniform spacing of aromatic residues throughout the PLD allows efficient liquid-liquid phase separation but prevents irreversible association that leads to aggregation. The PLD of TDP-43 has fewer and more unevenly spaced aromatic amino acids than other RNA-binding proteins, suggesting that reorganizing the spacing of aromatic residues throughout the PLD could prevent aggregation.

[0077] To test this, we designed and created TDP-43 variants in which the prion-like domain (PLD) of the TDP-43 variant was mutated to have more aromatic amino acids and / or more evenly spaced aromatic amino acids than the PLD from wild-type TDP-43. As an example, we created a PLDswap allele that replaced the PLD of TDP-43 with the PLD of hnRNPA2B1. Compared with the PLD of TDP-43, the PLD of hnRNPA2B1 is composed of evenly spaced aromatic residues. Embryonic stem (ES) cells and ES cell-derived motor neurons with the PLDswap allele as the only form of TDP-43 are viable and show normal TDP-43 subcellular distribution. Replacing the PLD of TDP-43 with the PLD of hNRNPA2B1 also rescued the splicing defects associated with the loss of functional TDP-43. This suggests that a potential therapeutic approach could be to remove wild-type TDP-43 and replace it with a modified anti-aggregation form. It is also possible that simply replacing the wild-type protein will rescue phenotypes associated with the loss of functional TDP-43.

[0078] II. TDP-43 variants

[0079] Variants of TAR DNA binding protein 43 (TDP-43) are provided herein. Human TDP-43 is designated as UniProt reference number Q13148. The human gene encoding TDP-43 (TARDBP or TDP43) is designated as NCBI GeneID 23435 and is present at position 1p36.22 on chromosome 1 (assembly: GRCh38.p14 (GCF_000001405.40); Position: NC_000001.11 (11012654..11030528)). At least two isoforms of human TDP-43 are known. The first isoform is 414 amino acids and is designated as UniProt reference number Q13148-1 and NCBI reference number NP_031401.1 (SEQ ID NO: 1). An exemplary coding sequence is designated as reference number CCDS122.1 (SEQ ID NO: 2), and an exemplary mRNA (cDNA) sequence is designated as reference number NM_007375.4 (SEQ ID NO: 3). A second isoform is 298 amino acids and is designated as UniProt reference number Q13148-2 (SEQ ID NO: 4).

[0080] Mouse TDP-43 is designated as UniProt reference number Q921F2. The mouse gene encoding TDP-43 (TARDBP or TDP43) is designated as NCBI GeneID 230908 and is present at position 4; 4E2 on chromosome 4 (assembly: GRCm39 (GCF_000001635.27); position: NC_000070.7 (148696839..148711672, complementary sequence)). The standard isoform is 414 amino acids and is designated as UniProt reference number Q921F2-1 and NCBI reference number NP_663531.1 (SEQ ID NO: 43). An exemplary coding sequence is designated as reference number CCDS38971.1 (SEQ ID NO: 44), and an exemplary mRNA (cDNA) sequence is designated as reference number NM_145556.4 (SEQ ID NO: 45).

[0081] TDP-43 is a highly conserved and ubiquitously expressed RNA / DNA binding protein that belongs to the heterogeneous nuclear ribonucleoprotein (hnRNP) family. TDP-43 is critical for a variety of cellular functions, including the regulation of RNA metabolism, mRNA transport, microRNA maturation, and stress granule formation. Consistent with its nuclear and cytoplasmic functions, TDP-43 can shuttle between the nucleus and the cytoplasm, but under normal physiological conditions, it is mainly located in the nucleus. Related to brain function, TDP-43 appears to be essential for the normal development of central neuronal cells in early embryogenesis.

[0082] The dysfunction of TDP-43 related pathways has been increasingly recognized as an important pathogenic mechanism in neurodegenerative diseases. Hyperphosphorylated and ubiquitinated TDP-43 cytoplasmic inclusions have been identified as pathological features of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Pathogenic missense mutations in the TARDBP gene encoding TDP-43 protein were subsequently identified as pathogenic genetic mutations in both ALS and FTLD, although only a small percentage in familial cases. The vast majority of patients with ALS and FTLD do not have TARDBP gene mutations, but show a wide range of abnormalities involving TDP-43. TDP-43 deposition has been associated with more and more neurodegenerative diseases, in which it has been identified as the main pathogenic factor, causing these diseases to be called TDP-43 protein diseases.

[0083] The protein structure of TDP-43 consists of an N-terminal region, a nuclear localization signal (NLS), two RNA recognition motifs: RRM1 and RRM2, a nuclear extracellular transfer signal (NES), and a C-terminal region containing a prion-like domain (PLD). PLD is a subset of low-complexity regions, rich in uncharged polar amino acids and glycine, similar to yeast prion protein, and can be defined using a hidden Markov algorithm. PLD is typically found in RNA-binding proteins, which drive protein aggregation in neurodegenerative diseases such as amyotrophic lateral sclerosis. Although primarily localized in the nucleus, TDP-43 shuttles between the nucleus and the cytoplasm, a process mediated by active and passive transport, in which it plays a physiological function. In addition, TDP-43 is localized in mitochondria, where it binds to the mitochondrial genome and is important in the respiratory chain pathway. The most famous function of TDP-43 is to regulate the splicing of cryptic exons and variable exons. Cryptic exons are intronic sequences that are mistakenly recognized as exons by the splicing machinery. GU-rich TDP-43 binding sites are often present near cryptic exons in large introns. TDP-43 inhibits the recognition of cryptic exons in large introns to promote normal splicing. The loss of TDP-43 leads to abnormal splicing with cryptic exons, causing the loss of normal RNA and protein. In addition, TDP-43 regulates the alternative splicing of specific target RNAs. This regulation includes exon gain or exon exclusion and can affect transcripts involved in the pathogenesis of ALS.

[0084] The N-terminal domain is important in the formation of functional homodimers, which is essential for the correct physiological function of TDP-43. Located in the N-terminal region is the NLS domain (amino acid 82-98), which mediates TDP-43 import into the nucleus, where it exerts its physiological function. RNA binding motifs (RRM1 (amino acid 106-176) and RRM2 (amino acid 191-262)) are necessary for TDP-43 protein to bind to RNA / DNA molecules, regulate transcription, translation, splicing and stability of mRNA, and mediate RNA export. In addition, TDP-43 forms ribonucleoprotein (RNP) particles, which are important for the transport of mRNA molecules and for promoting the biogenesis of non-coding RNAs such as microRNAs (miRNAs). Separately, the prion-like domain (PLD; amino acids 274-414; SEQ ID NO: 5 in human TDP-43; SEQ ID NO: 46 in mouse TDP-43) has been implicated in TDP-43 pathogenesis, as this region regulates protein solubility and mediates pathological aggregation. TDP-43 is also important for the formation of stress granules, which protect neuronal cells from cellular damage such as oxidative stress.

[0085] Biochemistry has shown that uniform spacing of aromatic residues throughout the PLD allows for efficient liquid-liquid phase separation, but prevents irreversible associations that lead to aggregation. The PLD of TDP-43 has fewer and more unevenly spaced aromatic amino acids than other RNA binding proteins, indicating that reorganizing the spacing of aromatic residues throughout the PLD can prevent aggregation. Provided herein are engineered TDP-43 variants that are less prone to aggregation than wild-type TDP-43. Provided herein are TDP-43 variants (e.g., human TDP-43 variants), wherein the PLD of the TDP-43 variant is mutated to have more aromatic amino acids and / or more evenly spaced aromatic amino acids than the PLD from wild-type TDP-43. The three aromatic amino acids are tyrosine, phenylalanine, and tryptophan. In some cases, the TDP-43 variants described herein are mutated to have more aromatic amino acids than the PLD from wild-type TDP-43. In some cases, the TDP-43 variants described herein are mutated to have more evenly spaced aromatic amino acids than the PLD from wild-type TDP-43. In some cases, the TDP-43 variants described herein are mutated to have more aromatic amino acids and more evenly spaced aromatic amino acids than the PLD from wild-type TDP-43.

[0086] In one example, the PLD of the TDP-43 variant is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the sequence shown in SEQ ID NO: 22. In one example, the PLD of the TDP-43 variant comprises the sequence shown in SEQ ID NO: 22. In one example, the PLD of the TDP-43 variant consists essentially of the sequence shown in SEQ ID NO: 22. In one example, the PLD of the TDP-43 variant consists of the sequence shown in SEQ ID NO: 22. In one example, the TDP-43 variant is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the sequence shown in SEQ ID NO: 23. In one example, the TDP-43 variant comprises the sequence shown in SEQ ID NO: 23. In one example, the TDP-43 variant consists essentially of the sequence shown in SEQ ID NO: 23. In one example, the TDP-43 variant consists of the sequence shown in SEQ ID NO:23.

[0087] In one example, the PLD of the TDP-43 variant is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the sequence shown in SEQ ID NO: 63. In one example, the PLD of the TDP-43 variant comprises the sequence shown in SEQ ID NO: 63. In one example, the PLD of the TDP-43 variant consists essentially of the sequence shown in SEQ ID NO: 63. In one example, the PLD of the TDP-43 variant consists of the sequence shown in SEQ ID NO: 63. In one example, the TDP-43 variant is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the sequence shown in SEQ ID NO: 64. In one example, the TDP-43 variant comprises the sequence shown in SEQ ID NO: 64. In one example, the TDP-43 variant consists essentially of the sequence shown in SEQ ID NO: 64. In one example, the TDP-43 variant consists of the sequence shown in SEQ ID NO:64.

[0088] In one example, a portion of the PLD from wild-type TDP-43 is replaced by at least a portion of the PLD from a different RNA binding protein in the variant TDP-43. In one example, the replaced portion of the PLD from wild-type TDP-43 and / or the portion of the PLD from the different RNA binding protein is at least about 10, at least about 15, at least about 20, at least about 25, or at least about 28 amino acids. In one example, the replaced portion of the PLD from wild-type TDP-43 and / or the portion of the PLD from the different RNA binding protein is about 10 to about 50, about 20 to about 40, about 25 to about 35, about 10 to about 45, about 10 to about 40, about 10 to about 35, about 10 to about 30, about 15 to about 50, about 20 to about 50, about 25 to about 50, or about 28 to about 50 amino acids. In another example, the substituted portion of the PLD from wild-type TDP-43 and / or the portion of the PLD from a different RNA binding protein is about 10 to about 140, about 20 to about 140, about 30 to about 140, about 40 to about 140, about 50 to about 140, about 60 to about 140, about 70 to about 140, about 80 to about 140, about 90 to about 140, about 100 to about 140, about 110 to about 140, about 120 to about 140, about 130 to about 140, about 28 to about 130, about 28 to about 120, about 28 to about 110, about 28 to about 100, about 28 to about 90, about 28 to about 80, about 28 to about 70, about 28 to about 60, about 28 to about 50, or about 28 to about 40 amino acids. In another example, the substituted portion of the PLD from wild-type TDP-43 and / or the portion of the PLD from a different RNA binding protein is about 10 to about 50, about 20 to about 40, or about 25 to about 35 amino acids. In another example, the substituted portion of the PLD from wild-type TDP-43 and / or the portion of the PLD from a different RNA binding protein is about 28 amino acids.

[0089] For example, a highly conserved twenty-eight amino acid sequence within the PLD of TDP-43, shown to be important for aggregation in yeast, can be substituted with a sequence from a PLD of a different RNA binding protein. For example, the substituted portion of the PLD from wild-type TDP-43 can include the sequence shown in SEQ ID NO:6. In another example, the substituted portion of the PLD from wild-type TDP-43 can consist essentially of the sequence shown in SEQ ID NO:6. In another example, the substituted portion of the PLD from wild-type TDP-43 can consist essentially of the sequence shown in SEQ ID NO:6. For example, the substituted portion of the PLD from wild-type TDP-43 can include the sequence shown in SEQ ID NO:47. In another example, the substituted portion of the PLD from wild-type TDP-43 can consist essentially of the sequence shown in SEQ ID NO:47. In another example, the substituted portion of the PLD from wild-type TDP-43 can consist of the sequence shown in SEQ ID NO:47.

[0090] In another example, a portion of the PLD from a different RNA binding protein is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the sequence shown in SEQ ID NO: 24. In another example, a portion of the PLD from a different RNA binding protein comprises the sequence shown in SEQ ID NO: 24. In another example, a portion of the PLD from a different RNA binding protein consists essentially of the sequence shown in SEQ ID NO: 24. In another example, a portion of the PLD from a different RNA binding protein consists of the sequence shown in SEQ ID NO: 24. In one example, the PLD of a TDP-43 variant is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the sequence shown in SEQ ID NO: 25. In one example, the PLD of a TDP-43 variant comprises the sequence shown in SEQ ID NO: 25. In one example, the PLD of the TDP-43 variant consists essentially of the sequence shown in SEQ ID NO: 25. In one example, the PLD of the TDP-43 variant consists of the sequence shown in SEQ ID NO: 25. In one example, the TDP-43 variant is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the sequence shown in SEQ ID NO: 26. In one example, the TDP-43 variant comprises the sequence shown in SEQ ID NO: 26. In one example, the TDP-43 variant consists essentially of the sequence shown in SEQ ID NO: 26. In one example, the TDP-43 variant consists of the sequence shown in SEQ ID NO: 26.

[0091] In another example, a portion of the PLD from a different RNA binding protein is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the sequence shown in SEQ ID NO:65. In another example, a portion of the PLD from a different RNA binding protein comprises the sequence shown in SEQ ID NO:65. In another example, a portion of the PLD from a different RNA binding protein consists essentially of the sequence shown in SEQ ID NO:65. In another example, a portion of the PLD from a different RNA binding protein consists of the sequence shown in SEQ ID NO:65. In one example, the PLD of a TDP-43 variant is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the sequence shown in SEQ ID NO:66. In one example, the PLD of a TDP-43 variant comprises the sequence shown in SEQ ID NO:66. In one example, the PLD of the TDP-43 variant consists essentially of the sequence shown in SEQ ID NO: 66. In one example, the PLD of the TDP-43 variant consists of the sequence shown in SEQ ID NO: 66. In one example, the TDP-43 variant is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the sequence shown in SEQ ID NO: 67. In one example, the TDP-43 variant comprises the sequence shown in SEQ ID NO: 67. In one example, the TDP-43 variant consists essentially of the sequence shown in SEQ ID NO: 67. In one example, the TDP-43 variant consists of the sequence shown in SEQ ID NO: 67.

[0092] In one example, the PLD from the wild-type TDP-43 is replaced by the PLD from a different RNA binding protein in the variant TDP-43. In one example, the PLD from the different RNA binding protein is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the sequence shown in SEQ ID NO: 13 or 27. In one example, the PLD from the different RNA binding protein comprises the sequence shown in SEQ ID NO: 13 or 27. In another example, the PLD from the different RNA binding protein consists essentially of the sequence shown in SEQ ID NO: 13 or 27. In another example, the PLD from the different RNA binding protein consists of the sequence shown in SEQ ID NO: 13 or 27. In one example, the TDP-43 variant is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the sequence shown in SEQ ID NO: 28. In one example, the TDP-43 variant comprises the sequence shown in SEQ ID NO: 28. In one example, the TDP-43 variant consists essentially of the sequence shown in SEQ ID NO: 28. In one example, the TDP-43 variant consists of the sequence shown in SEQ ID NO: 28.

[0093] In one example, the PLD from the wild-type TDP-43 is replaced by the PLD from the different RNA binding protein in the variant TDP-43. In one example, the PLD from the different RNA binding protein is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the sequence shown in SEQ ID NO: 58 or 68. In one example, the PLD from the different RNA binding protein comprises the sequence shown in SEQ ID NO: 58 or 68. In another example, the PLD from the different RNA binding protein consists essentially of the sequence shown in SEQ ID NO: 58 or 68. In another example, the PLD from the different RNA binding protein consists of the sequence shown in SEQ ID NO: 58 or 68. In one example, the TDP-43 variant is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the sequence shown in SEQ ID NO: 69. In one example, the TDP-43 variant comprises the sequence shown in SEQ ID NO: 69. In one example, the TDP-43 variant consists essentially of the sequence shown in SEQ ID NO: 69. In one example, the TDP-43 variant consists of the sequence shown in SEQ ID NO: 69.

[0094] In one example, the PLD from the wild-type TDP-43 is replaced by the PLD from a different RNA binding protein in the variant TDP-43. In one example, the PLD from the different RNA binding protein is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the sequence shown in SEQ ID NO: 21. In one example, the PLD from the different RNA binding protein comprises the sequence shown in SEQ ID NO: 21. In another example, the PLD from the different RNA binding protein consists essentially of the sequence shown in SEQ ID NO: 21. In another example, the PLD from the different RNA binding protein consists of the sequence shown in SEQ ID NO: 21.

[0095] In one example, the PLD from wild-type TDP-43 is replaced by the PLD from a different RNA binding protein in the variant TDP-43. In one example, the PLD from the different RNA binding protein is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the sequence shown in SEQ ID NO:73. In one example, the PLD from the different RNA binding protein comprises the sequence shown in SEQ ID NO:73. In another example, the PLD from the different RNA binding protein consists essentially of the sequence shown in SEQ ID NO:73. In another example, the PLD from the different RNA binding protein consists of the sequence shown in SEQ ID NO:73.

[0096] The different RNA binding protein may be a protein that is less prone to aggregation than wild-type TDP-43. Likewise, the different RNA binding protein may be an RNA binding protein that has more aromatic residues in the PLD and / or has more evenly spaced aromatic residues in the PLD compared to wild-type TDP-43.

[0097] An example of such RNA binding protein is heterogeneous nuclear ribonucleoprotein A2 / B1 (hnRNP A2 / B1 or hnRNPA2B1). hnRNPA2B1 is a heterogeneous nuclear ribonucleoprotein (hnRNP) that binds to nascent pre-mRNA and packages them into hnRNP particles. The arrangement of hnRNP particles on nascent hnRNA is non-random and sequence-dependent, and is used to condense and stabilize transcripts and minimize tangling and knotting. Packaging plays a role in multiple processes, such as transcription, pre-mRNA processing, RNA nuclear transfer, subcellular localization, mRNA translation, and the stability of mature mRNA. Human hnRNPA2B1 is designated as UniProt reference number P22626. The human gene encoding hnRNPA2B1 (HNRNPA2B1 or HNRPA2B1) is designated as NCBI GeneID 3181 and is present on chromosome 7 at position 7p15.2 (assembly: GRCh38.p14 (GCF_000001405.40); position: NC_000007.14 (26189927..26200746, complementary sequence)). At least two isoforms of human hnRNPA2B1 are known. The first isoform is designated as UniProt reference number P22626-1 and NCBI reference number NP_112533.1 (SEQ ID NO:7). An exemplary coding sequence is designated as reference number CCDS43557.1 (SEQ ID NO:8), and an exemplary mRNA (cDNA) sequence is designated as reference number NM_031243.3 (SEQ ID NO:9). The second isoform is designated as UniProt reference number P22626-2 and NCBI reference number NP_002128.1 (SEQ ID NO: 10). An exemplary coding sequence is designated as reference number CCDS5397.1 (SEQ ID NO: 11), and an exemplary mRNA (cDNA) sequence is designated as reference number NM_002137.3 (SEQ ID NO: 12). The PLD of human hnRNPA2B1 is shown in SEQ ID NO: 13 or 27.

