Humanized stathmin2 allele to track human TDP-43-dependent splicing changes caused by TDP-43 pathology

Abstract

Non-human animal genomes, non-human animal cells, and non-human animals comprising a humanized stathmin-2 (STMN2) locus and methods of making and using such non- human animal genomes, non-human animal cells, and non-human animals are provided. Non- human animal cells or non-human animals comprising a humanized STMN2 locus express a human STMN2 mRNA and a human stathmin-2 or a humanized stathmin-2. Optionally, non- human animal genomes, non-human animal cells, and non-human animals can further comprise a mutated Tardbp locus. Methods are provided for using such non-human animals comprising a humanized STMN2 locus to assess agents as regulators of human STMN2 mRNA splicing. Agents that affect splicing of STMN2 mRNA may be useful as amyotrophic lateral sclerosis (ALS) therapeutics.

Description

Attorney Docket No. 057766 / 640819HUMANIZED STATHMTN2 ALLELE TO TRACK HUMAN TDP-43-DEPENDENT SPLICING CHANGES CAUSED BY TDP-43 PATHOLOGYCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit under 35 USC 119(e) of U.S. Provisional Application No. 63 / 718,367, filed November 8, 2024, which is incorporated by reference in its entirety for all purposes.REFERENCE TO A SEQUENCE LISTING SUBMITTED AS AN XML FILE VIA PATENT CENTER

[0002] The Sequence Listing written in file 640819SEQLIST. xml is 292,461 bytes, was created on October 31, 2025, and is hereby incorporated by reference in its entirety.BACKGROUND

[0003] Stathmin-2 is a highly conserved cytosolic protein essential for axonal outgrowth and maintenance. STMN2 -knockout mice develop late-onset, predominantly motor axonopathy paralleling that seen in human amyotrophic lateral sclerosis (ALS). A cryptic splicing event in STMN2 mRNA mediated by human specific transactive response DNA-binding protein 43 (TDP- 43) has been shown to lead to loss of Stathmin-2 protein. TDP-43 is a highly conserved, ubiquitously expressed, RNA-binding protein that plays a critical role in essential cell functions such as transcription repression, RNA splicing, and translational regulation. Approximately 97% of ALS cases show a postmortem pathology of ubiquitinated, and hyper-phosphorylated cytosolic aggregates of TDP-43 and over 50 mutations in TDP-43 have also been linked to ALS, supporting TDP-43 dysfunction as a critical component in ALS. Depletion of the RNA-binding protein TDP-43 from the nucleus and its accumulation in cytoplasmic aggregates is a hallmark post-mortem pathological finding in nearly all cases of amyotrophic lateral sclerosis (ALS) and approximately 50% of frontotemporal dementia (FTD), a form of Alzheimer’s disease-related dementia.

[0004] Although it remains to be determined the phenotypic contribution of STMN2 loss to motor neuron degeneration in ALS, the TDP-43 -dependent cryptic exon in STMN2 nevertheless offers a unique and valuable molecular readout of disease-associated TDP-43 dysfunction. However, because TDP-43 binding sites are largely within introns, and intronic sequences are poorly conserved between species, the splicing events regulated by TDP-43 are generally notAttorney Docket No. 057766 / 640819 conserved between mouse and human, including STMN2. This presents a major barrier to modeling and characterizing the contribution of STMN2 mis-splicing to motor neuron disease in mouse models of TDP-43 pathology.SUMMARY

[0005] Non-human animals comprising a humanized endogenous STMN2 locus are provided, as well as methods of making and using such non-human animals. Non-human animal genomes or cells comprising a humanized endogenous STMN2 locus are also provided. Also provided are humanized endogenous STMN2 genes, nuclease agents and / or targeting vectors for use in humanizing a non-human animal Stmn2 gene, and methods of making and using such humanized STMN2 genes.