[0098] Mouse hnRNPA2B1 is designated as UniProt reference number O88569. The human gene encoding hnRNPA2B1 (Hnrnpa2b1 or Hnrpa2b1) is designated as NCBI GeneID 53379 and is present on chromosome 6 at position 6; 6B3 (assembly: GRCm39 (GCF_000001635.27); position: NC_000072.7 (51437414..51448054, complementary sequence)). At least three isoforms of mouse hnRNPA2B1 are known. A second isoform is designated as UniProt reference number O88569-1 and NCBI reference numbers NP_001361674.1 and XP_006506436.2 (SEQ ID NO: 48). The exemplary coding sequence is designated as reference number CCDS90046.1 (SEQ ID NO: 49), and the exemplary mRNA (cDNA) sequence is designated as reference number XM_006506373.3 (SEQ ID NO: 50) and NM_001374745.1 (SEQ ID NO: 51). The second isoform is designated as UniProt reference number O88569-2 and NCBI reference number NP_058086.2 (SEQ ID NO: 52). The exemplary coding sequence is designated as reference number CCDS51774.1 (SEQ ID NO: 53), and the exemplary mRNA (cDNA) sequence is designated as reference number NM_016806.3 (SEQ ID NO: 54). The third isoform is designated as UniProt reference number O88569-3 and NCBI reference number NP_872591.1 (SEQ ID NO: 55). An exemplary coding sequence is designated as reference number CCDS51773.1 (SEQ ID NO: 56), and an exemplary mRNA (cDNA) sequence is designated as reference number NM_182650.4 (SEQ ID NO: 57). The PLD of mouse hnRNPA2B1 is shown in SEQ ID NO: 58 or 68.

[0099] Another example of such an RNA binding protein is heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1). hnRNPA1 is involved in packaging pre-mRNA into hnRNP particles, transporting poly(A) mRNA from the nucleus to the cytoplasm, and regulating the selection of splice sites. Human hnRNPA1 is designated as UniProt reference number P09651. The human gene encoding hnRNPA1 (HNRNPA1 or HNRPA1) is designated as NCBI GeneID 3178 and is present at position 12q13.13 on chromosome 12 (assembly: GRCh38.p14 (GCF_000001405.40); position: NC_000012.12 (54280726..54287087)). At least three isoforms of human hnRNPA1 are known. The first isoform is designated as UniProt reference number P09651-1 and NCBI reference number NP_112420.1 (SEQ ID NO: 14). The exemplary coding sequence is designated as reference number CCDS44909.1 (SEQ ID NO: 15), and the exemplary mRNA (cDNA) sequence is designated as reference number NM_031157.4 (SEQ ID NO: 16). The second isoform is designated as UniProt reference number P09651-2 and NCBI reference number NP_002127.1 (SEQ ID NO: 17). The exemplary coding sequence is designated as reference number CCDS41793.1 (SEQ ID NO: 18), and the exemplary mRNA (cDNA) sequence is designated as reference number NM_002136.4 (SEQ ID NO: 19). The PLD of human hnRNPA2B1 (second isoform) is shown in SEQ ID NO: 21. The third isoform was designated UniProt reference number P09651-3 (SEQ ID NO: 20).

[0100] Mouse hnRNPA1 is assigned the UniProt reference number P49312. The human gene encoding hnRNPA1 (Hnrnpa1 or Hnrpa1) is assigned the NCBI GeneID 15382 and is present on chromosome 15 at position 15F3; 15 58.58 cM (assembly: GRCm39 (GCF_000001635.27); position: NC_000081.7 (103148370..103155125)). At least two isoforms of mouse hnRNPA1 are known. The first isoform is assigned the UniProt reference number P49312-1 and the NCBI reference number NP_034577.1 (SEQ ID NO: 59). An exemplary coding sequence is designated as reference CCDS37233.1 (SEQ ID NO: 60), and an exemplary mRNA (cDNA) sequence is designated as reference NM_010447.5 (SEQ ID NO: 61). A second isoform is designated as UniProt reference P49312-2 (SEQ ID NO: 62). The PLD of mouse hnRNPA2B1 is shown in SEQ ID NO: 73.

[0101] In some cases, TDP-43 variants are less prone to aggregation than wild-type TDP-43. In some cases, TDP-43 variants retain the function of wild-type TDP-43 in splicing regulation (e.g., retaining most of the function of wild-type TDP-43 in regulating the splicing of Adnp2, Dnajc5, Tsn, and / or Sortilin1). In some cases, TDP-43 variants are mainly nuclei. In some cases, TDP-43 variants retain the subcellular distribution of wild-type TDP-43. In some cases, TDP-43 variants retain the function of wild-type TDP-43 during embryonic development.

[0102] III. Nucleic Acids Encoding TDP-43 Variants

[0103] Provided herein are nucleic acids or nucleic acid constructs encoding TDP-43 variants. These nucleic acids or nucleic acid constructs can be isolated nucleic acid constructs.

[0104] In some cases, the nucleic acid encoding the TDP-43 variant can be codon-optimized (e.g., codon-optimized for expression in humans or in mice). For example, the nucleic acid can be modified to replace codons that have a higher frequency of usage in human cells, non-human cells, mammalian cells, rodent cells, mouse cells, rat cells, or any other host cell of interest.

[0105] The nucleic acid encoding the TDP-43 variant can be DNA or RNA. In some cases, the nucleic acid can be a messenger RNA (mRNA) encoding the TDP-43 variant. In some cases, the nucleic acid can be a complementary DNA (cDNA) encoding the TDP-43 variant. Examples of coding sequences of some TDP-43 variants described herein are shown, for example, in SEQ ID NOs: 70-72. For example, such nucleic acids may contain only coding sequences without any inserted introns. In other cases, the nucleic acid may contain one or more introns separating exons in the TDP-43 variant coding sequence. For example, the nucleic acid may contain a genomic sequence comprising both exons and introns.

[0106] In some cases, the nucleic acid is located in an expression construct comprising a nucleic acid encoding a TDP-43 variant operably connected to a promoter. The promoter can be any suitable promoter for in vivo expression in an animal or in vitro in an isolated cell. The promoter can be a constitutively active promoter (e.g., a CAG promoter or a U6 promoter), a conditional promoter, an inducible promoter, a time-limited promoter (e.g., a developmentally regulated promoter) or a spatially limited promoter (e.g., a cell-specific or tissue-specific promoter). Such promoters are well known and discussed elsewhere herein. In a specific example, the promoter is active in neurons. In a specific example, the promoter is active in glial cells. In a specific example, the promoter is active in muscle cells. In some cases, the promoter is a heterologous promoter (i.e., a promoter that is not naturally operably connected to a TDP-43 nucleic acid). In other cases, the promoter can be an endogenous promoter (i.e., a TDP-43 variant nucleic acid operably connected to a TDP-43 promoter). The heterologous promoter can be any type of promoter disclosed elsewhere herein. For example, the promoter can be a constitutive promoter, such as the EF1α promoter. Alternatively, the promoter can be a tissue-specific promoter or an inducible promoter. For example, the promoter can be a neuron-specific promoter. An example of a suitable neuron-specific promoter is the synapsin-1 promoter (e.g., the human synapsin-1 promoter). For example, the promoter can be a glial-specific promoter. For example, the promoter can be a muscle-specific promoter.

[0107] The nucleic acids and expression constructs disclosed herein may also comprise post-transcriptional regulatory elements, such as woodchuck hepatitis virus post-transcriptional regulatory elements.

[0108] The nucleic acids and expression constructs may also comprise one or more polyadenylation signal sequences. For example, the nucleic acid construct may comprise a polyadenylation signal sequence positioned at the 3' end of the TDP-43 variant coding sequence. Any suitable polyadenylation signal sequence may be used. The term polyadenylation signal sequence refers to any sequence that directs transcription termination and adds a poly-A tail to an mRNA transcript. In eukaryotes, transcription terminators are recognized by protein factors, and polyadenylation is followed by termination, which is the process of adding a poly(A) tail to an mRNA transcript in the presence of a poly(A) polymerase. Mammalian poly(A) signals are typically composed of a core sequence of about 45 nucleotides in length, which may be flanked by different auxiliary sequences for enhancing cleavage and polyadenylation efficiency. The core sequence consists of an upstream element (AATAAA or AAUAAA) that is highly conserved in mRNA, known as a poly A recognition motif or poly A recognition sequence, which is recognized by cleavage and polyadenylation specificity factor (CPSF); and a poorly defined downstream region (enriched in Us or Gs and Us) that is constrained by cleavage stimulating factor (CstF). Examples of transcription terminators that can be used include, for example, human growth hormone (HGH) polyadenylation signal, simian virus 40 (SV40) late polyadenylation signal, rabbit β-globin polyadenylation signal, bovine growth hormone (BGH) polyadenylation signal, phosphoglycerate kinase (PGK) polyadenylation signal, AOX1 transcription termination sequence, CYC1 transcription termination sequence, or any transcription termination sequence known to be suitable for regulating gene expression in eukaryotic cells.

[0109] The nucleic acid and expression construct may also include a polyadenylation signal sequence upstream of the TDP-43 variant coding sequence. The polyadenylation signal sequence upstream of the TDP-43 variant coding sequence may be flanked by a recombinase recognition site recognized by a site-specific recombinase. In some constructs, the recombinase recognition site is also flanked by a selection cassette comprising, for example, a coding sequence of a drug resistance protein. In other constructs, the recombinase recognition site is not flanked by a selection cassette. The polyadenylation signal sequence prevents transcription and expression of a protein or RNA encoded by the coding sequence. However, after exposure to a site-specific recombinase, the polyadenylation signal sequence will be excised and the protein or RNA can be expressed.

[0110] If the polyadenylation signal sequence is excised in a tissue-specific or developmental stage-specific manner, such configurations can achieve tissue-specific expression or developmental stage-specific expression. If the animal comprising the nucleic acid or expression construct also comprises a coding sequence of a site-specific recombinase operably linked to a tissue-specific or developmental stage-specific promoter, excision of the polyadenylation signal sequence in a tissue-specific or developmental stage-specific manner can be achieved. Then, the polyadenylation signal sequence will be excised only in those tissues or at those developmental stages, thereby achieving tissue-specific expression or developmental stage-specific expression. In one example, the TDP-43 variant encoded by the nucleic acid or expression construct can be expressed in a neuron-specific manner. In one example, the TDP-43 variant encoded by the nucleic acid or expression construct can be expressed in a glial-specific manner. In one example, the TDP-43 variant encoded by the nucleic acid or expression construct can be expressed in a muscle-specific manner.

[0111] Site-specific recombinases include enzymes that can promote recombination between recombinase recognition sites, wherein two recombination sites are physically separated in a single nucleic acid or on a separate nucleic acid. The example of recombinase includes Cre, Flp and Dre recombinases. An example of Cre recombinase gene is Crei, wherein two exons encoding Cre recombinase are separated by introns to prevent it from being expressed in prokaryotic cells. Such recombinases may further include a nuclear localization signal for promoting localization to the nucleus (e.g., NLS-Crei). The recombinase recognition site includes a nucleotide sequence that is recognized by the site-specific recombinase and can be used as a substrate for a recombination event. The example of recombinase recognition site includes FRT, FRT11, FRT71, attp, att, rox and lox sites, such as loxP, lox511, lox2272, lox66, lox71, loxM2 and lox5171.

[0112] Nucleic acids disclosed herein may include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), which may be single-stranded or double-stranded, and may be linear or circular. Nucleic acid constructs may be naked nucleic acids, or may be delivered by vectors such as AAV vectors, as described elsewhere herein. If in linear form, the ends of the nucleic acids may be protected by well-known methods (e.g., from exonucleolytic degradation). For example, one or more dideoxynucleotide residues may be added to the 3' end of a linear molecule and / or a self-complementary oligonucleotide may be attached to one or both ends. See, e.g., Chang et al., (1987) Proc. Natl. Acad. Sci. USA 84: 4959-4963 and Nehls et al., (1996) Science 272: 886-889, each of which is incorporated herein by reference in its entirety for all purposes. Other methods for protecting exogenous polynucleotides from degradation include, but are not limited to, adding terminal amino groups and using modified internucleotide bonds, such as phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues. In some cases, the nucleic acid or expression construct may include one or more of the following terminal structures: a hairpin, a loop, an inverted terminal repeat (ITR), or a torus. For example, the nucleic acid or expression construct may include an ITR.

[0113] The nucleic acid or expression construct may include modifications or sequences that provide additional desired features (e.g., modified or regulated stability; tracking or detection with fluorescent markers; binding sites for proteins or protein complexes, etc.). For example, one or more nucleosides within the mRNA may be modified. Examples of chemical modifications to mRNA nucleobases include pseudouridine, 1-methyl-pseudouridine, and 5-methyl-cytidine. The mRNA may also be capped. The mRNA may also be polyadenylated (to include a poly(A) tail). As an example, a capped and polyadenylated mRNA containing N1-methyl-pseudouridine may be used (e.g., it may be completely replaced with N1-methyl-pseudouridine). The nucleic acid construct may include one or more fluorescent markers, purification tags, epitope tags, or combinations thereof. For example, the nucleic acid construct may include one or more fluorescent markers (e.g., fluorescent proteins or other fluorophores or dyes), such as at least 1, at least 2, at least 3, at least 4, or at least 5 fluorescent markers. Exemplary fluorescent labels include fluorophores such as fluorescein (e.g., 6-carboxyfluorescein (6-FAM)), Texas Red, HEX, Cy3, Cy5, Cy5.5, Pacific Blue, 5-(and-6)-carboxytetramethylrhodamine (TAMRA), and Cy7. A variety of fluorescent dyes are commercially available for labeling oligonucleotides (e.g., from Integrated DNA Technologies). The tag or label can be located at the 5' end, 3' end, or internal to the nucleic acid construct. For example, the nucleic acid construct can be conjugated to a 5' end with a fluorescent dye from Integrated DNA Technologies ( 700) is conjugated with the IR700 fluorophore.

[0114] Nucleic acid or expression construct may also include conditional alleles. Conditional alleles may be multifunctional alleles, as described in US2011 / 0104799, which is incorporated herein by reference for all purposes. For example, conditional alleles may include: (a) a driver sequence in a sense orientation relative to the transcription of a target gene; (b) a drug selection cassette (DSC) in a sense or antisense orientation; (c) a nucleotide sequence of interest (NSI) in an antisense orientation; and (d) a conditional reversal module (COIN, which utilizes exon splitting introns and reversible gene trap-like modules) in the opposite orientation. See, for example, US2011 / 0104799. Conditional alleles may also include recombinant units, which recombinant units reorganize to form conditional alleles after exposure to a first recombinase, the conditional allele (i) lacking a promoter sequence and a DSC; and (ii) containing an NSI in a sense orientation and a COIN in an antisense orientation. See, e.g., US 2011 / 0104799.

[0115] Nucleic acid or expression construct may also include polynucleotides encoding selection markers. Alternatively, nucleic acid or expression construct may lack polynucleotides encoding selection markers. Selection markers may be included in selection boxes. Optionally, selection boxes may be self-deletion boxes. See, for example, US 8,697,851 and US 2013 / 0312129, each of which is incorporated herein by reference in its entirety for all purposes. As an example, self-deletion boxes may include Crei genes operably connected to mouse Prm1 promoter (comprising two exons separated by introns encoding Cre recombinase) and neomycin resistance genes operably connected to human ubiquitin promoter. By adopting Prm1 promoter, self-deletion boxes can be specifically deleted in male germ cells of F0 animals. Exemplary selection markers include neomycin phosphotransferase (neo r ), hygromycin B phosphotransferase (hyg r ), puromycin-N-acetyltransferase (puromycin r ), blasticidin-S deaminase (bsr r ), xanthine / guanine phosphoribosyltransferase (gpt) or herpes simplex virus thymidine kinase (HSV-k) or a combination thereof. The polynucleotide encoding the selection marker can be operably linked to a promoter that is active in the targeted cell. Examples of promoters are described elsewhere herein.

[0116] Nucleic acid or expression construct may also include reporter genes. Exemplary reporter genes include genes encoding: luciferase, beta-galactosidase, green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (eBFP), DsRed, ZsGreen, MmGFP, mPlum, mCherry, tdTomato, mStrawberry, J-Red, mOrange, mKO, mCitrine, Venus, YPet, emerald, CyPet, Cerulean, T- sky blue and alkaline phosphatase. Such reporter genes may be operably linked to a promoter active in the targeted cell. Examples of promoters are described elsewhere herein.

[0117] IV. Vector

[0118] Also provided herein are vectors comprising nucleic acids, nucleic acid constructs or expression constructs encoding TDP-43 variants. The vectors may include additional sequences, such as an origin of replication, a promoter, and genes encoding antibiotic resistance.

[0119] Some vectors can be circular. Alternatively, the vector can be linear. The vector can be packaged to be delivered by lipid nanoparticles, liposomes, non-lipid nanoparticles or viral capsids. Non-limiting exemplary vectors include plasmids, phagemids, cosmids, artificial chromosomes, minichromosomes, transposons, viral vectors and expression vectors.

[0120] Nucleic acid or expression construct can be located in a vector, such as a viral vector. The viral vector can be, for example, an adeno-associated virus (AAV) vector or a lentivirus (LV) vector (i.e., a recombinant AAV vector or a recombinant LV vector). Other exemplary viruses / viral vectors include retroviruses, adenoviruses, vaccinia viruses, poxviruses, and herpes simplex viruses. The virus can infect dividing cells, non-dividing cells, or both dividing cells and non-dividing cells. The virus can be integrated into the host genome, or alternatively not integrated into the host genome. Such viruses can also be engineered to have reduced immunity. The virus may have replication ability or may have replication defects (e.g., defects in one or more genes necessary for replication and / or packaging of additional rounds of virions). The virus can cause transient expression, long-term expression (e.g., at least 1 week, 2 weeks, 1 month, 2 months, or 3 months) or permanent expression. Exemplary viral titers (e.g., AAV titers) include about 10 12 vector genomes / mL, about 10 13 vector genomes / mL, about 10 14 vector genomes / mL, about 10 15 vector genomes / mL and about 10 16 Other exemplary viral titers (e.g., AAV titers) include about 10 12 vector genome (vg) / kg body weight, about 10 13 vg / kg body weight, about 10 14 vg / kg body weight, about 10 15 vg / kg body weight and about 10 16 vg / kg body weight.

[0121] In one example, the nucleic acid or expression construct is located in an AAV vector. AAV can be any suitable serotype and can be single-stranded AAV (ssAAV) or self-complementary AAV (scAAV). The ssDNA AAV genome consists of two open reading frames Rep and Cap, which are flanked by two reverse terminal repeats that allow synthesis of complementary DNA chains. When constructing an AAV transfer plasmid, the transgene is placed between the two ITRs, and Rep and Cap can be provided in trans. In addition to Rep and Cap, AAV may also require an auxiliary plasmid containing adenovirus genes. These genes (E4, E2a and VA) mediate AAV replication. For example, transfer plasmids, Rep / Cap and auxiliary plasmids can be transfected into HEK293 cells containing adenovirus genes E1+ to produce infectious AAV particles. Alternatively, Rep, Cap and adenovirus auxiliary genes can be combined into a single plasmid. Similar packaging cells and methods can be used for other viruses, such as retroviruses.