[0006] In one aspect, provided are non-human animal genomes, non-human animal cells, or non-human animals comprising a humanized endogenous STMN2 locus. Such non-human animal genomes, non-human animal cells, or non-human animals can comprise in their genome a humanized endogenous STMN2 locus in which a segment of the endogenous Stnm2 locus has been deleted and replaced with a corresponding human STMN2 sequence.

[0007] In some such non-human animal genomes, non-human animal cells, or non-human animals, a region of the endogenous Stmn2 locus comprising both coding sequence and noncoding sequence has been deleted and replaced with the corresponding human STMN2 sequence comprising both coding sequence and non-coding sequence.

[0008] In some such non-human animal genomes, non-human animal cells, or non-human animals, at least one intron and at least one exon of the endogenous Stmn2 locus have been deleted and replaced with the corresponding human STMN2 sequence. In some such non-human animal genomes, non-human animal cells, or non-human animals, exon 1, intron 1, and exon 2 of the endogenous Stmn2 locus have been deleted and replaced with the corresponding human STMN2 sequence.

[0009] In some such non-human animal genomes, non-human animal cells, or non-human animals, the humanized endogenous STMN2 locus encodes a human stathmin-2.

[0010] In some such non-human animal genomes, non-human animal cells, or non-human animals, the deleted segment of the endogenous Stnm2 locus comprises a region of endogenous Stmn2 genomic sequence from the start codon through the stop codon, and wherein theAttorney Docket No. 057766 / 640819 corresponding human STMN2 sequence comprises the human STMN2 genomic sequence from the start codon through the stop codon. In some such non-human animal genomes, non-human animal cells, or non-human animals, the endogenous Stmn2 5’ untranslated region has not been deleted and replaced with the corresponding human STMN2 sequence and / or wherein the humanized endogenous STMN2 locus comprises a human STMN2 3’ untranslated region.

[0011] In some such non-human animal genomes, non-human animal cells, or non-human animals, the humanized endogenous STMN2 locus comprises the endogenous Stmn2 promoter, wherein the human STMN2 sequence is operably linked to the endogenous Stmn2 promoter.

[0012] In some such non-human animal genomes, non-human animal cells, or non-human animals, the deleted segment of the endogenous Stmn2 locus comprises a region of endogenous Stmn2 genomic sequence from the start codon through the stop codon, and wherein the corresponding human STMN2 sequence comprises the human STMN2 genomic sequence from the start codon through the stop codon, wherein the endogenous Stnm2 5’ untranslated region has not been deleted and replaced with the corresponding human STMN2 sequence, wherein the humanized endogenous STMN2 locus comprises a human STMN2 3’ untranslated region, and wherein the humanized endogenous STMN2 locus comprises the endogenous Stmn2 promoter, wherein the human S1MN2 sequence is operably linked to the endogenous Stnm2 promoter.

[0013] In some such non-human animal genomes, non-human animal cells, or non-human animals, the humanized endogenous STMN2 locus comprises a human STMN2 sequence comprising, consisting essentially of, or consisting of a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO: 53 or 54. In some such non-human animal genomes, non-human animal cells, or non-human animals, the humanized endogenous STMN2 locus encodes a protein comprising, consisting essentially of, or consisting of a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO: 9, optionally wherein the humanized endogenous STMN2 locus encodes a protein comprising, consisting essentially of, or consisting of the sequence set forth in SEQ ID NO: 9. In some such non-human animal genomes, non-human animal cells, or non-human animals, the humanized endogenous STMN2 locus comprises a coding sequence comprising, consisting essentially of, or consisting of a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO: 10, optionally wherein theAttorney Docket No. 057766 / 640819 humanized endogenous STMN2 locus comprises a coding sequence comprising, consisting essentially of, or consisting of the sequence set forth in SEQ ID NO: 10. In some such nonhuman animal genomes, non-human animal cells, or non-human animals, the humanized endogenous STMN2 locus comprises a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO: 48 or 49, optionally wherein the humanized endogenous STMN2 locus comprises the sequence set forth in SEQ ID NO: 48 or 49.