[0122] A variety of serotypes of AAV have been identified. These serotypes differ in the cell types they infect (i.e., their tropism), allowing preferential transduction of specific cell types. The serotypes of CNS tissues include AAV1, AAV2, AAV4, AAV5, AAV8, and AAV9. For example, the selectivity of AAV serotypes for gene delivery in neurons is discussed in Hammond et al., (2017) PLoS One 12 (12): e0188830, which is incorporated herein by reference for all purposes. In a specific example, an AAV-PHP.eB vector is used. The AAV-PHP.eB vector exhibits a high ability to cross the blood-brain barrier, increasing its CNS transduction efficiency. In another specific example, an AAV9 vector is used. The serotypes used for skeletal muscle transduction include, for example, AAV1, AAV2, AAV6, and AAV9. See, e.g., Riaz et al., (2015) Skeletal Muscle, Vol. 5, No. 37, which is incorporated herein by reference in its entirety for all purposes.

[0123] Tropism can be further refined by pseudotypes, which are mixtures of capsids and genomes from different viral serotypes. For example, AAV2 / 5 indicates a virus containing a serotype 2 genome packaged in a capsid from serotype 5. The use of pseudotyped viruses can improve transduction efficiency as well as alter tropism. Hybrid capsids derived from different serotypes can also be used to alter viral tropism. For example, AAV-DJ contains hybrid capsids from eight serotypes and exhibits high infectivity in a wide range of in vivo cell types. AAV-DJ8 is another example showing the properties of AAV-DJ, but with enhanced brain uptake. AAV serotypes can also be modified by 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.

[0124] To accelerate transgene expression, 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, transgene expression may be delayed. To address this delay, scAAV containing complementary sequences that can spontaneously anneal after infection can be used, thereby eliminating the need for host cell DNA synthesis. However, single-stranded AAV (ssAAV) vectors can also be used.

[0125] To increase packaging capacity, a longer transgene can be split between two AAV transfer plasmids, the first with a 3' splice donor and the second with a 5' splice acceptor. Upon co-infection of the cells, these viruses form concatemers, splice together, and the full-length transgene can be expressed. While this allows for expression of longer transgenes, the expression efficiency is low. A similar approach for increasing capacity utilizes homologous recombination. For example, a transgene can be split between two transfer plasmids but with a large amount of sequence overlap so that co-expression induces homologous recombination and expression of the full-length transgene.

[0126] V. Lipid Nanoparticles

[0127] Also provided herein are lipid nanoparticles comprising a TDP-43 variant or a nucleic acid, nucleic acid construct, expression construct or vector encoding a TDP-43 variant.

[0128] Lipid formulations can protect biomolecules from degradation while improving their cellular uptake. Lipid nanoparticles are particles comprising multiple lipid molecules physically associated with each other by intermolecular forces. These particles include microspheres (including single-layer and multi-layer vesicles, for example, liposomes), dispersed phases in emulsions, micelles or internal phases in suspensions. Such lipid nanoparticles can be used to encapsulate one or more nucleic acids or proteins for delivery. Formulations containing cationic lipids can be used to deliver polyanions such as nucleic acids. Other lipids that can be included are neutral lipids (i.e., uncharged or zwitterionic lipids), anionic lipids, auxiliary lipids for enhancing transfection, and stealth lipids that increase the length of time that nanoparticles can exist in vivo. Examples of suitable cationic lipids, neutral lipids, anionic lipids, auxiliary lipids, and stealth lipids can be found in WO 2016 / 010840 A1, which is incorporated herein by reference as a whole for all purposes. Exemplary lipid nanoparticles can include cationic lipids and one or more other components. In one example, other components can include auxiliary lipids such as cholesterol. In another example, other components can include auxiliary lipids such as cholesterol and neutral lipids such as DSPC. In another example, other components may include a helper lipid such as cholesterol, an optional neutral lipid such as DSPC, and a stealth lipid such as S010, S024, S027, S031, or S033.

[0129] LNPs may include one or more or all of the following: (i) lipids for encapsulation and endosomal escape; (ii) neutral lipids for stability; (iii) auxiliary lipids for stability; and (iv) stealth lipids. See, for example, Finn et al., (2018) Cell Rep. 22(9): 2227-2235 and WO 2017 / 173054 A1, each of which is incorporated herein by reference in its entirety for all purposes. Specific examples of using LNPs for delivery to the brain are disclosed in Nabhan et al., (2016) Sci. Rep. 6: 20019, which is incorporated herein by reference in its entirety for all purposes.

[0130] VI. Composition

[0131] Also provided herein are compositions comprising TDP-43 variants or nucleic acids, nucleic acid constructs, expression constructs, vectors or lipid nanoparticles disclosed herein. Such compositions can be used, for example, to administer TDP-43 variants to cells or subjects, or to express TDP-43 variants in cells or subjects. Such compositions can be used, for example, to inhibit or reduce TDP-43 aggregation in cells or subjects. Such compositions can be used, for example, to rescue abnormal TDP-43 shear regulation in cells or subjects. Such compositions can be used, for example, to rescue abnormal subcellular distribution of endogenous TDP-43 in cells or subjects. Such compositions can be used, for example, to treat TDP-43 protein diseases in subjects. Such compositions can be used, for example, to prevent TDP-43 protein diseases in subjects.

[0132] VII. Cells or Animals

[0133] Also provided are cells or subjects (eg, animals or non-human animals) comprising a TDP-43 variant or nucleic acid, nucleic acid construct, expression construct, vector or lipid nanoparticle disclosed herein. These cells or subjects may express a TDP-43 variant.

[0134] These cells or subjects can be, for example, mammals, non-human mammals and humans. Mammals can be, for example, non-human mammals, humans, rodents, rats, mice or hamsters. Other non-human mammals include, for example, non-human primates, monkeys, apes, cats, dogs, rabbits, horses, bulls, deer, bison, livestock (e.g., bovine species, such as cows, bulls, etc.; ovine species, such as sheep, goats, etc.; and porcine species, such as pigs and wild boars). The term "non-human" does not include humans.

[0135] The cell can be an isolated cell (e.g., in vitro), or can be in a subject (e.g., an animal or mammal). The cell can also be in any type of undifferentiated or differentiated state. In one example, the cell is a neuron. In one example, the cell is a glial cell. In one example, the cell is a muscle cell.

[0136] The cells provided herein can be normal healthy cells, or can be diseased cells comprising TDP-43 aggregates or abnormal TDP-43 function (e.g., abnormal TDP-43 splicing regulation or abnormal TDP-43 subcellular localization). These cells can be, for example, susceptible to TDP-43 aggregation, or they can have pre-existing TDP-43 aggregation.

[0137] In one example, the cell is a human cell, a rodent cell, a mouse cell or a rat cell, such as a human neuron, a rodent neuron, a mouse neuron or a rat neuron, or a human glial cell, a rodent glial cell, a mouse glial cell or a rat glial cell, or a human muscle cell, a rodent muscle cell, a mouse muscle cell or a rat muscle cell. In a specific example, the cell is a human neuron. In a specific example, the cell is a human glial cell. In a specific example, the cell is a human muscle cell. In a specific example, the cell is in a subject (e.g., a neuron in a subject's brain or a glial cell in a subject's brain or a muscle cell in a subject's body).

[0138] In some such cells or animals, endogenous TDP-43 is not expressed in cells or animals. For example, the endogenous TARDBP locus may include a mutation that prevents endogenous TDP-43 from being expressed in cells or animals. Similarly, cells or animals may include a preparation that reduces or eliminates endogenous TDP-43 expression in cells. Such preparations are described in more detail below. For example, the preparation may include an antisense oligonucleotide or RNAi preparation targeting endogenous TARDBP messenger RNA, or a nucleic acid encoding the antisense oligonucleotide or RNAi preparation. The preparation may also include a nuclease preparation targeting endogenous TARDBP locus or one or more nucleic acids encoding a nuclease preparation. For example, a nuclease preparation may be a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or a clustered regularly spaced short palindromic repeat (CRISPR)-related (Cas) protein and guide RNA. In a specific example, the nuclease preparation is a Cas protein and a guide RNA (e.g., a Cas9 protein and a guide RNA). Other examples of suitable preparations are described in more detail elsewhere herein.

[0139] Some cells or animals have genetically modified endogenous TARDBP loci, wherein nucleic acids encoding TDP-43 variants are integrated into the endogenous TARDBP loci. Cells or animals can be heterozygous or homozygous for the integrated nucleic acids. The integrated nucleic acids can be operably connected to the endogenous TARDBP promoter, or it can be operably connected to an exogenous promoter. In one example, TDP-43 variants are expressed from the endogenous TARDBP loci and replace the expression of endogenous TDP-43.

[0140] Such cells or animals may have any phenotype associated with a TDP-43 variant described herein. For example, the cell or animal has reduced TDP-43 aggregation compared to a control cell that does not contain a TDP-43 variant or nucleic acid.

[0141] Such cells or animals can be prepared by any suitable method. For example, a method of preparing such cells or animals may include administering a TDP-43 variant or nucleic acid to the cells or animals. Suitable administration methods are described in more detail elsewhere herein.

[0142] Provided are various methods for preparing non-human animal genomes, non-human animal cells and non-human animals including genetically modified endogenous TARDBP loci as described elsewhere herein. Any convenient method or scheme for producing a genetically modified organism is applicable to producing this genetically modified non-human animal. See, for example, Cho et al., (2009) Current Protocols in Cell Biology 42:19.11:19.11.1-19.11.22, and GamaSosa et al., (2010) Brain Struct.Funct.214 (2-3): 91-109, each of these documents is incorporated herein by reference as a whole for all purposes. Such genetically modified non-human animals can be generated by performing gene knock-in on the TARDBP locus of targeting.

[0143] For example, the method of generating a non-human animal including a genetically modified endogenous TARDBP locus may include: (1) modifying the genome of a pluripotent cell to include a genetically modified endogenous TARDBP locus; (2) identifying or selecting a genetically modified pluripotent cell including a genetically modified endogenous TARDBP locus; (3) introducing the genetically modified pluripotent cell into a non-human animal host embryo; and (4) incubating the host embryo in a surrogate mother. Optionally, a host embryo comprising a modified pluripotent cell (e.g., a non-human ES cell) can be incubated before being implanted in a surrogate mother and incubated therein to produce an F0 non-human animal, until the blastocyst stage. The surrogate mother can then produce an F0 generation non-human animal including a genetically modified endogenous TARDBP locus.

[0144] The methods may further include identifying cells or animals having a modified target genomic locus. Various methods can be used to identify cells and animals having targeted genetic modifications.

[0145] The step of modifying genome can be for example modified using exogenous donor nucleic acid (for example, targeting vector) to TARDBP locus to include genetically modified endogenous TARDBP locus disclosed herein.For example, targeting vector can be used to produce genetically modified endogenous TARDBP locus, wherein targeting vector includes 5' homology arms of 5' target sequences at 5' target sequences at targeting endogenous TARDBP locus and 3' homology arms of 3' target sequences at 3' target sequences at targeting endogenous TARDBP locus.Exogenous donor nucleic acid can also include nucleic acid insets, which include DNA segments to be integrated into the locus.Integrating nucleic acid insets in the TARDBP locus can cause the addition of the nucleic acid sequence of interest in the TARDBP locus, the deletion of the nucleic acid sequence of interest in the TARDBP locus or the replacement (that is, deletion and insertion) of the nucleic acid sequence of interest in the TARDBP locus.Homologous arms can flank the insertion nucleic acids of nucleic acids including encoding TDP-43 variants to produce genetically modified endogenous TARDBP locus.

[0146] Exogenous donor nucleic acid can be used for insertion or homologous recombination mediated by non-homologous end joining. Exogenous donor nucleic acid can include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), which can be single-stranded or double-stranded, and it can be linear or circular form. For example, the repair template can be a single-stranded oligodeoxynucleotide (ssODN).

[0147] The exogenous donor nucleic acid may also include a heterologous sequence that is not present at a non-targeted endogenous TARDBP locus.For example, the exogenous donor nucleic acid may include a selection cassette, such as a selection cassette flanked by recombinase recognition sites.

[0148] The term "homologous arms" refers to the homology arms of the exogenous donor nucleic acid. Some exogenous donor nucleic acids include homology arms. If the exogenous donor nucleic acid also includes a nucleic acid inset, the homology arms can be flanked by the nucleic acid inset. For ease of reference, homology arms are referred to herein as 5' and 3' (i.e., upstream and downstream) homology arms. This term relates to the relative position of the homology arms and the nucleic acid inset in the exogenous donor nucleic acid. 5' and 3' homology arms correspond to the region in the TARDBP locus, which is referred to herein as "5' target sequence" and "3' target sequence", respectively.

[0149] When the homology arms and target sequence share a sufficient level of sequence identity with each other, the two regions "correspond or correspond" to each other to serve as substrates for a homologous recombination reaction. The term "homology" includes DNA sequences that are identical to or share sequence identity with corresponding sequences. The sequence identity between a given target sequence and the corresponding homology arms present in an exogenous donor nucleic acid can be any degree of sequence identity that allows homologous recombination to occur. In some embodiments, the amount of the sequence identity shared by the homology arm of exogenous donor nucleic acid (or its fragment) and target sequence (or its fragment) can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, so that sequence experience homologous recombination.In addition, the homology region corresponding between homology arm and corresponding target sequence can have any length that is enough to promote homologous recombination.In some targeting vectors, the expected sudden change in endogenous TARDBP locus is included in the insertion nucleic acid with homology arm on the side.

[0150] In cells other than one-cell stage embryos, the exogenous donor nucleic acid can be a "large targeting vector" or "LTVEC" comprising a targeting vector, which includes homology arms corresponding to and derived from nucleic acid sequences larger than nucleic acid sequences commonly used by other methods for performing homologous recombination in cells. LTVEC also includes such targeting vectors, which include nucleic acid inserts, the nucleic acid sequences of which are larger than nucleic acid sequences commonly used by other methods for performing homologous recombination in cells. For example, LTVEC can modify large loci that cannot be accommodated by traditional plasmid-based targeting vectors due to their size restrictions. For example, in the absence of a nick or double-strand break induced by a nuclease agent (e.g., Cas protein), the targeting locus can be (i.e., 5' and 3' homology arms can correspond to) a locus of a cell that cannot be targeted or can only be mistargeted or only targeted with significantly low efficiency using conventional methods. LTVEC can be of any length, and is generally at least 10 kb in length. The sum of 5' homology arms and 3' homology arms in LTVEC is generally at least 10 kb.

[0151] The screening step can include, for example, a quantitative determination of the modification of the allele (MOA) for evaluating the parental chromosome. For example, quantitative determination can be performed via quantitative PCR such as real-time PCR (qPCR). Real-time PCR can utilize a first primer set identifying the target locus and a second primer set identifying a non-targeted reference locus. Primer sets can include a fluorescent probe identifying an amplified sequence.

[0152] Other examples of suitable quantitative assays include fluorescence mediated in situ hybridization (FISH), comparative genomic hybridization, isothermal DNA amplification, quantitative hybridization with immobilized probes, Probe, Molecular beacon probe or ECLIPSE TM Probe technology (see, e.g., US 2005 / 0144655, which is incorporated herein by reference in its entirety for all purposes).

[0153] The example of suitable pluripotency cell is embryonic stem (ES) cell (for example, mouse ES cell or rat ES cell). Modified pluripotency cell can be recombinantly produced, for example, by the following manner: (a) one or more exogenous donor nucleic acids (for example, targeting vector) including insertion nucleic acid are introduced into the cell, and the insertion nucleic acid is flanked by 5' and 3' homology arms corresponding to 5' and 3' target sites, wherein the insertion nucleic acid includes a nucleic acid encoding TDP-43 variant to produce a genetically modified endogenous TARDBP locus; and (b) identifying at least one cell (that is, identifying at least one cell including a genetically modified endogenous TARDBP locus) comprising an insertion nucleic acid integrated at the endogenous TARDBP locus in its genome.

[0154] Alternatively, the modified pluripotent unicellular cell can be produced by: (a) introducing into the cell: (i) a nuclease preparation or a nucleic acid encoding the nuclease preparation, wherein the nuclease preparation induces a nick or double-strand break at a target site within the endogenous TARDBP locus; and (ii) one or more exogenous donor nucleic acids (e.g., a targeting vector) comprising an insertion nucleic acid, the insertion nucleic acid being flanked by, for example, 5' and 3' homology arms corresponding to 5' and 3' target sites located sufficiently close to the nuclease target site, wherein the insertion nucleic acid comprises a nucleic acid encoding a TDP-43 variant to produce a genetically modified endogenous TARDBP locus; and (c) identifying at least one cell comprising an insertion nucleic acid integrated into the endogenous TARDBP locus in its genome (i.e., identifying at least one cell comprising a genetically modified endogenous TARDBP locus). Any nuclease agent that induces a nick or double-strand break into the desired recognition site can be used. Examples of suitable nucleases include transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), meganucleases, and clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) systems (e.g., CRISPR / Cas9 systems) or components of such systems (e.g., CRISPR / Cas9). See, e.g., US2013 / 0309670 and US2015 / 0159175, each of which is incorporated herein by reference in its entirety for all purposes.

[0155] Donor cells can be introduced into host embryos at any stage, such as the blastocyst stage or the pre-morula stage (i.e., the 4-cell stage or the 8-cell stage). Produce offspring that can pass the genetic modification through the germline. See, for example, U.S. Patent No. 7,294,754, which is incorporated herein by reference in its entirety for all purposes.

[0156] Alternatively, a method of producing a non-human animal as described elsewhere herein may include: (1) modifying the genome of a one-cell stage embryo to include a genetically modified endogenous TARDBP locus; (2) selecting the genetically modified embryo; and (3) gestating the genetically modified embryo in a surrogate mother, thereby producing offspring that are capable of transmitting the genetic modification through the germline.

[0157] Nuclear transfer techniques can also be used to produce non-human mammals. In short, the method for nuclear transfer can include the following steps: (1) enucleating an oocyte or providing an enucleated oocyte; (2) isolating or providing a donor cell or nucleus combined with the enucleated oocyte; (3) inserting the cell or nucleus into the enucleated oocyte to form a reconstructed cell; (4) implanting the reconstructed cell into the uterus of an animal to form an embryo; and (5) allowing the embryo to develop. In such methods, oocytes are usually obtained from dead animals, but oocytes can also be isolated from the fallopian tubes and / or ovaries of living animals. Before enucleation, oocytes can be matured in various well-known culture media. Enucleation of oocytes can be performed in a variety of well-known ways. Donor cells or nuclei can be inserted into enucleated oocytes to form recombinant cells by microinjecting donor cells under the zona pellucida before fusion. Fusion can be induced by applying DC electric pulses (electrofusion) on the contact / fusion plane, by exposing cells to chemicals that promote fusion such as polyethylene glycol, or by inactivating viruses such as Sendai virus. Before, during and / or after the nuclear donor and the recipient oocyte fusion, the recombinant cell can be activated by electrical and / or non-electrical means. The activation method comprises electric pulse, chemically induced shock, sperm penetration, increases the level of divalent cations in the oocyte and reduces the phosphorylation (such as by means of kinase inhibitors) of cellular proteins in the oocyte. The recombinant cell or embryo of activation can be cultivated in well-known culture medium, and then transferred to the uterus of the animal. See, for example, US2008 / 0092249, WO 1999 / 005266, US2004 / 0177390, WO2008 / 017234 and U.S. Patent No. 7,612,250, each document in these documents is incorporated herein by reference as a whole for all purposes.