[0014] In some such non-human animal genomes, non-human animal cells, or non-human animals, the humanized endogenous STMN2 locus does not comprise a selection cassette or a reporter gene.

[0015] In some such non-human animal genomes, non-human animal cells, or non-human animals, the non-human animal or non-human animal cell is homozygous for the humanized endogenous STMN2 locus. In some such non-human animal genomes, non-human animal cells, or non-human animals, the non-human animal or non-human animal cell is heterozygous for the humanized endogenous STMN2 locus. In some such non-human animal genomes, non-human animal cells, or non-human animals, the non-human animal or non-human animal cell is compound heterozygous or hemizygous for the humanized endogenous STMN2 locus.

[0016] In some such non-human animal genomes, non-human animal cells, or non-human animals: (I) (i) the first allele of the humanized endogenous STMN2 locus comprises a human STMN2 sequence comprising, consisting essentially of, or consisting of a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO: 53 or 54; or (ii) the first allele of the humanized endogenous STMN2 locus encodes a protein comprising, consisting essentially of, or consisting of a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO: 9, optionally wherein the first allele of the humanized endogenous STMN2 locus encodes a protein comprising, consisting essentially of, or consisting of the sequence set forth in SEQ ID NO: 9; or (iii) the first allele of the humanized endogenous STMN2 locus comprises a coding sequence comprising, consisting essentially of, or consisting of a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO: 10, optionally wherein the first allele of the humanized endogenous STMN2 locus comprises a coding sequence comprising,Attorney Docket No. 057766 / 640819 consisting essentially of, or consisting of the sequence set forth in SEQ ID NO: 10; or (iv) the first allele of the humanized endogenous STMN2 locus comprises a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO: 48 or 49, optionally wherein the first allele of the humanized endogenous STMN2 locus the sequence set forth in SEQ ID NO: 48 or 49; and (II) (i) the second allele of the humanized endogenous STMN2 locus is an inactivated allele comprising a deletion.

[0017] In some such non-human animal genomes, non-human animal cells, or non-human animals, the non-human animal genome, non-human animal or non-human animal cell further comprises a mutation in the Tardbp locus. In some such non-human animal genomes, non- human animal cells, or non-human animals, the mutation in the Tardbp locus causes mislocalization of encoded TAR DNA-binding protein 43 (TDP-43) protein.

[0018] In some such non-human animal genomes, non-human animal cells, or non-human animals the encoded TDP-43 protein comprises a mutation in the nuclear localization signal (NLS), nuclear export signal (NES), or prion-like domain (PLD) of TDP-43.

[0019] In some such non-human animal genomes, non-human animal cells, or non-human animals (I) the mutation in the NLS comprises point mutations at positions 82-84, 95, 97, and 98, optionally wherein the TDP-43 protein is a mouse TDP-43 protein, and the mutation comprises K82A, R83A, K84A, K95A, K97A, and R98A point mutations; (II) the mutation in the NES comprises a deletion of between positions 239 and 250, optionally wherein the TDP-43 protein is a mouse TDP-43 protein, and the mutation comprises a deletion between V239 and 1250; or (III) the mutation in the PLD comprises a deletion between positions 274 and 414, optionally wherein the TDP-43 protein is a mouse TDP-43 protein, and the mutation comprises a deletion between G274 and M414.

[0020] In some such non-human animal genomes, non-human animal cells, or non-human animals, the non-human animal is a mammal. In some such non-human animal genomes, non- human animal cells, or non-human animals, the mammal is a rodent, optionally wherein the rodent is a rat or a mouse. In some such non-human animal genomes, non-human animal cells, or non-human animals, the mammal is the mouse.

[0021] In some such non-human animal genomes, non-human animal cells, or non-human animals, RNA transcripts from the humanized endogenous STMN2 locus include a cryptic exon when TDP-43 is disrupted by mutations that lead to mislocalization and loss of TDP-43 splicingAttorney Docket No. 057766 / 640819 activity. In some such non-human animal genomes, non-human animal cells, or non-human animals, RNA transcripts from the humanized endogenous STMN2 locus do not include the cryptic exon when wild type mouse TDP-43 is present.