[0158] The various methods provided herein allow for the generation of genetically modified non-human F0 animals, wherein the cells of the genetically modified F0 animals comprise genetically modified endogenous TARDBP loci. It should be appreciated that, depending on the method used to generate the F0 animals, the number of cells having genetically modified endogenous TARDBP loci within the F0 animals will vary. By, for example Method is introduced into donor ES cell in the morula pre-embryo from corresponding organism (for example, 8-cell stage mouse embryo) and allows the cell mass of F0 animal of greater percentage to include the cell with the nucleotide sequence of interest including targeted gene modification.For example, at least 50%, 60%, 65%, 70%, 75%, 85%, 86%, 87%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cell contribution of non-human F0 animal can include the cell mass with targeted modification.

[0159] Cells of the genetically modified F0 animal can be heterozygous for the genetically modified endogenous TARDBP locus, or can be homozygous for the genetically modified endogenous TARDBP locus.

[0160] VIII. Methods

[0161] Provided herein is a method of administering a TDP-43 variant as described herein to a cell or subject, or administering a nucleic acid encoding a TDP-43 variant to a cell or subject so that a TDP-43 variant is expressed. Also provided is a method of reducing or inhibiting TDP-43 aggregation in a cell or subject, a method of rescuing abnormal TDP-43 splicing regulation in a cell or subject, or a method of rescuing abnormal subcellular distribution of TDP-43 in a cell or subject. Also provided is a method of treating a subject's TDP-43 protein disease or a method of preventing a subject's TDP-43 protein disease. Such methods may include administering a TDP-43 variant as described herein (e.g., a therapeutically effective amount) to a cell or subject, or administering a nucleic acid encoding a TDP-43 variant (e.g., a therapeutically effective amount) to a cell or subject so that a TDP-43 variant is expressed. A therapeutically effective amount is an amount that produces the desired effect of administering it. The exact amount will depend on the purpose of treatment, and will be determined by those skilled in the art using known techniques. See, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding.

[0162] Some such methods include administering a TDP-43 variant to the cell or subject. Some such methods include administering a nucleic acid encoding a TDP-43 variant to the cell or subject. The nucleic acid can be a nucleic acid construct described in more detail elsewhere herein. In some cases, the nucleic acid encoding the TDP-43 variant can be codon-optimized (e.g., codon-optimized for expression in humans or in mice). For example, the nucleic acid can be modified to replace codons with a higher frequency of usage in human cells, non-human cells, mammalian cells, rodent cells, mouse cells, rat cells, or any other host cell of interest.

[0163] The nucleic acid encoding the TDP-43 variant may be DNA or RNA. In some cases, the nucleic acid may be a messenger RNA (mRNA) encoding the TDP-43 variant. In some cases, the nucleic acid may be a complementary DNA (cDNA) encoding the TDP-43 variant. For example, such nucleic acids may contain only coding sequences without any inserted introns. Examples of coding sequences of some TDP-43 variants described herein are shown, for example, in SEQ ID NOs: 70-72. In other cases, the nucleic acid may include one or more introns separating exons in the TDP-43 variant coding sequence. For example, the nucleic acid may include a TDP-43 variant genomic sequence including both exons and introns.

[0164] In some methods, the nucleic acid is located in an expression construct, which comprises a nucleic acid encoding a TDP-43 variant operably connected to a promoter. The promoter can be any suitable promoter for in vivo expression in an animal or in vitro in an isolated cell. The promoter can be a constitutively active promoter (e.g., a CAG promoter or a U6 promoter), a conditional promoter, an inducible promoter, a time-limited promoter (e.g., a developmentally regulated promoter) or a spatially limited promoter (e.g., a cell-specific or tissue-specific promoter). Such promoters are well known and discussed elsewhere herein. In a specific example, the promoter is active in neurons. In a specific example, the promoter is active in glial cells. In a specific example, the promoter is active in muscle cells. In some cases, the promoter is a heterologous promoter (i.e., a promoter that fails to be naturally operably connected to a TDP-43 nucleic acid). In other cases, the promoter can be an endogenous promoter (i.e., a TDP-43 variant nucleic acid operably connected to a TARDBP promoter). The heterologous promoter can be any type of promoter disclosed elsewhere herein. For example, the promoter can be a constitutive promoter, such as the EF1α promoter. Alternatively, the promoter can be a tissue-specific promoter or an inducible promoter. For example, the promoter can be a neuron-specific promoter. An example of a very specific suitable neuron-specific promoter with a low expression level is the synapsin-1 promoter (e.g., the human synapsin-1 promoter). For example, the promoter can be a glial-specific promoter. For example, the promoter can be a muscle-specific promoter.

[0165] The nucleic acids and expression constructs disclosed herein may also comprise post-transcriptional regulatory elements, such as woodchuck hepatitis virus post-transcriptional regulatory elements.For example, the promoter may be a glial-specific promoter.

[0166] The nucleic acids and expression constructs may also comprise one or more polyadenylation signal sequences. For example, the nucleic acid construct may comprise a polyadenylation signal sequence positioned at the 3' end of the TDP-43 variant coding sequence. Any suitable polyadenylation signal sequence may be used. The term polyadenylation signal sequence refers to any sequence that directs transcription termination and adds a poly-A tail to an mRNA transcript. In eukaryotes, transcription terminators are recognized by protein factors, and polyadenylation is followed by termination, which is the process of adding a poly(A) tail to an mRNA transcript in the presence of a poly(A) polymerase. Mammalian poly(A) signals are typically composed of a core sequence of about 45 nucleotides in length, which may be flanked by different auxiliary sequences for enhancing cleavage and polyadenylation efficiency. The core sequence consists of an upstream element (AATAAA or AAUAAA) that is highly conserved in mRNA, known as a poly A recognition motif or poly A recognition sequence, which is recognized by cleavage and polyadenylation specificity factor (CPSF); and a poorly defined downstream region (enriched in Us or Gs and Us) that is constrained by cleavage stimulating factor (CstF). Examples of transcription terminators that can be used include, for example, human growth hormone (HGH) polyadenylation signal, simian virus 40 (SV40) late polyadenylation signal, rabbit β-globin polyadenylation signal, bovine growth hormone (BGH) polyadenylation signal, phosphoglycerate kinase (PGK) polyadenylation signal, AOX1 transcription termination sequence, CYC1 transcription termination sequence, or any transcription termination sequence known to be suitable for regulating gene expression in eukaryotic cells.

[0167] The nucleic acid and expression construct may also optionally include a polyadenylation signal sequence upstream of the TDP-43 variant coding sequence. The polyadenylation signal sequence upstream of the TDP-43 variant coding sequence may be flanked by a recombinase recognition site recognized by a site-specific recombinase. In some constructs, the recombinase recognition site is also flanked by a selection cassette comprising, for example, a coding sequence of a drug resistance protein. In other constructs, the recombinase recognition site is not flanked by a selection cassette. The polyadenylation signal sequence prevents transcription and expression of a protein or RNA encoded by the coding sequence. However, after exposure to a site-specific recombinase, the polyadenylation signal sequence will be excised and the protein or RNA can be expressed.

[0168] If the polyadenylation signal sequence is excised in a tissue-specific or developmental stage-specific manner, such configurations can achieve tissue-specific expression or developmental stage-specific expression. If the animal comprising the nucleic acid or expression construct also comprises a coding sequence of a site-specific recombinase operably linked to a tissue-specific or developmental stage-specific promoter, excision of the polyadenylation signal sequence in a tissue-specific or developmental stage-specific manner can be achieved. Then, the polyadenylation signal sequence will be excised only in those tissues or at those developmental stages, thereby achieving tissue-specific expression or developmental stage-specific expression. In one example, the TDP-43 variant encoded by the nucleic acid or expression construct can be expressed in a neuron-specific manner. In one example, the TDP-43 variant encoded by the nucleic acid or expression construct can be expressed in a glial-specific manner. In one example, the TDP-43 variant encoded by the nucleic acid or expression construct can be expressed in a muscle-specific manner.

[0169] Site-specific recombinases include enzymes that can promote recombination between recombinase recognition sites, wherein two recombination sites are physically separated in a single nucleic acid or on a separate nucleic acid. The example of recombinase includes Cre, Flp and Dre recombinases. An example of Cre recombinase gene is Crei, wherein two exons encoding Cre recombinase are separated by introns to prevent it from being expressed in prokaryotic cells. Such recombinases may further include a nuclear localization signal for promoting localization to the nucleus (e.g., NLS-Crei). The recombinase recognition site includes a nucleotide sequence that is recognized by the site-specific recombinase and can be used as a substrate for a recombination event. The example of recombinase recognition site includes FRT, FRT11, FRT71, attp, att, rox and lox sites, such as loxP, lox511, lox2272, lox66, lox71, loxM2 and lox5171.

[0170] Nucleic acids disclosed herein may include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), which may be single-stranded or double-stranded, and may be linear or circular. Nucleic acid constructs may be naked nucleic acids, or may be delivered by vectors such as AAV vectors, as described elsewhere herein. If in linear form, the ends of the nucleic acids may be protected by well-known methods (e.g., from exonucleolytic degradation). For example, one or more dideoxynucleotide residues may be added to the 3' end of a linear molecule and / or a self-complementary oligonucleotide may be attached to one or both ends. See, e.g., Chang et al., (1987) Proc. Natl. Acad. Sci. USA 84: 4959-4963 and Nehls et al., (1996) Science 272: 886-889, each of which is incorporated herein by reference in its entirety for all purposes. Other methods for protecting exogenous polynucleotides from degradation include, but are not limited to, adding terminal amino groups and using modified internucleotide bonds, such as phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues. In some cases, the nucleic acid or expression construct may include one or more of the following terminal structures: a hairpin, a loop, an inverted terminal repeat (ITR), or a torus. For example, the nucleic acid or expression construct may include an ITR.

[0171] The nucleic acid or expression construct may include modifications or sequences that provide additional desired features (e.g., modified or regulated stability; tracking or detection with fluorescent markers; binding sites for proteins or protein complexes, etc.). For example, one or more nucleosides within the mRNA may be modified. Examples of chemical modifications to mRNA nucleobases include pseudouridine, 1-methyl-pseudouridine, and 5-methyl-cytidine. The mRNA may also be capped. The mRNA may also be polyadenylated (to include a poly(A) tail). As an example, a capped and polyadenylated mRNA containing N1-methyl-pseudouridine may be used (e.g., it may be completely replaced with N1-methyl-pseudouridine). The nucleic acid construct may include one or more fluorescent markers, purification tags, epitope tags, or combinations thereof. For example, the nucleic acid construct may include one or more fluorescent markers (e.g., fluorescent proteins or other fluorophores or dyes), such as at least 1, at least 2, at least 3, at least 4, or at least 5 fluorescent markers. Exemplary fluorescent labels include fluorophores such as fluorescein (e.g., 6-carboxyfluorescein (6-FAM)), Texas Red, HEX, Cy3, Cy5, Cy5.5, Pacific Blue, 5-(and-6)-carboxytetramethylrhodamine (TAMRA), and Cy7. A variety of fluorescent dyes are commercially available for labeling oligonucleotides (e.g., from Integrated DNA Technologies). The tag or label can be located at the 5' end, 3' end, or internal to the nucleic acid construct. For example, the nucleic acid construct can be conjugated to a 5' end with a fluorescent dye from Integrated DNA Technologies ( 700) is conjugated with the IR700 fluorophore.

[0172] Nucleic acid or expression construct may also include conditional alleles. Conditional alleles may be multifunctional alleles, as described in US2011 / 0104799, which is incorporated herein by reference for all purposes. For example, conditional alleles may include: (a) a driver sequence in a sense orientation relative to the transcription of a target gene; (b) a drug selection cassette (DSC) in a sense or antisense orientation; (c) a nucleotide sequence of interest (NSI) in an antisense orientation; and (d) a conditional reversal module (COIN, which utilizes exon splitting introns and reversible gene trap-like modules) in the opposite orientation. See, for example, US2011 / 0104799. Conditional alleles may also include recombinant units, which recombinant units reorganize to form conditional alleles after exposure to a first recombinase, the conditional allele (i) lacking a promoter sequence and a DSC; and (ii) containing an NSI in a sense orientation and a COIN in an antisense orientation. See, e.g., US 2011 / 0104799.

[0173] Nucleic acid or expression construct may also include polynucleotides encoding selection markers. Alternatively, nucleic acid or expression construct may lack polynucleotides encoding selection markers. Selection markers may be included in selection boxes. Optionally, selection boxes may be self-deletion boxes. See, for example, US 8,697,851 and US 2013 / 0312129, each of which is incorporated herein by reference in its entirety for all purposes. As an example, self-deletion boxes may include Crei genes operably connected to mouse Prm1 promoter (comprising two exons separated by introns encoding Cre recombinase) and neomycin resistance genes operably connected to human ubiquitin promoter. By adopting Prm1 promoter, self-deletion boxes can be specifically deleted in male germ cells of F0 animals. Exemplary selection markers include neomycin phosphotransferase (neo r ), hygromycin B phosphotransferase (hyg r ), puromycin-N-acetyltransferase (puromycin r ), blasticidin-S deaminase (bsr r ), xanthine / guanine phosphoribosyltransferase (gpt) or herpes simplex virus thymidine kinase (HSV-k) or a combination thereof. The polynucleotide encoding the selection marker can be operably linked to a promoter that is active in the targeted cell. Examples of promoters are described elsewhere herein.

[0174] Nucleic acid or expression construct may also include reporter genes. Exemplary reporter genes include genes encoding: luciferase, beta-galactosidase, green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (eBFP), DsRed, ZsGreen, MmGFP, mPlum, mCherry, tdTomato, mStrawberry, J-Red, mOrange, mKO, mCitrine, Venus, YPet, emerald, CyPet, Cerulean, T- sky blue and alkaline phosphatase. Such reporter genes may be operably linked to a promoter active in the targeted cell. Examples of promoters are described elsewhere herein.

[0175] The nucleic acid or expression construct may be located in a vector, such as a viral vector. The vector may include additional sequences, such as an origin of replication, a promoter, and genes encoding antibiotic resistance.

[0176] Some vectors can be circular. Alternatively, the vector can be linear. The vector can be packaged to be delivered by lipid nanoparticles, liposomes, non-lipid nanoparticles or viral capsids. Non-limiting exemplary vectors include plasmids, phagemids, cosmids, artificial chromosomes, minichromosomes, transposons, viral vectors and expression vectors.

[0177] Nucleic acid or expression construct can be located in a vector, such as a viral vector. The viral vector can be, for example, an adeno-associated virus (AAV) vector or a lentivirus (LV) vector (i.e., a recombinant AAV vector or a recombinant LV vector). Other exemplary viruses / viral vectors include retroviruses, adenoviruses, vaccinia viruses, poxviruses, and herpes simplex viruses. The virus can infect dividing cells, non-dividing cells, or both dividing cells and non-dividing cells. The virus can be integrated into the host genome, or alternatively not integrated into the host genome. Such viruses can also be engineered to have reduced immunity. The virus may have replication ability or may have replication defects (e.g., defects in one or more genes necessary for replication and / or packaging of additional rounds of virions). The virus can cause transient expression, long-term expression (e.g., at least 1 week, 2 weeks, 1 month, 2 months, or 3 months) or permanent expression. Exemplary viral titers (e.g., AAV titers) include about 10 12 vector genomes / mL, about 10 13 vector genomes / mL, about 10 14 vector genomes / mL, about 10 15 vector genomes / mL and about 10 16 Other exemplary viral titers (e.g., AAV titers) include about 10 12 vector genome (vg) / kg body weight, about 10 13 vg / kg body weight, about 10 14 vg / kg body weight, about 10 15 vg / kg body weight and about 10 16 vg / kg body weight.

[0178] In one example, the nucleic acid or expression construct is located in an AAV vector. AAV can be any suitable serotype and can be single-stranded AAV (ssAAV) or self-complementary AAV (scAAV). The ssDNA AAV genome consists of two open reading frames Rep and Cap, which are flanked by two reverse terminal repeats that allow synthesis of complementary DNA chains. When constructing an AAV transfer plasmid, the transgene is placed between the two ITRs, and Rep and Cap can be provided in trans. In addition to Rep and Cap, AAV may also require an auxiliary plasmid containing adenovirus genes. These genes (E4, E2a and VA) mediate AAV replication. For example, transfer plasmids, Rep / Cap and auxiliary plasmids can be transfected into HEK293 cells containing adenovirus genes E1+ to produce infectious AAV particles. Alternatively, Rep, Cap and adenovirus auxiliary genes can be combined into a single plasmid. Similar packaging cells and methods can be used for other viruses, such as retroviruses.

[0179] A variety of serotypes of AAV have been identified. These serotypes differ in the cell types they infect (i.e., their tropism), allowing preferential transduction of specific cell types. The serotypes of CNS tissues include AAV1, AAV2, AAV4, AAV5, AAV8, and AAV9. For example, the selectivity of AAV serotypes for gene delivery in neurons is discussed in Hammond et al., (2017) PLoS One 12 (12): e0188830, which is incorporated herein by reference for all purposes. In a specific example, an AAV-PHP.eB vector is used. The AAV-PHP.eB vector exhibits a high ability to cross the blood-brain barrier, increasing its CNS transduction efficiency. In another specific example, an AAV9 vector is used. The serotypes used for skeletal muscle transduction include, for example, AAV1, AAV2, AAV6, and AAV9. See, e.g., Riaz et al., (2015) Skeletal Muscle, Vol. 5, No. 37, which is incorporated herein by reference in its entirety for all purposes.

[0180] Tropism can be further refined by pseudotypes, which are mixtures of capsids and genomes from different viral serotypes. For example, AAV2 / 5 indicates a virus containing a serotype 2 genome packaged in a capsid from serotype 5. The use of pseudotyped viruses can improve transduction efficiency as well as alter tropism. Hybrid capsids derived from different serotypes can also be used to alter viral tropism. For example, AAV-DJ contains hybrid capsids from eight serotypes and exhibits high infectivity in a wide range of in vivo cell types. AAV-DJ8 is another example showing the properties of AAV-DJ, but with enhanced brain uptake. AAV serotypes can also be modified by 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.

[0181] To accelerate transgene expression, 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, transgene expression may be delayed. To address this delay, scAAV containing complementary sequences that can spontaneously anneal after infection can be used, thereby eliminating the need for host cell DNA synthesis. However, single-stranded AAV (ssAAV) vectors can also be used.

[0182] To increase packaging capacity, a longer transgene can be split between two AAV transfer plasmids, the first with a 3' splice donor and the second with a 5' splice acceptor. Upon co-infection of the cells, these viruses form concatemers, splice together, and the full-length transgene can be expressed. While this allows for expression of longer transgenes, the expression efficiency is low. A similar approach for increasing capacity utilizes homologous recombination. For example, a transgene can be split between two transfer plasmids but with a large amount of sequence overlap so that co-expression induces homologous recombination and expression of the full-length transgene.

[0183] In some methods, TDP-43 variants or nucleic acids encoding TDP-43 variants are associated with lipid nanoparticles. Lipid formulations can protect biomolecules from degradation while improving their cellular uptake. Lipid nanoparticles are particles comprising multiple lipid molecules physically associated with each other by intermolecular forces. These particles include microspheres (including single-layer and multi-layer vesicles, for example, liposomes), dispersed phases in emulsions, micelles or internal phases in suspensions. Such lipid nanoparticles can be used to encapsulate one or more nucleic acids or proteins for delivery. Formulations containing cationic lipids can be used to deliver polyanions such as nucleic acids. Other lipids that can be included are neutral lipids (i.e., uncharged or zwitterionic lipids), anionic lipids, auxiliary lipids for enhancing transfection, and stealth lipids that increase the length of time that nanoparticles can exist in vivo. Examples of suitable cationic lipids, neutral lipids, anionic lipids, auxiliary lipids, and stealth lipids can be found in WO 2016 / 010840 A1, which is incorporated herein by reference as a whole for all purposes. Exemplary lipid nanoparticles can include cationic lipids and one or more other components. In one example, other components may include helper lipids such as cholesterol. In another example, other components may include helper lipids such as cholesterol and neutral lipids such as DSPC. In another example, other components may include helper lipids such as cholesterol, optional neutral lipids such as DSPC, and stealth lipids such as S010, S024, S027, S031, or S033.

[0184] LNPs may include one or more or all of the following: (i) lipids for encapsulation and endosomal escape; (ii) neutral lipids for stability; (iii) auxiliary lipids for stability; and (iv) stealth lipids. See, for example, Finn et al., (2018) Cell Rep. 22(9): 2227-2235 and WO 2017 / 173054 A1, each of which is incorporated herein by reference in its entirety for all purposes. Specific examples of using LNPs for delivery to the brain are disclosed in Nabhan et al., (2016) Sci. Rep. 6: 20019, which is incorporated herein by reference in its entirety for all purposes.

[0185] A TDP-43 variant or a nucleic acid encoding a TDP-43 variant can be administered to a cell or subject by any suitable means. Provided herein are various methods and compositions that allow for the introduction of a molecule (eg, a nucleic acid or a protein) into a cell or a subject.

[0186] The methods provided herein do not rely on a particular method for introducing nucleic acid or protein into cells, but only rely on nucleic acid or protein to enter the interior of cells. Methods for introducing nucleic acid and protein into various cell types are known in the art, including, for example, stable transfection methods, transient transfection methods, and virus-mediated methods.

[0187] Transfection protocols and protocols for introducing molecules (e.g., nucleic acids or proteins) into cells can vary. Non-limiting transfection methods include chemical-based transfection methods using liposomes; nanoparticles; calcium phosphate (Graham et al., (1973) Virology 52 (2): 456-67, Bacchetti et al., (1977) Proc. Natl. Acad. Sci. USA 74 (4): 1590-4, and Kriegler, M (1991). Transfer and Expression: A Laboratory Manual. New York: WH Freeman and Company. pp. 96-97, each of which is incorporated herein by reference in its entirety for all purposes); dendrimers; or cationic polymers such as DEAE-dextran or polyethyleneimine. Non-chemical methods include electroporation, sonoporation, and phototransfection. Particle-based transfection includes the use of a gene gun or magnet-assisted transfection (Bertram (2006), Current Pharmaceutical Biotechnology 7, 277-28), which is incorporated herein by reference in its entirety for all purposes. Viral methods can also be used for transfection.

[0188] The introduction of molecules (e.g., nucleic acids or proteins) into cells can also be mediated by electroporation, intracytoplasmic injection, viral infection, adenovirus, adeno-associated virus, lentivirus, retrovirus, transfection, lipid-mediated transfection, or nuclear transfection. Nuclear transfection is an improved electroporation technique that enables nucleic acid substrates to be delivered not only to the cytoplasm but also to pass through the nuclear membrane into the nucleus. In addition, the use of nuclear transfection in the methods disclosed herein generally requires far fewer cells than conventional electroporation (e.g., only about 2 million compared to 7 million for conventional electroporation). In one example, using NUCLEOFECTOR TM System for nuclear transfection.

[0189] Molecules (e.g., nucleic acids or proteins) can also be introduced into cells by microinjection. Microinjection of mRNA preferably enters the cytoplasm (e.g., to deliver mRNA directly to the translation machinery), while microinjection of protein or DNA encoding the protein preferably enters the nucleus. Alternatively, microinjection can be performed by injection into both the nucleus and the cytoplasm: a needle can first be introduced into the nucleus, and a first amount can be injected, and when the needle is removed from the cell, a second amount can be injected into the cytoplasm. Methods for performing microinjection are well known. See, e.g., 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); Ryals et al., (2010) Proc. Natl. Acad. Sci. USA 107: 15022-15026 and Meyer et al., (2012) Proc. Natl. Acad. Sci. USA 109: 9354-9359, each of which is incorporated herein by reference in its entirety for all purposes.

[0190] Other methods for introducing molecules (e.g., nucleic acids or proteins) into cells may 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. Methods for administering nucleic acids or proteins to subjects to modify cells in vivo are disclosed elsewhere herein. As a specific example, molecules (e.g., nucleic acids or proteins) can be introduced into cells or non-human animals with carriers such as poly (lactic acid) (PLA) microspheres, poly (D, L-lactic acid-ethanol-acid) (PLGA) microspheres, liposomes, micelles, reverse micelles, lipid helices, or lipid microtubules. Some specific examples of delivery to non-human animals include hydrodynamic delivery, viral-mediated delivery (e.g., lentiviral-mediated delivery or adeno-associated virus (AAV)-mediated delivery) and lipid nanoparticle-mediated delivery.

[0191] In one example, the TDP-43 variant or a nucleic acid encoding a TDP-43 variant can be administered via viral transduction such as lentiviral transduction or adeno-associated viral transduction. In another example, the TDP-43 variant or a nucleic acid encoding a TDP-43 variant can be administered via lipid nanoparticle (LNP)-mediated delivery.

[0192] In vivo administration can be by any suitable route so that the TDP-43 variant or nucleic acid encoding the TDP-43 variant reaches the intended target cells (e.g., neurons in the subject's brain and / or glial cells in the subject's brain and / or myocytes of the subject) or target tissues (e.g., brain or muscle). Examples of routes of administration include parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal or intramuscular. Systemic administration includes, for example, oral and parenteral routes. Examples of parenteral routes include intravenous, intraarterial, intraosseous, intramuscular, intradermal, subcutaneous, intranasal and intraperitoneal routes. A specific example is intravenous infusion. Nasal drops and intravitreal injections are other specific examples. Local modes of administration include, for example, intrathecal, intraventricular, intraparenchymal (e.g., local intraparenchymal delivery to the striatum (e.g., into the caudate nucleus or into the putamen), cerebral cortex, precentral gyrus, hippocampus (e.g., into the dentate gyrus or CA3 region), temporal cortex, amygdala, frontal cortex, thalamus, cerebellum, medulla, hypothalamus, tectum, tegmentum, or substantia nigra), intraocular, intraorbital, subconjunctival, intravitreal, subretinal, and transscleral routes. When administered locally (e.g., intraparenchymal or intravitreal) compared to systemic administration (e.g., intravenous), significantly smaller amounts of the components (compared to systemic methods) can be exerted. Local modes of administration can also reduce or eliminate the incidence of potential toxic side effects that may occur when therapeutically effective amounts of the components are administered systemically. For example, the TDP-43 variant or a nucleic acid encoding the TDP-43 variant can be administered directly to the brain of a subject or to neurons or glial cells in the brain of a subject. In one specific example, administration to the subject is by intrathecal injection or by intracranial injection (e.g., stereotactic surgery or intraventricular injection for injection in the hippocampus and other brain regions). In one specific example, administration to the subject is by intraventricular injection. In another specific example, administration to the subject is by intracranial injection. In another specific example, administration to the subject is by intrathecal injection. In another example, the TDP-43 variant or a nucleic acid encoding a TDP-43 variant can be administered directly to the muscle of the subject or to the muscle cells of the subject (e.g., intramuscular injection).

[0193] The frequency of administration and the number of doses may depend on factors such as the half-life of the composition administered and the route of administration. The introduction of nucleic acids or proteins into cells or non-human animals may be performed once or multiple times over a period of time. For example, the introduction may be performed at the following frequencies: at least twice over a period of time, at least three times over a period of time, at least four times over a period of time, at least five times over a period of time, at least six times over a period of time, at least seven times over a period of time, at least eight times over a period of time, at least nine times over a period of time, at least ten times over a period of time, at least eleven times over a period of time, at least twelve times over a period of time, at least thirteen times over a period of time, at least fourteen times over a period of time, at least fifteen times over a period of time, at least sixteen times over a period of time, at least seventeen times over a period of time, at least eighteen times over a period of time, at least nineteen times over a period of time, or at least twenty times over a period of time.

[0194] The cell or subject in these methods can be, for example, a mammal, a non-human mammal, and a human. The mammal can be, for example, a non-human mammal, a human, a rodent, a rat, a mouse, or a hamster. Other non-human mammals include, for example, non-human primates, monkeys, apes, cats, dogs, rabbits, horses, bulls, deer, bison, livestock (e.g., cattle species, such as cows, bulls, etc.; sheep species, such as sheep, goats, etc.; and pig species, such as pigs and wild boars). The term "non-human" does not include humans. In a specific example, the cell or subject is a human.

[0195] The cell can be an isolated cell (e.g., in vitro), or can be in a subject (e.g., an animal or mammal). The cell can also be in any type of undifferentiated or differentiated state. In one example, the cell is a neuron. In one example, the cell is a glial cell. In one example, the cell is a muscle cell.

[0196] The cells provided herein can be normal healthy cells, or can be diseased cells comprising TDP-43 aggregates or abnormal TDP-43 function (e.g., abnormal TDP-43 splicing regulation or abnormal TDP-43 subcellular localization). These cells can be, for example, susceptible to TDP-43 aggregation, or they can have pre-existing TDP-43 aggregation.

[0197] In one example, the cell is a human cell, a rodent cell, a mouse cell or a rat cell, such as a human neuron, a rodent neuron, a mouse neuron or a rat neuron, or a human glial cell, a rodent glial cell, a mouse glial cell or a rat glial cell, or a human muscle cell, a rodent muscle cell, a mouse muscle cell or a rat muscle cell. In a specific example, the cell is a human neuron. In a specific example, the cell is a human glial cell. In a specific example, the cell is a human muscle cell. In a specific example, the cell is in a subject (e.g., a neuron or glial cell in a subject's brain or a muscle cell in a subject's body). For example, such methods can be methods for inhibiting or reducing TDP-43 aggregation, methods for reducing TDP-43 phosphorylation, methods for reducing or rescuing abnormal regulation of TDP-43 splicing, or methods for reducing or rescuing abnormal TDP-43 subcellular localization (e.g., reducing or rescuing abnormal nuclear depletion of TDP-43) in a subject's cell (e.g., a neuron or glial cell in a subject's brain or a muscle cell of a subject).

[0198] The methods described herein may also include administering to a cell or subject a formulation that reduces or eliminates endogenous TDP-43 expression in a cell or subject. Any suitable formulation may be used to reduce or inhibit the expression of endogenous TDP-43. Examples of formulations that can reduce the expression of endogenous TDP-43 include nuclease preparations (e.g., ZFN, TALEN or CRISPR / Cas), DNA binding proteins fused to transcription repressors (e.g., transcription repressors, such as catalytically inactive or dead Cas (dCas) (dCas-KRAB) fused to a KRAB domain) or antisense oligonucleotides, siRNA, shRNA or antisense RNA.

[0199] Nuclease preparations can be used to reduce the expression of endogenous TDP-43. For example, such nuclease preparations can be designed to target and cut the region of the expression of the TARDBP gene that will destroy the TARDBP gene. As a specific example, nuclease preparations can be designed to cut the region near the start codon of TARDBP. For example, the target sequence can be located within about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500 or 1,000 nucleotides of the start codon, and the cutting performed by the nuclease agent may destroy the start codon. Alternatively, it is possible to use a nuclease agent designed to cut the region near the start codon and the stop codon, so as to delete the coding sequence between the two nuclease target sequences. The DNA binding protein fused to the transcription repressor domain can also be used to reduce the expression of endogenous TDP-43. For example, a DNA binding protein fused to a transcriptional repressor domain (e.g., a catalytically inactive Cas fused to a KRAB transcriptional repressor domain) can be designed to target a region of TARDBP near the start codon, such as within about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, or 1,000 nucleotides of the start codon).

[0200] The cutting of the nuclease agent can result in double-strand breaks that can be repaired by non-homologous end joining (NHEJ). NHEJ comprises repairing double-strand breaks in nucleic acids by directly connecting the broken ends to each other or to an exogenous sequence without the need for a homologous template. Connecting non-continuous sequences by NHEJ usually results in deletions, insertions or translocations near the double-strand break site. These insertions and deletions (indels) can disrupt the expression of the target gene by, for example, frameshift mutations or the destruction of the start codon.

[0201] Any nuclease agent that induces a nick or double-strand break into a desired recognition site can be used in the methods and compositions disclosed herein. Naturally occurring or natural nuclease agents can be used, as long as the nuclease agent induces a nick or double-strand break in the desired recognition site. Alternatively, a modified or engineered nuclease preparation "engineered nuclease agent" can be used to include a nuclease that is engineered from its native form (modified or derived from the native form) to specifically recognize and induce a nick or double-strand break in the desired recognition site. Therefore, an engineered nuclease preparation can be derived from a natural, naturally occurring nuclease preparation, or can be artificially produced or synthesized. Engineered nucleases can induce nicks or double-strand breaks in, for example, a recognition site, wherein the recognition site is not a sequence recognized by a natural (non-engineered or unmodified) nuclease agent. The modification of a nuclease preparation can be only one amino acid in a protein cutting agent or one nucleotide in a nucleic acid cutting agent. Producing a gap or double-strand break in a recognition site or other DNA can be referred to herein as "cutting" or "cleaving" a recognition site or other DNA.

[0202] Active variants and fragments of exemplary recognition sites are also provided. Such active variants may include at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a given recognition site, wherein the active variant retains biological activity and can therefore be recognized and cut by a nuclease agent in a sequence-specific manner. The determination method of double-strand breaks in the recognition site by a nuclease preparation is known in the art (e.g., qPCR assay, Frendewey et al., (2010) Methods in Enzymology 476:295-307, which is incorporated herein by reference in its entirety for all purposes).

[0203] One type of nuclease preparation is a transcription activator-like effector nuclease (TALEN). TAL effector nucleases are a class of sequence-specific nucleases that can be used to make double-strand breaks at specific target sequences in prokaryotic or eukaryotic genomes. TAL effector nucleases are produced by fusing natural or engineered transcription activator-like (TAL) effectors or their functional parts with the catalytic domain of, for example, an endonuclease. The unique modular TAL effector DNA binding domain allows the design of proteins with potential recognition specificity for any given DNA. Therefore, the DNA binding domain of the TAL effector nuclease can be engineered to recognize specific DNA target sites and, therefore, be used to make double-strand breaks at the desired target sequence. See WO 2010 / 079430; Morbitzer et al., (2010) Proc. Natl. Acad. Sci. USA 107(50):21617-21622; Scholze & Boch (2010) Virulence 1:428-432; Christian et al., Genetics (2010) 186:757-761; Li et al., (2010) Nucleic Acids Res. (2011) 39(1):359-372; and Miller et al., (2011) Nature Biotechnology 29:143-148, each of which is incorporated herein by reference in its entirety for all purposes.

[0204] Examples of suitable TAL nucleases and methods for making suitable TAL nucleases are disclosed in, for example, US2011 / 0239315 A1, US2011 / 0269234 A1, US2011 / 0145940 A1, US 2003 / 0232410 A1, US2005 / 0208489 A1, US2005 / 0026157 A1, US 2005 / 0064474 A1, US2006 / 0188987 A1, and US2006 / 0063231 A1, each of which is incorporated herein by reference in its entirety for all purposes. In various embodiments, the TAL effector nuclease is engineered to cut in or near a target nucleic acid sequence, for example in a locus of interest or a genomic locus of interest, wherein the target nucleic acid sequence is at or near a sequence to be modified by a targeting vector. TAL nucleases suitable for use with the various methods and compositions provided herein include those specifically designed to bind at or near a target nucleic acid sequence to be modified by a targeting vector as described herein.

[0205] In some TALENs, each monomer of the TALEN includes 33-35 TAL repeats that recognize a single base pair through two hypervariable residues. In some TALENs, the nuclease agent is a chimeric protein comprising a TAL repeat-based DNA binding domain operably linked to an independent nuclease such as a FokI nuclease. For example, a nuclease preparation may include a first TAL repeat-based DNA binding domain and a second TAL repeat-based DNA binding domain, wherein each of the first TAL repeat-based DNA binding domain and the second TAL repeat-based DNA binding domain is operably linked to a FokI nuclease, wherein the first TAL repeat-based DNA binding domain and the second TAL repeat-based DNA binding domain recognize two consecutive target DNA sequences in each chain of a target DNA sequence separated by spacer sequences of different lengths (12-20bp), and wherein the FokI nuclease subunits dimerize to produce an active nuclease that breaks double strands on the target sequence.

[0206] The nuclease agent used in the various methods and compositions disclosed herein may further include a zinc finger nuclease (ZFN). In some ZFNs, each monomer of the ZFN includes 3 or more zinc finger-based DNA binding domains, wherein each zinc finger-based DNA binding domain binds to a 3bp subsite. In other ZFNs, the ZFN is a chimeric protein including a zinc finger-based DNA binding domain, and the binding domain is operably connected to an independent nuclease such as a FokI nuclease endonuclease. For example, a nuclease preparation may include a first ZFN and a second ZFN, wherein each of the first ZFN and the second ZFN is operably connected to a FokI nuclease subunit, wherein the first ZFN and the second ZFN recognize two continuous target DNA sequences in each chain of a target DNA sequence separated by an approximately 5-7bp spacer, and wherein the FokI nuclease subunit dimerizes to produce an active nuclease that breaks a double strand. See, e.g., US20060246567; US20080182332; US20020081614; US20030021776; WO / 2002 / 057308A2; US20130123484; US20100291048; WO / 2011 / 017293A2; and Gaj et al., (2013) Trends in Biotechnology, 31(7):397-405, each of which is incorporated herein by reference in its entirety for all purposes.