[0022] In some such non-human animals, the non-human animal comprises the humanized endogenous STMN2 locus in its germline.

[0023] In some such non-human animal cells, the non-human animal cell is a neuron, optionally wherein the non-human animal cell is an embryonic-stem-cell-derived motor neuron (ESMN), optionally wherein the ESMN is a mouse ESMN. In some such non-human animal cells, the cell is in vitro.

[0024] In another aspect, provided are targeting vectors for generating a humanized endogenous STMN2 locus in which a segment of the endogenous Stmn2 locus has been deleted and replaced with a corresponding human STMN2 sequence, wherein the targeting vector comprises an insert nucleic acid comprising the corresponding human STMN2 sequence flanked by a 5’ homology arm targeting a 5’ target sequence at the endogenous Stnm2 locus and a 3’ homology arm targeting a 3’ target sequence at the endogenous Stmn2 locus.

[0025] In another aspect, provided are nucleic acids comprising humanized non-human animal Stmn2 gene in which a segment of the non-human animal Stmn2 gene has been deleted and replaced with a corresponding human STMN2 sequence.

[0026] In another aspect, provided are methods of assessing the effectiveness of an agent as a regulator of human STMN2 messenger RNA (mRNA) splicing, comprising: (a) administering the agent to any of the non-human animals herein or any of the non-human animal cells herein; (b) measuring the splicing of human STMN2 RNA transcripts encoded by the humanized endogenous STMN2 locus in the non-human animal or non-human animal cell, and (c) comparing the measured splicing of human STMN2 RNA transcripts in the non-human animal or non-human animal cell with the splicing of human STMN2 RNA transcripts in a control non- human animal or non-human animal cell, wherein splicing is measured as an amount of full- length STMN2 mRNA or as an amount of full-length stathmin-2 protein, wherein an increase in full-length STMN2 mRNA or an increase in full-length stathmin-2 protein in the non-human animal or non-human animal cell administered the agent relative to that in the control non-human animal or non-human animal cell indicates that the agent is effective as a regulator of human STMN2 mRNA splicing.Attorney Docket No. 057766 / 640819

[0027] In some such methods, the splicing is measured as the amount of full-length STMN2 mRNA encoded by the humanized endogenous STMN2 locus. In some such methods, the assessing further comprises measuring expression of a cryptic spliced STMN2 mRNA encoded by the humanized endogenous STMN2 locus. In some such methods, the assessing comprises measuring expression of a full-length STMN2 mRNA encoded by the humanized endogenous STMN2 locus and a cryptic spliced STMN2 mRNA encoded by the humanized endogenous STMN2 locus.

[0028] In some such methods, the splicing is measured as the amount of full-length stathmin- 2 protein. In some such methods, the assessing further comprises measuring expression of a truncated human stathmin-2 encoded by the humanized endogenous STMN2 locus. In some such methods, the assessing comprises measuring expression of the full-length human stathmin-2 encoded by the humanized endogenous STMN2 locus and the truncated human stathmin-2 encoded by the humanized endogenous STMN2 locus.

[0029] In some such methods, the agent is a TDP-43 variant. In some such methods, the agent is an amyotrophic lateral sclerosis (ALS) therapeutic agent or a candidate ALS therapeutic agent.

[0030] In another aspect, provided are methods of making any of the non-human animals herein, the method comprising: (I) (a) modifying the genome of a non-human animal embryonic stem (ES) cell to comprise in its genome the humanized endogenous STMN2 locus; (b) identifying or selecting the genetically modified non-human animal ES cell comprising in its genome the humanized endogenous STMN2 locus; (c) introducing the genetically modified non- human animal ES 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 comprise in its genome the humanized endogenous STMN2 locus; (b) selecting the genetically modified non-human animal one-cell stage embryo comprising in its genome the humanized endogenous STMN2 locus; and (c) gestating the genetically modified non-human animal one-cell stage embryo in a surrogate mother.