[0207] The methods and compositions disclosed herein can utilize clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) systems or components of such systems to modify the genome or change the expression of genes in cells. The CRISPR / Cas system comprises transcripts and other elements related to Cas gene expression or guiding its activity. The CRISPR / Cas system can be, for example, a type I, type II, type III system, or a type V system (e.g., VA subtype or VB subtype). The methods and compositions disclosed herein can employ the CRISPR / Cas system by utilizing a CRISPR complex (including a guide RNA (gRNA) complexed with a Cas protein) for site-directed binding or cutting of nucleic acids. See, e.g., WO 2013 / 176772, WO 2014 / 065596, WO 2014 / 089290, WO 2014 / 093622, WO 2014 / 099750, WO 2013 / 142578, and WO 2014 / 131833, each of which is herein incorporated by reference in its entirety for all purposes.

[0208] The CRISPR / Cas systems used in the compositions and methods disclosed herein can be non-naturally occurring. A "non-naturally occurring" system includes anything that indicates artificiality, such as one or more components of the system being altered or mutated from their naturally occurring state, at least substantially free of at least one other component with which the component is naturally associated in nature or associated with at least one other component with which the component is not naturally associated. For example, some CRISPR / Cas systems employ non-naturally occurring CRISPR complexes including gRNAs and Cas proteins that do not naturally occur together, employ non-naturally occurring Cas proteins, or employ non-naturally occurring gRNAs.

[0209] Also provided are active variants and fragments of nuclease preparations (i.e., engineered nuclease preparations).Such active variants can include and have at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with natural nuclease agents, wherein active variants retain the ability to cut at the desired recognition site and therefore retain nick or double-strand break inducing activity.For example, any nuclease agent in the nuclease agent described herein can be modified from the natural endonuclease sequence, and is designed to recognize and induce nick or double-strand break at the recognition site not recognized by the natural nuclease agent.Therefore, some engineered nucleases have the specificity of inducing nick or double-strand break at the recognition site different from the corresponding natural nuclease agent recognition site.The determination of nick or double-strand break inducing activity is known, and the overall activity and specificity of endonucleases to DNA substrates containing recognition sites are usually measured.

[0210] Nuclease preparation can be introduced into the cell by any known means. The polypeptide encoded by the nuclease preparation can be directly introduced into the cell. Alternatively, one or more nucleic acids encoded by the nuclease preparation can be introduced into the cell. When the nucleic acid encoded by the nuclease preparation is introduced into the cell, the nuclease preparation can be expressed transiently, conditionally or constitutively in the cell. Therefore, the nucleic acid encoded by the nuclease preparation can be included in the expression cassette and can be operably connected to a conditional promoter, an inducible promoter, a constitutive promoter or a tissue-specific promoter. This type of promoter of concern is discussed in further detail elsewhere herein. Alternatively, the nuclease preparation is introduced into the cell as the mRNA encoded by the nuclease preparation.

[0211] The nucleic acid that the nuclease preparation is encoded can be stably integrated in the genome of the cell, and can be operably connected to the promoter that has activity in the cell.Alternatively, the nucleic acid that the nuclease preparation is encoded can be in the targeting vector (for example, including the targeting vector of inserting polynucleotide, or in the carrier or plasmid separated from the targeting vector including inserting polynucleotide).

[0212] When providing a nuclease preparation to a cell by introducing a nucleic acid encoding a nuclease preparation, the nucleic acid encoding the nuclease preparation can be modified to replace a codon with a higher frequency of use in the cell of interest compared to the naturally occurring polynucleotide sequence encoding the nuclease preparation. For example, the polynucleotide encoding the nuclease preparation can be modified to replace a codon with a higher frequency of use in a given prokaryotic or eukaryotic cell of interest comprising a bacterial cell, a yeast cell, a human cell, a non-human cell, a mammalian cell, a rodent cell, a mouse cell, a rat cell, or any other host cell of interest compared to a naturally occurring polynuclear polynucleotide sequence.

[0213] Antisense oligonucleotides, antisense RNA, small interfering RNA (siRNA) or short hairpin RNA (shRNA) can also be used to reduce the expression of endogenous TDP-43. Such antisense RNA, siRNA or shRNA can be designed to target any region of TARDBP mRNA.

[0214] The term "antisense RNA" refers to a single-stranded RNA complementary to a messenger RNA strand transcribed in a cell. The term "small interfering RNA (siRNA)" refers to a typical double-stranded RNA molecule that induces RNA interference (RNAi) pathways. The length of these molecules can vary (usually between 18-30 base pairs) and contain varying degrees of complementarity with the target mRNA in its antisense strand. Some but not all siRNAs have unpaired protruding bases on the 5' end or 3' end of the sense strand and / or antisense strand. The term "siRNA" comprises a duplex of two separate chains, and a single strand of a hairpin structure that can form a duplex region. The length of the double-stranded structure can be, for example, less than 20, 25, 30, 35, 40, 45 or 50 nucleotides. For example, the length of the double-stranded structure can be about 21-23 nucleotides, about 19-25 nucleotides or about 19-23 nucleotides. The term "short hairpin RNA (shRNA)" refers to a single-stranded RNA base that self-hybridizes in a hairpin structure and can induce RNA interference (RNAi) pathways when processed. The length of these molecules can vary (e.g., for shRNA adapted to microRNAs, typically about 50-90 nucleotides in length, or in some cases greater than 250 nucleotides in length). The shRNA molecule is processed in the cell to form siRNA, which in turn can knock down gene expression. The shRNA can be integrated into a vector. The term "shRNA" also refers to a DNA molecule from which a short hairpin RNA molecule can be transcribed.

[0215] Antisense oligonucleotides and RNAi agents can also be used to reduce the expression of endogenous TDP-43. Such antisense oligonucleotides or RNAi agents can be designed to target any region of TARDBP mRNA.

[0216] "RNAi agent" is a composition comprising a small double-stranded RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that can promote the degradation or inhibition of the translation of target RNAs such as messenger RNA (mRNA) in a sequence-specific manner. The oligonucleotide in the RNAi agent is a polymer of linked nucleosides, each of which can be independently modified or unmodified. The RNAi agent works through an RNA interference mechanism (i.e., by interacting with the RNA interference pathway mechanism (RNA-induced silencing complex or RISC) of mammalian cells to induce RNA interference). Although the term RNAi agent as used herein is considered to work primarily through an RNA interference mechanism, the disclosed RNAi agent is not subject to the constraints or limitations of any particular pathway or mechanism of action. The RNAi agent disclosed herein includes a sense strand and an antisense strand, and includes but is not limited to short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA) and dicer substrates. The antisense strand of the RNAi agents described herein is at least partially complementary to a sequence (ie, consecutive or ordered nucleobases or nucleotides, described by consecutive letters using standard nomenclature) in a target RNA.

[0217] The basic principle shared by single-stranded antisense oligonucleotides (ASOs) and RNA interference (RNAi) is that oligonucleotides bind to target RNA through Watson-Crick base pairing. Without wishing to be bound by theory, during RNAi, small RNA duplexes (RNAi agents) are associated with RNA-induced silencing complexes (RISC), one strand (follower strand) is lost, and the remaining strand (guide strand) cooperates with RISC to bind complementary RNA. Then, the catalytic component of RISC, Argonaute 2 (Ago2), cuts the target RNA. The guide strand is always associated with a complementary sense strand or protein (RISC). In contrast, ASOs must survive and function as single strands. ASOs bind to target RNA and block ribosomes or other factors such as splicing factors from binding to RNA or recruiting proteins such as nucleases. Different modifications and target regions are selected for ASOs according to the desired mechanism of action. Gapmers are ASO oligonucleotides containing 2-5 chemically modified nucleotides (eg, LNA or 2'-MOE) flanking a central 8-10 base gap of DNA on each end. After binding to the target RNA, the DNA-RNA hybrid acts as a substrate for RNase H.

[0218] ASO is a DNA oligomer, usually 15-25 bases long, designed to be antisense oriented relative to the RNA of interest. Examples of ASOs targeting TARDBP are provided in US2020-0165610, which is incorporated herein by reference in its entirety for all purposes. Hybridization of ASO with target RNA mediates RNase H cleavage of RNA, which can prevent protein translation of mRNA. In order to increase nuclease resistance, phosphorothioate (PS) modifications can be added to oligonucleotides. Phosphorothioate bonds also promote binding to serum proteins, which increases the bioavailability of ASOs and promotes productive cellular uptake. In phosphorothioates, sulfur atoms replace non-bridge oxygens in the oligophosphate backbone. ASOs can be chimeras including both DNA and modified RNA bases. The use of modified RNAs such as 2'-O-methoxy-ethyl (2'-MOE) RNA, 2'-O-methyl (2'OMe) RNA, or affinity-plus-locked nucleobases in chimeric antisense designs increases nuclease stability and affinity of antisense oligonucleotides for target RNAs (T m ) both. However, these modifications do not activate RNase H cleavage (i.e., ASOs are composed entirely of sugar-modified RNA-like nucleotides (such as 2'-MOE), however, do not support RNase H cleavage of complementary RNA). Therefore, an antisense strategy is a "gapmer" design, which incorporates 2'-O-modified RNA or affinity-locked nucleic acid bases into chimeric antisense oligonucleotides that retain an RNase-H activation domain. Standard gapmers retain a central region of PS-modified DNA bases sufficient to induce RNase H cleavage. These bases are flanked on both sides by 2'-modified blocks that will increase binding affinity with the target. For example, a gapmer can contain a central segment of deoxynucleotides that allow induction of RNase H cleavage, wherein the central portion is flanked by a block of 2'-O-alkyl-modified ribonucleotides that protect the central segment from nuclease degradation. Once delivered to the cell, the ASO enters the nucleus and binds to its complementary endogenous RNA target. Hybridization of the ASO gapmer to the target RNA forms a DNA:RNA heteroduplex in the central region, which becomes a substrate for cleavage by the enzyme RNase H1.

[0219] In one example, the preparation for reducing or eliminating endogenous TDP-43 expression includes antisense oligonucleotides or RNAi preparations targeting endogenous TARDBP messenger RNA, or nucleic acids encoding the antisense oligonucleotides or RNAi preparations. In one example, the preparation includes a nuclease preparation targeting an endogenous TARDBP locus or one or more nucleic acids encoding a nuclease preparation. Nuclease preparations can be, for example, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or clustered regularly spaced short palindromic repeats (CRISPR) related (Cas) proteins and guide RNAs. In a specific example, the nuclease preparation is a Cas protein and a guide RNA, and optionally wherein the Cas protein is a Cas9 protein. For example, the method described herein may also include administering a nucleic acid encoding a TDP-43 variant, wherein the nuclease preparation cuts the endogenous TARDBP locus, and the nucleic acid encoding the TDP-43 variant is inserted into the cut endogenous TARDBP locus or recombined with it, wherein the TDP-43 variant is expressed from the endogenous TARDBP locus and replaces the expression of endogenous TDP-43.

[0220] Such methods may also include screening cells or subjects to confirm the presence of a TDP-43 variant or a nucleic acid encoding a TDP-43 variant. Screening cells or subjects for a TDP-43 variant or a nucleic acid encoding a TDP-43 variant may be performed by any known means. Such methods may also include screening cells or subjects to confirm the expression of a nucleic acid encoding a TDP-43 variant. Screening cells or subjects for expression of a nucleic acid encoding a TDP-43 variant may be performed by any known means. For example, methods for measuring protein expression and for measuring the expression of mRNA encoded by a coding sequence are well known.

[0221] One example of an assay that can be used is BASESCOPE TM RNA in situ hybridization (ISH) assay, a method that can quantify cell-specific edited transcripts, including single nucleotide changes, in intact fixed tissues. BASESCOPE TM RNA ISH assays can complement the use of NGS and qPCR in the characterization of gene editing. While NGS / qPCR can provide quantitative averages of wild-type and edited sequences, it does not provide information about the heterogeneity or percentage of edited cells within a tissue. BASESCOPE TM ISH assays can provide a landscape of the entire tissue and quantify wild-type versus edited transcripts at single-cell resolution, where the actual number of cells containing edited mRNA transcripts in the target tissue can be quantified. TMThe assay uses paired oligonucleotide ("ZZ") probes to amplify the signal in the absence of nonspecific background, thus enabling single-molecule RNA detection. TM The probe design and signal amplification system utilizes the 1ZZ probe to achieve single-molecule RNA detection and can differentially detect single nucleotide edits and mutations in intact fixed tissues.

[0222] As another example, a reporter gene can be used for screening. For example, a nucleic acid encoding a TDP-43 variant can encode a TDP-43 variant fused to a reporter gene, such as a fluorescent protein. Exemplary reporter genes include genes encoding: luciferase, β-galactosidase, green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (eBFP), DsRed, ZsGreen, MmGFP, mPlum, mCherry, tdTomato, mStrawberry, J-Red, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, Cerulean, T-Sky Blue, and alkaline phosphatase.

[0223] As another example, a selection marker can be used to screen for cells having a TDP-43 variant or a nucleic acid encoding a TDP-43 variant. Exemplary selection markers include neomycin phosphotransferase (neo r ), hygromycin B phosphotransferase (hyg r ), puromycin-N-acetyltransferase (puromycin r ), blasticidin S deaminase (bsr r ), xanthine / guanine phosphoribosyltransferase (gpt), or herpes simplex virus thymidine kinase (HSV-k).

[0224] These methods may also include assessing one or more signs or symptoms of TDP-43 proteinopathy by any suitable means. The example of such signs and symptoms is discussed in more detail elsewhere herein, and includes, for example, TDP-43 hyperphosphorylation, TDP-43 aggregation, abnormal splicing regulation of TDP-43 and abnormal TDP-43 subcellular distribution (e.g., abnormal nuclear depletion). This may be, for example, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks or longer after introducing TDP-43 variants or nucleic acids encoding TDP-43 variants. For example, it may be assessed from about 2 weeks to about 6 weeks or about 3 weeks to about 5 weeks after introducing TDP-43 variants or nucleic acids encoding TDP-43 variants.

[0225] The methods described herein can, for example, reduce the amount of new TDP-43 aggregate formation (new TDP-43 aggregation) in a cell or subject, and / or can reduce the amount of existing TDP-43 aggregate formation (pre-existing TDP-43 aggregation) in a cell or subject. For example, the methods described herein can prevent new TDP-43 aggregate formation, and / or can reverse existing TDP-43 aggregate formation.

[0226] The methods described herein can, for example, reduce the amount of abnormal regulation of splicing by TDP-43 in a cell or subject. For example, the methods described herein can rescue abnormal regulation of splicing by TDP-43.

[0227] The methods described herein can, for example, reduce abnormal subcellular localization of TDP-43 in a cell or subject (e.g., reduce abnormal nuclear depletion of TDP-43). For example, the methods described herein can rescue abnormal subcellular localization of TDP-43 (e.g., rescue abnormal nuclear depletion of TDP-43).

[0228] The methods described herein can, for example, reduce the level of phospho-TDP-43 in a cell or a subject.

[0229] In some methods described herein, these methods are used to treat or prevent TDP-43 proteinopathy in a subject. Therapeutic or pharmaceutical compositions comprising the compositions disclosed herein can be administered with suitable carriers, excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerance, etc. A variety of suitable formulations can be found in formulas known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. See also Powell et al., "Compendium of excipients for parenteral formulations", PDA (1998) J. Pharm. Sci. Technol. 52: 238-311. The compositions disclosed herein can be administered to alleviate or prevent or reduce the severity of one or more signs or symptoms of TDP-43 proteinopathy described in more detail elsewhere herein.

[0230] In some methods (for example, methods for the treatment of), the subject has one or more signs or symptoms of TDP-43 protein disease. For example, the subject may have pre-existing TDP-43 aggregates formed in one or more cells. TDP-43 protein disease is classified based on the degree of modified TDP-43 inclusion bodies, and includes increasing neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration with ubiquitin immunoreactivity, tau negative inclusion bodies (FTLD-U) and concurrent motor neuron disease FTLD (FTLD-MND). In addition, TDP-43 inclusion bodies have also been identified in many other neurodegenerative diseases, including Alzheimer's disease, cortical basal degeneration, Lewy body disease and Pick's disease (Pick's disease). TDP-43 protein disease is usually characterized by abnormal phosphorylation, ubiquitination, cutting and / or nuclear depletion of TDP-43 in neurons and glial cells. Various major neurodegenerative diseases show similar TDP-43 pathological manifestations in neurons and even glia, including the accumulation of detergent-resistant, ubiquitinated or hyperphosphorylated TDP-43 inclusion bodies in the cytoplasm, usually accompanied by TDP-43 depletion from the nucleus. These features of TDP-43-related pathological features are generally referred to as TDP-43 protein diseases. See, for example, Gao et al., (2018) J. Neurochem. doi: 10.1111 / jnc.14327, which is incorporated herein by reference in its entirety for all purposes.

[0231] TDP-43 proteinopathy includes a wide range of neurodegenerative diseases and phenotypes, which can be inherited or significantly distributed in a Mendelian pattern. It has been found that TDP-43 aggregates in several diseases, including Alzheimer's disease (brain), LATE (brain) and inclusion body myositis (muscle). A large number of genes and diseases have been associated with TDP-43 proteinopathy (Table 2). See, for example, de Boer et al., (2021) J. Neurol. Neurosurg. Psychiatry 92: 86-95, which is incorporated herein by reference in its entirety for all purposes.

[0232] Table 2. Diseases associated with TDP-43 lesions .

[0233]

[0234]

[0235] *Multisystem proteinopathy - a familial disorder in which patients present with ALS, FTLD, inclusion body myositis, Paget's disease of bone, or a combination of these phenotypes.

[0236] ALS, amyotrophic lateral sclerosis; bi, behavioral disorders; CARTS, brain age-related TDP-43 with sclerosis; ci, cognitive impairment; CTE, chronic traumatic encephalopathy; FOSMN, facial-onset sensorimotor neuronopathy; FTLD, frontotemporal lobar degeneration; HS, hippocampal sclerosis; LATE, predominantly limbic age-related TDP-43 encephalopathy; na, not applicable; PPA, primary progressive aphasia; sIBM, sporadic inclusion body myositis; TDP-43, TAR DNA-binding protein 43.

[0237] TDP-43 aggregation is evident in ~97% of all cases of amyotrophic lateral sclerosis (ALS). These TDP-43 inclusions are evident in both demented and non-demented patients with ALS and increase in density as the disease progresses (particularly with the development of cognitive impairment). In ALS, three major cell type-specific patterns of TDP-43 lesions have been identified, including (1) glial (22% of cases); (2) mixed neuronal and glial (59% of cases); (3) neuronal (7% of cases). The extent of TDP-43 lesions distinguishes ALS-FTLD from ALS without FTLD, and the presence of TDP-43 lesions in extramotor areas is associated with cognitive impairment in ALS, as can be assessed by the Edinburgh Cognitive and Behavioral ALS Screen (ECAS). TDP-43 lesions in the orbitofrontal cortex, dorsolateral prefrontal cortex, medial prefrontal cortex, and ventral anterior cingulate gyrus are associated with executive dysfunction. Language dysfunction was associated with TDP-43 lesions in the inferior frontal gyrus, lateral temporal region, middle and inferior temporal gyri, and angular gyrus. Verbal fluency dysfunction was associated with TDP-43 lesions in the prefrontal cortex, inferior frontal gyrus, ventral anterior cingulate gyrus, and lateral temporal region. However, behavioral abnormalities were associated with TDP-43 lesions in the orbitofrontal cortex and prefrontal cortex and ventral anterior cingulate gyrus.

[0238] Methods described herein can alleviate one or more signs and symptoms of TDP-43 protein diseases of cells or experimenters.At the cellular level, some examples of the signs and symptoms of TDP-43 protein diseases are included in the accumulation of detergent resistance, ubiquitination or hyperphosphorylated TDP-43 inclusion bodies in neurons or neuroglial cells or myocytes, abnormal phosphorylation, ubiquitination, cutting and / or nuclear depletion or cytoplasm of TDP-43, which are usually accompanied by TDP-43 depletion from the nucleus.Other signs and symptoms at organism level can include neurodegeneration, and the term refers to the progressive loss of neuronal structure and function.

[0239] All patent applications, websites, other publications, accession numbers, etc. cited above or below are incorporated herein by reference in their entirety for all purposes, to the extent that each individual project is specifically and individually indicated to be incorporated by reference in this manner. If different versions of a sequence are associated with accession numbers at different times, it is intended to refer to the version associated with the accession number on the effective filing date of the present application. The effective filing date means the earlier date in the actual filing date or the filing date of the priority application (where applicable) that mentions the accession number. Similarly, if different versions of publications, websites, etc. are published at different times, unless otherwise specified, it is intended to refer to the version published most recently on the effective filing date of the present application. Unless otherwise specifically specified, any feature, step, element, embodiment, or aspect of the present invention may be used in combination with any other feature, step, element, embodiment, or aspect. Although the present invention has been described in detail by way of illustration and example for the sake of clarity and understanding, it is apparent that certain changes and modifications may be made within the scope of the appended claims.

[0240] Brief description of sequence

[0241] The nucleotide and amino acid sequences listed in the attached sequence table are shown using the standard letter abbreviations of nucleotide bases and the three-letter code of amino acid. The nucleotide sequence follows the standard specification starting from the 5' end of the sequence and continuing forward (that is, from left to right in each row) to the 3' end. Although only one chain of each nucleotide sequence is shown, it should be understood that any reference to the chain shown includes the complementary chain. When the nucleotide sequence encoding the amino acid sequence is provided, it should be understood that the degenerate codon variants of the same amino acid sequence encoded therein are also provided. The amino acid sequence follows the standard specification starting from the amino terminal of the sequence and continuing forward (that is, from left to right in each row) to the carboxyl terminal.

[0242] Table 3. Description of sequences

[0243]

[0244]

[0245] Example

[0246] Example 1. Replacement of the prion-like domain of TDP-43 with the PLD of hnRNPA2B1

[0247] Several structural features of the TDP-43 protein have been identified, including a nuclear localization signal (NLS), two RNA recognition motifs (RRM1 and RRM2), a putative nuclear extracellular transport signal (NES), and a large domain in the carboxyl-terminal half of the protein that has been described as a low-complexity, weakly ordered, or prion-like domain (PLD). Of the TDP-43 mutations associated with familial cases of ALS, the majority are found in the PLD. The PLD of TDP-43 has been critically implicated in mediating the propensity of TDP-43 to form aggregates. Biochemistry has shown that the uniform spacing of aromatic residues throughout the PLD allows for efficient liquid-liquid phase separation but prevents irreversible associations that lead to aggregation. The PLD of TDP-43 has fewer and more unevenly spaced aromatic amino acids than other RNA-binding proteins ( Figure 1 ), suggesting that reorganizing the spacing of aromatic residues throughout PLD may prevent aggregation. To test this hypothesis, a PLDswap allele (amino acid sequence set forth in SEQ ID NO:69; coding sequence set forth in SEQ ID NO:70) was created that replaces the PLD of mouse TDP-43 with the PLD of mouse hnRNPA2B1 ( Figure 2B Compared with the PLD of TDP-43, the PLD of hnRNPA2B1 is composed of evenly spaced aromatic residues ( Figure 1 Western blot analysis of subcellular fractions obtained from ES cell-derived motor neurons harboring the PLDswap allele as the sole form of TDP-43 demonstrated normal TDP-43 subcellular distribution ( Figure 3 ). To determine if this chimeric protein could replace TDP-43, attempts were made to generate mice in which this was the sole form of TDP-43. It has been previously demonstrated that genetic ablation of TDP-43 results in embryonic lethality at e3.5-e5.5, and mice with a mutated NLS (ΔNLS) or PLD deletion (ΔPLD) die at embryonic days 12.5 and 15.5, respectively. Rather surprisingly, mice with the PLDswap allele as the sole form of TDP-43 died at birth, suggesting that this form of TDP-43 can replace the role of TDP-43 during embryogenesis ( Figure 4 ).

[0248] Finally, it was desired to determine whether the chimeric protein could retain the function of TDP-43. The most fully characterized function of TDP-43 is to regulate RNA splicing. Specifically, TDP-43 binds to intronic sequences to inhibit cryptic exons from being abnormally included in mRNA transcripts, and can also control alternative splicing events. In order to determine TDP-43 function, semi-quantitative RT-PCR was performed on specific splicing events in Adnp2, Dnajc5, Poldip3, Tsn, and Sortilin1 that were determined to be TDP-43 dependent. It has been previously shown that these events are disrupted by mutations that render NLS non-functional (ΔNLS), extranuclear transfer signal missing (ΔNES), and prion-like domain missing (ΔPLD). Surprisingly, in ES cell-derived motor neurons, replacing the PLD of TDP-43 with the PLD of hnRNPA2B1 TDP-43 retained splicing function to the greatest extent ( Figure 5 ).

[0249] Together, these data demonstrate that the prion-like domain of TDP-43 can be engineered, suggesting that removing wild-type TDP-43 and replacing it with a modified, aggregation-resistant form could prove to be of therapeutic value. It is also possible that simply replacing the wild-type protein will rescue phenotypes associated with the loss of functional TDP-43.

[0250] method

[0251] Cell culture. The chimeric TDP-43-hnRNP-A2B1PLDswap (as the only form of protein expressed by the cell) supports the viability of embryonic stem (ES) cells and motor neurons (ESMN) derived from them by differentiation in culture. ES cells were cultured for 2 days in embryonic stem cell medium (ESM; DMEM + 15% fetal bovine serum + penicillin / streptomycin + glutamine + non-essential amino acids + nucleosides + β-mercaptoethanol + sodium pyruvate + LIF), during which the medium was changed every day. One hour before trypsinization, the ES medium was replaced with 7mL ADFNK medium (advanced DMEM / F12 + neuronal basal medium + 10% knockout serum + penicillin / streptomycin + glutamine + β-mercaptoethanol). The ADFNK medium was aspirated and the ESC was trypsinized with 0.05% trypsin-EDTA. The precipitated cells were resuspended in 12mL ADFNK and grown in suspension for two days. Cells were cultured for another 4 days in ADFNK supplemented with retinoic acid (RA), smooth receptor agonist and purple morphine to obtain limb-like motor neurons (ESMN). Dissociated motor neurons were plated and cultured in embryonic stem cell-derived motor neuron culture medium (ESMN; Neuronal basal culture medium + 2% horse serum + B27 + glutamine + penicillin / streptomycin + β-mercaptoethanol + 10ng / mL GDNF, BDNF, CNTF) to mature. The cre recombinase delivered by electroporation was used to activate the conditional knockout allele at the ES cell stage and ESMN stage.

[0252] Subcellular fractionation of TDP-43. The subcellular localization of the chimeric TDP-43-PLDswap protein was analyzed using antibodies (Proteintech, Rosemont, IL) that recognize the N-terminus of the TDP-43 polypeptide (α-TDP-43N-term) and the C-terminal prion-like domain (α-TDP-43C-term) of the TDP-43 polypeptide. Soluble cytoplasmic protein extracts were prepared by incubating ES cell-derived MNs in ice-cold lysis buffer (10mM KCl, 10mM Tris-HCl, pH 7.4, 1mM MgCl2, 1mM DTT, 0.01% NP-40) supplemented with protease and phosphatase inhibitors (Roche) on ice for 10 minutes. The cells were then passed through a 27-gauge syringe five times. After centrifugation at 4000rpm 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 an equal volume of protease and phosphatase. An equal volume of 2X SDS sample buffer was added to each fraction and the samples were heated to 90° C. An equal volume of each fraction was then loaded onto a 10% SDS gel and electrophoresed at 225 V for 50 minutes, followed by Western blot analysis of TDP-43 using either an α-TDP-43-N-term antibody or an α-TDP-43-C-term antibody, the latter of which will not recognize the PLDswap mutant.

[0253] Mice expressing chimeric TDP-43-hnRNPA2B1 'PLDswap' proteins were generated. Although the loss of TDP-43 resulted in embryonic lethality, embryonic stem cells expressing only mutant ΔNLS TDP-43 or mutant ΔPLD TDP-43 from the endogenous TARDBP locus were viable and could be differentiated into motor neurons in vitro. These data raise the possibility that embryonic stem cells expressing mutant TDP-43 polypeptides lacking functional domains from the endogenous TARDBP locus may be viable and useful in creating animal models of TDP-43 protein diseases. For example, such embryonic stem cells can be used to generate non-human animals, such as mice, expressing mutant TDP-43 proteins lacking functional domains to examine the role of TDP-43 domains in normal and pathological biological processes.

[0254] To generate embryos or animals expressing chimeric TDP-43-hnRNPA2B1 protein, Method (Dechiara (2009) Methods Mol.Biol.530:311-324 and Poueymirou et al., (2007) Nat.Biotechnol.25:91-99, each of which is incorporated herein by reference in its entirety for all purposes). Targeted ES cells include (i) a TARDBP gene including a chimeric TDP-43-hnRNPA2B1'PLDswap' allele at the endogenous TARDBP locus, and (ii) an empty TDP-43 allele generated by Cre-mediated floxed exon 3 deletion (-), and the targeted ES cells are injected into non-compacted 8-cell stage Swiss Webster embryos. The viability of the embryos after fertilization is examined, and the ability to produce live F0 generation mice is assessed.

[0255] RT-PCR of splicing events. Total RNA was isolated from ES cell-derived motor neurons using Trizol reagent and subsequently treated with DNA enzyme. For cDNA, 1 μg of total RNA was used as a template for cDNA synthesis, which was synthesized using SuperScript IV first-strand synthesis system (ThermoFisher catalog number 18091050). The reaction was carried out in a volume of 20 μL, and then after cDNA synthesis was completed, the final volume was brought to 100 μL. PCR was performed using Q5 2X MasterMix (NEB) with 2 μL cDNA templates, using each 1 mM of forward primer and reverse primer, with a total reaction volume of 25 μL. For each transcript, PCR was first optimized to determine the number of cycles that allowed amplification, which remained unsaturated and within the linear range. The reaction was performed on a 1.8% agarose gel in 1X TAE, and the bands were visualized using SybrSafe. The primers used and the corresponding number of cycles are listed below.

[0256] Table 4. Primers and corresponding cycle numbers .

[0257]

[0258] Example 2. Effects of TDP-43PLDswap in neonatal mice

[0259] The requirement for TDP-43 function during embryogenesis and fetal development is essential and can include TDP-43 functions that are irrelevant to postnatal neuronal survival in vivo. Therefore, to further characterize the function of the PLDswap form of TDP-43, while also circumventing any requirement for prenatal TDP-43, a set of experiments was performed to test whether the PLDswap form of TDP-43 could compensate for wild-type TDP-43 in postnatal mice. To this end, mice carrying an exon 3 floxed conditional knockout (cKO) allele ("flEx3") were utilized that underwent Cre-mediated recombination to generate a ΔEx3 knockout allele when Cre was present. This conditional allele was then mutated with a PLDswap mutant (TDP-43 flEx3 / PLDswap ) to allow conditional removal of WT TDP-43, leaving PLDswap as the sole form of the protein in Cre-expressing cells. As negative and positive controls, the flEx3 conditional allele (TDP-43 flEx3 / flEx3 ) and mice homozygous for the conditional allele (TDP-43 flEx3 / WT ) mice with one WT allele. To induce conditional knockout of the floxed WT allele, PHP.eB.AAV viruses expressing the Cre-2A-mCherry cassette under the control of the ubiquitin CAG promoter (PHP.eB.AAV-CAG-Cre-2A-mCherry) or the neuron-specific human synapsin promoter (PHP.eB.AAV-hSYN-Cre-2A-mCherry) were injected into P0 newborn mouse pups of all three genotypes by intracerebroventricular (icv) injection. Fig. 6A Given the essential requirement for TDP-43, survival was used as a readout for TDP-43 function in these animals. As expected, since TDP-43 is essential, survival was not significantly increased for the cKO allele (TDP-43 ΔEx3 / ΔEx3 Mice homozygous for either ubiquitin (CAG) or neuron-specific (SYN) all died 5 weeks after Cre delivery. flEx3 / WT and TDP-43 flEx3 / PLDswap The mice survived significantly longer and to a very similar extent, indicating that the PLDswap form of TDP-43 is able to serve as a long-term replacement for wild-type TDP-43 protein in neurons. Figure 6B .

[0260] One caveat of this experimental system is that high expression of Cre recombinase in neurons may show toxicity, which may lead to overexpression of the positive control TDP-43. flEx3 / WTThe mice survived shorter than expected, particularly in the CAG-Cre environment, where expression is hyperexpressed. Thus, this system may underestimate the extent to which the PLDswap form of TDP-43 can compensate for TDP-43 function in vivo.

[0261] To support the survival data, the role of the PLDswap protein in TDP-43-dependent splicing events was also directly tested. To this end, spinal cord tissue was collected from mice with or without Cre-mediated removal of the conditional wild-type TDP-43 allele and semi-quantitative RT-PCR was performed for specific TDP-43-dependent splicing events. Two mRNAs known to undergo TDP-43 splicing were tested: Adnp2 mRNA and Tsn mRNA. While all animals without CAG-Cre expression showed normal transcript processing, animals with the conditional allele (and TDP-43 ΔEx3 / ΔEx3 ) triggered mis-splicing of both transcripts (i.e., cryptic exon addition in Adnp2 and exon 5 skipping in Tsn). In contrast, both transcripts were altered upon Cre-treatment of TDP-43 ΔEx3 / WT In Cre-treated TDP-43 flEx3 / PLDswap In mice, there was a trend toward rescue of splicing defects, particularly in the increase of cryptic exons in Adnp2. Figure 7 This analysis of two genes that are dependent on TDP-43 for their normal splicing provides molecular support for the notion that the chimeric PLDswap form of TDP-43 has the ability to function like normal TDP-43 to some extent.

[0262] Example 3. Replacing a portion of the prion-like domain of TDP-43 with a portion of the PLD of hnRNPA2B1

[0263] As explained in Example 1, the PLD of TDP-43 has fewer and more unevenly spaced aromatic amino acids than other RNA binding proteins ( Figure 1 ), suggesting that reorganizing the spacing of aromatic residues throughout the PLD could prevent aggregation. To test this hypothesis, the PLD28aa allele (amino acid sequence as shown in SEQ ID NO:67; coding sequence as shown in SEQ ID NO:71) was created, which replaces a highly conserved 28-amino acid sequence within the PLD of mouse TDP-43 that has been shown to be important for aggregation in yeast with a sequence from the PLD of mouse hnRNPA2B1. Compared to the PLD of TDP-43, the PLD of hnRNPA2B1 is composed of evenly spaced aromatic residues ( Figure 1). The PLD28aa allele was confirmed to be active as the sole form of TDP-43 in mouse embryonic stem cells (data not shown). As in Example 1, subcellular fractions obtained from ES cell-derived motor neurons were subjected to Western blot analysis with the PLD28aa allele as the sole form of TDP-43 to analyze the subcellular distribution of TDP-43. As in Example 1, mice were generated in which this was the sole form of TDP-43 in order to determine whether this chimeric protein could replace TDP-43. Embryonic development was assessed in mice. Experiments were also performed in Example 1 to determine whether this chimeric protein could retain the function of TDP-43, particularly by assessing the TDP-43 splicing function in ES cell-derived motor neurons.

[0264] Example 4. Introduction of evenly spaced aromatic residues ("tags") into the prion-like domain of TDP-43 to Promotes liquid-liquid phase separation (LLPS) but inhibits aggregation

[0265] As explained in Example 1, the PLD of TDP-43 has fewer and more unevenly spaced aromatic amino acids than other RNA binding proteins ( Figure 1 ), suggesting that reorganizing the spacing of aromatic residues throughout PLD can prevent aggregation. To test this hypothesis, a PLDaromatic allele (amino acid sequence as shown in SEQ ID NO:64; coding sequence as shown in SEQ ID NO:72) was created that introduces evenly spaced aromatic residues ("tags") within the prion-like domain of TDP-43 to promote liquid-liquid phase separation (LLPS) but inhibit aggregation. The PLDaromatic allele was confirmed to be viable as the sole form of TDP-43 in mouse embryonic stem cells (data not shown). As in Example 1, subcellular fractions obtained from ES cell-derived motor neurons were subjected to Western blot analysis with the PLDaromatic allele as the sole form of TDP-43 to analyze the subcellular distribution of TDP-43. As in Example 1, mice were generated in which this was the sole form of TDP-43 to determine whether this chimeric protein could replace TDP-43. Embryonic development was assessed in mice. Experiments were also performed in Example 1 to determine whether the chimeric protein could retain the function of TDP-43, specifically by assessing TDP-43 splicing function in ES cell-derived motor neurons.

Claims

1. A TAR DNA-binding protein 43 (TDP-43) variant, wherein the prion-like domain (PLD) of the TDP-43 variant is mutated to have more aromatic amino acids and / or more evenly spaced aromatic amino acids than the PLD from wild-type TDP-43.

2. The TDP-43 variant according to claim 1, wherein the PLD is mutated to have more aromatic amino acids.

3. The TDP-43 variant according to claim 1 or 2, wherein the PLD is mutated to have more evenly spaced aromatic amino acids than the PLD from the wild-type TDP-43.

4. The TDP-43 variant according to any one of claims 1 to 3, wherein the PLD is mutated to have more aromatic amino acids and / or more evenly spaced aromatic amino acids than the PLD from the wild-type TDP-43.

5. The TDP-43 variant according to any one of claims 1 to 4, wherein the PLD in the TDP-43 variant is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the sequence shown in SEQ ID NO: 22, or wherein the PLD in the TDP-43 variant is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the sequence shown in SEQ ID NO:

63.

6. The TDP-43 variant according to any one of claims 1 to 5, wherein the PLD in the TDP-43 variant comprises, consists essentially of, or consists of the sequence shown in SEQ ID NO: 22, or wherein the PLD in the TDP-43 variant comprises, consists essentially of, or consists of the sequence shown in SEQ ID NO:

63.