[0031] In another aspect, provided are methods of making any of the non-human animal cells herein, the method comprising modifying the genome of a non-human animal cell to comprise in its genome the humanized endogenous STMN2 locus. In some such methods, the non-humanAttorney Docket No. 057766 / 640819 animal cell is an embryonic stem (ES) cell. In some such methods, the non-human animal is a mouse or a rat. In some such methods, the non-human animal is the mouse.BRIEF DESCRIPTION OF THE FIGURES

[0032] Figure 1 (not to scale) shows a schematic of the mouse Stmn2 locus, the human STMN2 locus, the wild type humanized STMN2 allele with a drug selection cassette (targeted allele, MAID 8636), and the wild type humanized STMN2 allele without the drug selection cassette (targeted allele, cassette removed, MAID 8637).

[0033] Figure 2 (not to scale) shows schematics of the targeting and screening assays used to assess humanization of the mouse Stmn2 locus, including loss-of-allele assays (8636mTU, 8636mTD, 8636mTD2), gain-of-allele assays (8636hTU, 8636hTD, 8636hTD2), CRISPR retention assays (90108mretU, 90108mretU2, 90108mretD, 90108mretD2), and CRISPR assay (90108mTM).

[0034] Figure 3 shows an alignment of mouse stathmin-2 (mStmn2 protein), human stathmin-2 (hSTMN2 protein), humanized STMN2 protein (humanized), and humanized STMN2 protein where alternate exon is used (hSTMN2 using alt. exon). The positions of boundary between exon 1 and exon 2, membrane association domain (boxed with broken line), phosphorylation domain (boxed with solid line), and coiled-coil domain (underlined with blue line) are indicated.

[0035] Figure 4 shows a diagram depicting the described functional domain architecture of mouse TDP-43 and mutations predicted to abolish functionality of these domains. TDP-43 comprises an N-terminal region, nuclear localization signal (NLS), two RNA recognition motifs: RRM1 and RRM2, nuclear export signal (NES), and a C-terminal region encompassing a prionlike domain (PLD).

[0036] Figure 5 illustrates a protocol used to differentiate embryonic stem (ES) cells into motor neurons.

[0037] Figure 6 shows mutations in different functional domains of TDP-43 disrupt pre- mRNA splicing, cryptic exon repression and autoregulation. (A) Semi-quantitative RT-PCR analysis of known TDP-43 mRNA targets indicates that TDP-43 function in both cryptic and alternative splicing is strongly disrupted by TDP-43 mis-localization driven by both ANLS and APLD mutants. (B) Semi -quantitative RT-PCR analysis of TDP-43 mRNA shows an increase inAttorney Docket No. 057766 / 640819 the presence of a short isoform in ESCMNs with ANLS and ANES as the only form of TDP-43. (C) Schematic depicting Stathmin-2 humanization. Stathmin-2 undergoes cryptic exon inclusion in ALS patients leading to a loss of Stathmin-2 protein. (D) qRT-PCR analysis of ES-derived motor neurons indicates that in ANLS, ANES, and APLD cells where there is mTDP-43 dysfunction, we see inclusion of the STMN2 cryptic exon. Inclusion of the cryptic exon leads to a loss of STMN2 mRNA. (E) qRT-PCR analysis of ES-derived motor neurons indicates that in ANLS, ANES, and APLD cells where there is mTDP-43 dysfunction, we see reduction in amount of full-length STMN2 mRNA. (F and G) Automated capillary-based western blotting analysis indicates that loss of mTDP-43 function, and cryptic exon inclusion leads to a reduction in STMN2 protein. (F) shows a western blot, and (G) shows a bar graph of quantified western blot data.

[0038] Figure 7 shows splicing events regulated by TDP-43 in mouse and human are poorly conserved. (A) Schematic comparing splicing in mouse and human. (B) Schematic of model of human TDP-43 splicing by humanizing the gene encoding Stathmin2, which undergoes cryptic exon inclusion in ALS patients as a result of TDP-43 pathology. CE = cryptic exon.

[0039] Figure 8 shows an in vitro assay to screen for TDP-43 variants using hSTMN2 lines.DEFINITIONS

[0040] The terms “protein,” “polypeptide,” and “peptide,” used interchangeably herein, include polymeric forms of amino acids of any length, including coded and non-coded amino acids and chemically or biochemically modified or derivatized amino acids. The terms also include polymers that have been modified, such as polypeptides having modified peptide backbones. The term “domain” refers to any part of a protein or polypeptide having a particular function or structure.

[0041] Proteins are said to have an “N-terminus” and a “C-terminus.” The term “N- terminus” relates to the start of a protein or polypeptide, terminated by an amino acid with a free amine group (-NH2). The term “C-terminus” relates to the end of an amino acid chain (protein or polypeptide), terminated by a free carboxyl group (-COOH).

[0042] The terms “nucleic acid” and “polynucleotide,” used interchangeably herein, include polymeric forms of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, or analogs or modified versions thereof. They include single-, double-, and multi-stranded DNAAttorney Docket No. 057766 / 640819 or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers comprising purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural, or derivatized nucleotide bases.

[0043] Nucleic acids are said to have “5’ ends” and “3’ ends” because mononucleotides are reacted to make oligonucleotides in a manner such that the 5’ phosphate of one mononucleotide pentose ring is attached to the 3’ oxygen of its neighbor in one direction via a phosphodiester linkage. An end of an oligonucleotide is referred to as the “5’ end” if its 5’ phosphate is not linked to the 3’ oxygen of a mononucleotide pentose ring. An end of an oligonucleotide is referred to as the “3’ end” if its 3’ oxygen is not linked to a 5’ phosphate of another mononucleotide pentose ring. A nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5’ and 3’ ends. In either a linear or circular DNA molecule, discrete elements are referred to as being “upstream” or 5’ of the “downstream” or 3’ elements.

[0044] The term “genomically integrated” refers to a nucleic acid that has been introduced into a cell such that the nucleotide sequence integrates into the genome of the cell. Any protocol may be used for the stable incorporation of a nucleic acid into the genome of a cell.

[0045] The term “targeting vector” refers to a recombinant nucleic acid that can be introduced by homologous recombination, non-homologous-end-joining-mediated ligation, or any other means of recombination to a target position in the genome of a cell.

[0046] The term “viral vector” refers to a recombinant nucleic acid that includes at least one element of viral origin and includes elements sufficient for or permissive of packaging into a viral vector particle. The vector and / or particle can be utilized for the purpose of transferring DNA, RNA, or other nucleic acids into cells in vitro ex vivo, or in vivo. Numerous forms of viral vectors are known.

[0047] The term “isolated” with respect to cells, tissues, proteins, and nucleic acids includes cells, tissues, proteins, and nucleic acids that are relatively purified with respect to other bacterial, viral, cellular, or other components that may normally be present in situ, up to and including a substantially pure preparation of the cells, tissues, proteins, and nucleic acids. The term “isolated” also includes cells, tissues, proteins, and nucleic acids that have no naturally occurring counterpart, have been chemically synthesized and are thus substantially uncontaminated by other cells, tissues, proteins, and nucleic acids, or has been separated orAttorney Docket No. 057766 / 640819 purified from most other components (e.g., cellular components) with which they are naturally accompanied (e.g., other cellular proteins, polynucleotides, or cellular components).

[0048] The term “wild type” includes entities having a structure and / or activity as found in a normal (as contrasted with mutant, diseased, altered, or so forth) state or context. Wild type genes and polypeptides often exist in multiple different forms (e.g., alleles).

[0049] The term “endogenous sequence” refers to a nucleic acid sequence that occurs naturally within a cell or non-human animal. For example, an endogenous Stmn2 sequence of a non-human animal refers to a native Stmn2 sequence that naturally occurs at the Stmn2 locus in the non-human animal.