7. The TDP-43 variant of any one of claims 1-6, wherein the TDP-43 variant is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the sequence shown in SEQ ID NO: 23, or wherein the TDP-43 variant is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the sequence shown in SEQ ID NO:

64.

8. The TDP-43 variant according to any one of claims 1 to 7, wherein the TDP-43 variant comprises, consists essentially of, or consists of the sequence shown in SEQ ID NO: 23, or wherein the TDP-43 variant comprises, consists essentially of, or consists of the sequence shown in SEQ ID NO:

64.

9. The TDP-43 variant according to any one of claims 1 to 4, wherein a portion of the PLD from the wild-type TDP-43 is replaced by a different RNA from the variant TDP-43. At least a portion of the PLD of the binding protein is replaced, optionally wherein: (i) the portion of the PLD from the wild-type TDP-43 that is substituted is at least about 10, at least about 15, at least about 20, at least about 25, or at least about 28 amino acids; (ii) the portion of the PLD from the wild-type TDP-43 that is substituted is from about 10 to about 50, from about 20 to about 40, or from about 25 to about 35 amino acids; (iii) the portion of the PLD that is substituted is about 28 amino acids; or (iv) the portion of the PLD that is substituted comprises, consists essentially of, or consists of SEQ ID NO:6, or the portion of the PLD that is substituted comprises, consists essentially of, or consists of SEQ ID NO:

47.

10. The TDP-43 variant according to claim 9, wherein the different RNA binding protein is hnRNPA2B1.

11. The TDP-43 variant of claim 10, wherein the portion of the PLD from hnRNPA2B1 is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the sequence shown in SEQ ID NO: 24, or wherein the portion of the PLD from hnRNPA2B1 is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the sequence shown in SEQ ID NO:

65.

12. The TDP-43 variant according to claim 10 or 11, wherein the portion of the PLD from hnRNPA2B1 comprises, consists essentially of, or consists of the sequence shown in SEQ ID NO: 24, or wherein the portion of the PLD from hnRNPA2B1 comprises SEQ ID The sequence shown in NO:65, consists essentially of the sequence, or consists of the sequence.

13. The TDP-43 variant according to any one of claims 10 to 12, wherein the PLD in the TDP-43 variant is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the sequence shown in SEQ ID NO: 25, or wherein the PLD in the TDP-43 variant is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the sequence shown in SEQ ID NO:

66.

14. The TDP-43 variant according to any one of claims 10 to 13, wherein the PLD in the TDP-43 variant comprises, consists essentially of, or consists of the sequence shown in SEQ ID NO: 25, or wherein the PLD in the TDP-43 variant comprises, consists essentially of, or consists of the sequence shown in SEQ ID NO:

66.

15. The TDP-43 variant of any one of claims 10-14, wherein the TDP-43 variant is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the sequence shown in SEQ ID NO: 26, or wherein the TDP-43 variant is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the sequence shown in SEQ ID NO:

67.

16. The TDP-43 variant according to any one of claims 10 to 15, wherein the TDP-43 variant comprises, consists essentially of, or consists of the sequence shown in SEQ ID NO: 26, or wherein the TDP-43 variant comprises, consists essentially of, or consists of the sequence shown in SEQ ID NO:

67.

17. The TDP-43 variant according to any one of claims 1 to 4, wherein the PLD from the wild-type TDP-43 is replaced by a PLD from a different RNA binding protein in the variant TDP-43.

18. The TDP-43 variant according to claim 17, wherein the different RNA binding protein is hnRNPA2B1.

19. The TDP-43 variant of claim 18, wherein the PLD in the TDP-43 variant is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the sequence shown in SEQ ID NO: 27 or 13, or wherein the PLD in the TDP-43 variant is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the sequence shown in SEQ ID NO: 68 or 58.

20. The TDP-43 variant according to claim 18 or 19, wherein the PLD in the TDP-43 variant comprises, consists essentially of, or consists of the sequence shown in SEQ ID NO: 27 or 13, or wherein the PLD in the TDP-43 variant comprises, consists essentially of, or consists of the sequence shown in SEQ ID NO: 68 or 58.

21. The TDP-43 variant of any one of claims 18-20, wherein the TDP-43 variant is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the sequence shown in SEQ ID NO: 28, or wherein the TDP-43 variant is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the sequence shown in SEQ ID NO:

69.

22. The TDP-43 variant according to any one of claims 18 to 21, wherein the TDP-43 variant comprises, consists essentially of, or consists of the sequence shown in SEQ ID NO: 28, or wherein the TDP-43 variant comprises, consists essentially of, or consists of the sequence shown in SEQ ID NO:

69.

23. The TDP-43 variant of any one of claims 1-22, wherein the TDP-43 variant is less prone to aggregation than the wild-type TDP-43.

24. The TDP-43 variant according to any one of claims 1-23, wherein the TDP-43 variant retains the function of wild-type TDP-43 in splicing regulation.

25. The TDP-43 variant according to any one of claims 1-24, wherein the TDP-43 variant is predominantly nuclear and / or retains the subcellular distribution of wild-type TDP-43.

26. The TDP-43 variant according to any one of claims 1-25, wherein the TDP-43 variant retains the function of wild-type TDP-43 during embryonic development.

27. The TDP-43 variant according to any one of claims 1-26, wherein the TDP-43 variant is a human TDP-43 variant.

28. The TDP-43 variant according to any one of claims 1-27, for use in the treatment of a TDP-43 proteinopathy in a subject, optionally wherein the TDP-43 proteinopathy is amyotrophic lateral sclerosis (ALS).

29. The TDP-43 variant according to any one of claims 1-27, for use in the prevention of a TDP-43 proteinopathy in a subject, optionally wherein the TDP-43 proteinopathy is amyotrophic lateral sclerosis (ALS).

30. Use of a TDP-43 variant according to any one of claims 1 to 27 for the manufacture of a medicament for treating a TDP-43 proteinopathy, optionally wherein the TDP-43 proteinopathy is amyotrophic lateral sclerosis (ALS).

31. Use of the TDP-43 variant according to any one of claims 1-27 for the manufacture of a medicament for preventing a TDP-43 proteinopathy, optionally wherein the TDP-43 proteinopathy is amyotrophic lateral sclerosis (ALS).

32. A nucleic acid encoding the TDP-43 variant according to any one of claims 1-27.

33. The nucleic acid of claim 32, wherein the nucleic acid is messenger RNA.

34. The nucleic acid of claim 32, wherein the nucleic acid comprises DNA, optionally wherein the DNA comprises complementary DNA (cDNA).

35. The nucleic acid of claim 34, wherein the nucleic acid is in an expression construct comprising a promoter operably linked to the nucleic acid encoding the TDP-43 variant, optionally wherein the promoter is a neuron-specific promoter or a constitutive promoter.

36. The nucleic acid of claim 35, wherein the promoter is a constitutive promoter, a tissue-specific promoter, or an inducible promoter.

37. The nucleic acid of claim 36, wherein the promoter is a neuron-specific promoter, optionally wherein the promoter is a synapsin-1 promoter, and optionally wherein the promoter is a human synapsin-1 promoter.

38. The nucleic acid of any one of claims 34-37, wherein the nucleic acid is in a vector.

39. The nucleic acid of claim 38, wherein the vector is a viral vector.

40. The nucleic acid of claim 38, wherein the viral vector is a lentiviral vector or an adeno-associated virus (AAV) vector.

41. The nucleic acid of claim 40, wherein the vector is the AAV vector, optionally wherein the AAV vector is an AAV-PHP.eB vector.

42. The nucleic acid of any one of claims 32-41, wherein the nucleic acid is codon optimized for expression in human cells.

43. A cell comprising: (i) a TDP-43 variant according to any one of claims 1 to 27; or (ii) The nucleic acid of any one of claims 32-42, wherein the TDP-43 variant is expressed.

44. The cell of claim 43, wherein the cell is a mammalian cell.

45. The cell of claim 44, wherein the mammalian cell is a human cell, a rodent cell, a mouse cell, or a rat cell.

46. ​​The cell of claim 45, wherein the cell is the human cell.

47. The cell of any one of claims 43-46, wherein the cell is a neuron, a glial cell, or a muscle cell.

48. The cell of any one of claims 43-47, wherein the cell is in a subject.

49. The cell of claim 48, wherein the cell is a neuron in the subject's brain.

50. The cell of any one of claims 43-47, wherein endogenous TDP-43 is not expressed in the cell.

51. The cell of claim 50, wherein the endogenous TARDBP locus comprises a mutation that prevents endogenous TDP-43 from being expressed in the cell.

52. The cell of any one of claims 43-51, further comprising an agent that reduces or eliminates endogenous TDP-43 expression in the cell.

53. The cell of claim 52, wherein the agent comprises an antisense oligonucleotide or RNAi agent targeting endogenous TARDBP messenger RNA, or a nucleic acid encoding the antisense oligonucleotide or RNAi agent.

54. The cell of claim 52, wherein the preparation comprises a nuclease preparation or one or more nucleic acids encoding the nuclease preparation that targets the endogenous TARDBP locus.

55. The cell of claim 54, wherein the nuclease agent is a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) protein and guide RNA.

56. The cell of claim 55, wherein the nuclease preparation is the Cas protein and the guide RNA, optionally wherein the Cas protein is a Cas9 protein.

57. The cell of any one of claims 43-56, wherein the cell has a genetically modified endogenous TARDBP locus, wherein the nucleic acid is integrated into the endogenous TARDBP locus.

58. The cell of claim 57, wherein the cell is heterozygous for the integrated nucleic acid.

59. The cell of claim 57, wherein the cell is homozygous for the integrated nucleic acid.

60. The cell of any one of claims 57-59, wherein the nucleic acid is operably linked to the endogenous TARDBP promoter.

61. The cell of any one of claims 57-60, wherein the TDP-43 variant is expressed from the endogenous TARDBP locus and replaces the expression of the endogenous TDP-43.

62. The cell of any one of claims 43-61, wherein the cell has reduced TDP-43 aggregation compared to a control cell lacking the TDP-43 variant or the nucleic acid.

63. A non-human animal, comprising: (i) a TDP-43 variant according to any one of claims 1 to 27; or (ii) The nucleic acid of any one of claims 32-42, wherein the TDP-43 variant is expressed.

64. The non-human animal of claim 63, wherein the non-human animal is a mammal.

65. The non-human animal of claim 64, wherein the non-human animal is a rodent, rat or mouse.

66. The non-human animal of claim 65, wherein the non-human animal is the mouse.

67. The non-human animal of any one of claims 63-66, wherein the TDP-43 variant or the nucleic acid is present in a neuron, a glial cell, or a muscle cell of the non-human animal.

68. The non-human animal of any one of claims 63-67, wherein the TDP-43 variant or the nucleic acid is present in the neuron.

69. The non-human animal of claim 68, wherein the neuron is located in the brain of the non-human animal.

70. The non-human animal of any one of claims 63-67, wherein endogenous TDP-43 is not expressed in the non-human animal.

71. The non-human animal of claim 70, wherein the endogenous TARDBP locus comprises a mutation that prevents endogenous TDP-43 from being expressed in the non-human animal.

72. The non-human animal of any one of claims 63-71, further comprising an agent that reduces or eliminates endogenous TDP-43 expression in the non-human animal.

73. The non-human animal of claim 72, wherein the agent comprises an antisense oligonucleotide or an RNAi agent targeting endogenous TARDBP messenger RNA, or a nucleic acid encoding the antisense oligonucleotide or the RNAi agent.

74. The non-human animal of claim 72, wherein the preparation comprises a nuclease preparation that targets the endogenous TARDBP locus or one or more nucleic acids encoding the nuclease preparation.

75. A non-human animal according to claim 74, wherein the nuclease preparation is a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN) or a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) protein and a guide RNA.

76. The non-human animal of claim 75, wherein the nuclease preparation is the Cas protein and the guide RNA, optionally wherein the Cas protein is a Cas9 protein.

77. The non-human animal of any one of claims 63-76, wherein the non-human animal has a genetically modified endogenous TARDBP locus, wherein the nucleic acid is integrated at the endogenous TARDBP locus.

78. The non-human animal of claim 77, wherein the non-human animal is heterozygous for the integrated nucleic acid.

79. The non-human animal of claim 77, wherein the non-human animal is homozygous for the integrated nucleic acid.

80. The non-human animal of any one of claims 77-79, wherein the nucleic acid is operably linked to the endogenous TARDBP promoter.

81. The non-human animal of any one of claims 77-80, wherein the TDP-43 variant is expressed from the endogenous TARDBP locus and replaces the expression of the endogenous TDP-43.

82. The non-human animal of any one of claims 63-81, wherein the non-human animal has reduced TDP-43 aggregation compared to a control non-human animal without the TDP-43 variant or the nucleic acid.

83. A method of making a non-human animal according to any one of claims 63-83, the method comprising administering the TDP-43 variant or the nucleic acid to the non-human animal.

84. A method of preparing a non-human animal according to any one of claims 77-82, the method comprising: (I)(a) modifying the genome of a pluripotent non-human animal cell to include a genetically modified endogenous TARDBP locus; (b) identifying or selecting the genetically modified pluripotent non-human animal cell comprising the genetically modified endogenous TARDBP locus; (c) introducing the genetically modified pluripotent non-human animal cell into a non-human animal host embryo; as well as (d) gestating the non-human animal host embryo in a surrogate mother; or (II)(a) modifying the genome of a one-cell stage embryo of a non-human animal to include the genetically modified endogenous TARDBP locus; (b) selecting the genetically modified non-human animal one-cell stage embryo comprising the genetically modified endogenous TARDBP locus; and (c) gestating the genetically modified non-human animal one-cell stage embryo in a surrogate mother.

85. A method comprising administering to a cell: (i) a TDP-43 variant according to any one of claims 1 to 27; or (ii) The nucleic acid of any one of claims 32-42, wherein the TDP-43 variant is expressed.

86. The method of claim 85, wherein the cell is a mammalian cell.

87. The method of claim 86, wherein the mammalian cell is a human cell, a rodent cell, a mouse cell, or a rat cell.

88. The method of claim 87, wherein said cell is said human cell.

89. The method of any one of claims 85-88, wherein the cell is a neuron, a glial cell, or a muscle cell.

90. The method of any one of claims 85-89, wherein the cell is in a subject.

91. The method of claim 90, wherein the cell is a neuron in the subject's brain.

92. The method of claim 90 or 91, wherein the TDP-43 variant or the nucleic acid is administered to the subject via intracerebroventricular injection, intracranial injection, or intrathecal injection.

93. The method of any one of claims 85-92, further comprising administering to the cell an agent that reduces or eliminates endogenous TDP-43 expression in the cell.

94. The method of claim 93, wherein the agent comprises an antisense oligonucleotide or RNAi agent targeting endogenous TARDBP messenger RNA, or a nucleic acid encoding the antisense oligonucleotide or the RNAi agent.

95. The method of claim 93, wherein the preparation comprises a nuclease preparation targeted to the endogenous TARDBP locus or one or more nucleic acids encoding the nuclease preparation.

96. The method of claim 95, wherein the nuclease preparation is a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) protein and a guide RNA.

97. The method of claim 96, wherein the nuclease preparation is the Cas protein and the guide RNA, optionally wherein the Cas protein is a Cas9 protein.

98. The method of any one of claims 95-97, wherein the method comprises administering a nucleic acid encoding the TDP-43 variant, wherein the nuclease preparation cleaves the endogenous TARDBP locus, the nucleic acid encoding the TDP-43 variant is inserted into or recombined with the cleaved endogenous TARDBP locus, wherein the TDP-43 variant is expressed from the endogenous TARDBP locus and replaces the expression of the endogenous TDP-43.

99. The method of any one of claims 85-98, wherein endogenous TDP-43 in the cell is susceptible to aggregation, and wherein the method reduces TDP-43 aggregation in the cell.

100. The method of any one of claims 85-99, wherein there is aberrant splicing regulation of endogenous TDP-43 in the cell, and wherein the TDP-43 variant rescues the aberrant TDP-43 splicing regulation in the cell.

101. The method of any one of claims 85-100, wherein there is an aberrant subcellular distribution of endogenous TDP-43 in the cell, and wherein the TDP-43 variant rescues the aberrant subcellular distribution of endogenous TDP-43 in the cell.

102. A method of treating a TDP-43 proteinopathy in a subject, the method comprising administering to one or more cells in the subject: (i) a TDP-43 variant according to any one of claims 1 to 27; or (ii) The nucleic acid of any one of claims 32-42, wherein the TDP-43 variant is expressed in one or more cells in the subject.

103. A method of preventing TDP-43 proteinopathy in a subject, the method comprising administering to one or more cells in the subject: (i) a TDP-43 variant according to any one of claims 1 to 27; or (ii) The nucleic acid of any one of claims 32-42, wherein the TDP-43 variant is expressed in one or more cells in the subject.

104. The method of claim 102 or 103, wherein the TDP-43 proteinopathy is amyotrophic lateral sclerosis (ALS).

105. The method of any one of claims 102-104, wherein the subject is a mammal.

106. The method of any one of claims 102-105, wherein the subject is a human.

107. The method of any one of claims 102-106, wherein the one or more cells comprise neurons in the subject's brain.

108. The method of any one of claims 102-107, wherein the TDP-43 variant or the nucleic acid is administered to the subject via intracerebroventricular injection, intracranial injection, or intrathecal injection.

109. The method of any one of claims 102-108, further comprising administering to the one or more cells an agent that reduces or eliminates endogenous TDP-43 expression in the one or more cells.

110. The method of claim 109, wherein the agent comprises an antisense oligonucleotide or an RNAi agent targeting endogenous TARDBP messenger RNA, or a nucleic acid encoding the antisense oligonucleotide or the RNAi agent.

111. The method of claim 109, wherein the preparation comprises a nuclease preparation or one or more nucleic acids encoding the nuclease preparation that targets the endogenous TARDBP locus.

112. The method of claim 111, wherein the nuclease preparation is a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) protein and a guide RNA.

113. The method of claim 112, wherein the nuclease preparation is the Cas protein and the guide RNA, optionally wherein the Cas protein is a Cas9 protein.

114. The method of any one of claims 111-113, wherein the method comprises administering a nucleic acid encoding the TDP-43 variant, wherein the nuclease preparation cleaves the endogenous TARDBP locus in the one or more cells, the nucleic acid encoding the TDP-43 variant is inserted into or recombined with the cleaved endogenous TARDBP locus, wherein the TDP-43 variant is expressed from the endogenous TARDBP locus and replaces the expression of the endogenous TDP-43.

115. The method of any one of claims 102-114, wherein endogenous TDP-43 in the one or more cells is susceptible to aggregation, and wherein the method reduces TDP-43 aggregation in the one or more cells.

116. The method of any one of claims 102-115, wherein there is aberrant splicing regulation of endogenous TDP-43 in the one or more cells, and wherein the TDP-43 variant rescues the aberrant splicing regulation of TDP-43 in the one or more cells.

117. The method of any one of claims 102-116, wherein there is an aberrant subcellular distribution of endogenous TDP-43 in the one or more cells, and wherein the TDP-43 variant rescues the aberrant subcellular distribution of endogenous TDP-43 in the one or more cells.

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