[0050] “Exogenous” molecules or sequences include molecules or sequences that are not normally present in a cell in that form or location (e.g., genomic locus). Normal presence includes presence with respect to the particular developmental stage and environmental conditions of the cell. An exogenous molecule or sequence, for example, can include a mutated version of a corresponding endogenous sequence within the cell, such as a humanized version of the endogenous sequence, or can include a sequence corresponding to an endogenous sequence within the cell but in a different form (i.e., not within a chromosome). In contrast, endogenous molecules or sequences include molecules or sequences that are normally present in that form and location in a particular cell at a particular developmental stage under particular environmental conditions.

[0051] The term “heterologous” when used in the context of a nucleic acid or a protein indicates that the nucleic acid or protein comprises at least two segments that do not naturally occur together in the same molecule. For example, the term “heterologous,” when used with reference to segments of a nucleic acid or segments of a protein, indicates that the nucleic acid or protein comprises two or more sub-sequences that are not found in the same relationship to each other (e.g., joined together) in nature. As one example, a “heterologous” region of a nucleic acid vector is a segment of nucleic acid within or attached to another nucleic acid molecule that is not found in association with the other molecule in nature. For example, a heterologous region of a nucleic acid vector could include a coding sequence flanked by sequences not found in association with the coding sequence in nature. Likewise, a “heterologous” region of a protein is a segment of amino acids within or attached to another peptide molecule that is not found in association with the other peptide molecule in nature (e.g., a fusion protein, or a protein with aAttorney Docket No. 057766 / 640819 tag). Similarly, a nucleic acid or protein can comprise a heterologous label or a heterologous secretion or localization sequence.

[0052] “Codon optimization” (i.e., a “codon optimized” sequence) takes advantage of the degeneracy of codons, as exhibited by the multiplicity of three-base pair codon combinations that specify an amino acid, and generally includes a process of modifying a nucleic acid sequence for enhanced expression in particular host cells by replacing at least one codon of the native sequence with a codon that is more frequently or most frequently used in the genes of the host cell while maintaining the native amino acid sequence. For example, a nucleic acid encoding a Stathmin-2 protein can be modified to substitute codons having a higher frequency of usage in a given prokaryotic or eukaryotic cell, including a bacterial cell, a yeast cell, a human cell, a nonhuman cell, a mammalian cell, a rodent cell, a mouse cell, a rat cell, a hamster cell, or any other host cell, as compared to the naturally occurring nucleic acid sequence. Codon usage tables are readily available, for example, at the “Codon Usage Database.” These tables can be adapted in several ways. See Nakamura et al. (2000) Nucleic Acids Research 28:292, herein incorporated by reference in its entirety for all purposes. Computer algorithms for codon optimization of a sequence for expression in a particular host are also available (see, e.g., Gene Forge).

[0053] The term “locus” refers to a specific location of a gene (or significant sequence), DNA sequence, polypeptide-encoding sequence, or position on a chromosome of the genome of an organism. For example, a “stathmin-2 locus” or “Stmn2 locus” may refer to the specific location of & Slmn2 gene, Stmn2 DNA sequence, stathmin-2 -encoding sequence, or Stmn2 position on a chromosome of the genome of an organism that has been identified as to where such a sequence resides. A "'Stmn2 locus” may comprise a regulatory element of a 57 / w?2gene, including, for example, an enhancer, a promoter, 5’ and / or 3’ untranslated region (UTR), or a combination thereof.

[0054] The term “gene” refers to DNA sequences in a chromosome that may contain, if naturally present, at least one coding and at least one non-coding region. The DNA sequence in a chromosome that codes for a product (e.g., but not limited to, an RNA product and / or a polypeptide product) can include the coding region interrupted with non-coding introns and sequence located adjacent to the coding region on both the 5’ and 3’ ends such that the gene corresponds to the full-length mRNA (including the 5’ and 3’ untranslated sequences). Additionally, other non-coding sequences including regulatory sequences (e.g., but not limitedAttorney Docket No. 057766 / 640819 to, promoters, enhancers, and transcription factor binding sites), polyadenylation signals, internal ribosome entry sites, silencers, insulating sequence, and matrix attachment regions may be present in a gene. These sequences may be close to the coding region of the gene (e.g., but not limited to, within 10 kb) or at distant sites, and they influence the level or rate of transcription and translation of the gene.

[0055] The term “allele” refers to a variant form of a gene. Some genes have a variety of different forms, which are located at the same position, or genetic locus, on a chromosome. A diploid organism has two alleles at each genetic locus. Each pair of alleles represents the genotype of a specific genetic locus. Genotypes are described as homozygous if there are two identical alleles at a particular locus and as heterozygous if the two alleles differ. Genotypes are described as compound heterozygous (e.g., hemizygous) if there is a combination of a humanized allele and an inactivated allele (e.g., a CRISPR-induced deletion allele). The term “compound heterozygosity” refers includes situations in which both alleles of the target locus (i.e., the alleles on both homologous chromosomes) have been modified, but they have been modified in different ways (e.g., a targeted modification in one allele and inactivation or disruption of the other allele), for example, a biallelic modification can result in compound heterozygosity if the cell has one allele with the targeted modification and another allele that is not capable of being expressed or is not otherwise functional. Compound heterozygosity includes hemizygosity. Hemizygosity includes situations in which only one allele (i.e., an allele on one of two homologous chromosomes) of the target locus is present, for example, if the targeted modification occurs in one allele with a corresponding loss or deletion of the other allele. In an exemplary compound heterozygous (hemizygous) genotype, there is a combination of a humanized allele and an in activated allele (e.g., a deletion allele or collapsed allele, such as a CRISPR-induced deletion allele). Some CRISPR-induced deletion alleles result from genome collapsing, whereby a large nucleic acid sequence (e.g., from near the start codon to near the stop codon) is deleted from a chromosome between two cleavage sites.

[0056] A “promoter” is a regulatory region of DNA usually comprising a TATA box capable of directing RNA polymerase II to initiate RNA synthesis at the appropriate transcription initiation site for a particular polynucleotide sequence. A promoter may additionally comprise other regions which influence the transcription initiation rate. The promoter sequences disclosed herein modulate transcription of an operably linked polynucleotide. A promoter can be active inAttorney Docket No. 057766 / 640819 one or more of the cell types disclosed herein (e.g., a eukaryotic cell, a non-human mammalian cell, a human cell, a rodent cell, a pluripotent cell, a one-cell stage embryo, a differentiated cell, or a combination thereof). A promoter can be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a temporally restricted promoter (e.g., a developmentally regulated promoter), or a spatially restricted promoter (e.g., a cell-specific or tissue-specific promoter). Examples of promoters can be found, for example, in WO 2013 / 176772, herein incorporated by reference in its entirety for all purposes.

[0057] 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., an alcohol dehydrogenase (alcA) gene promoter), tetracycline-regulated promoters (e.g., a tetracycline-responsive promoter, a tetracycline operator sequence (tetO), a tet-On promoter, or a tet-Off promoter), steroid regulated promoters (e.g., a rat glucocorticoid receptor, a promoter of an estrogen receptor, or a promoter of an ecdysone receptor), or metal -regulated promoters (e.g., a metalloprotein promoter). Physically regulated promoters include, for example temperature-regulated promoters (e.g., a heat shock promoter) and light-regulated promoters (e.g., a light-inducible promoter or a light-repressible promoter).

[0058] Tissue-specific promoters can be, for example, neuron-specific promoters, gliaspecific promoters, muscle cell-specific promoters, heart cell-specific promoters, kidney cellspecific promoters, bone cell-specific promoters, endothelial cell-specific promoters, or immune cell-specific promoters (e.g., a B cell promoter or a T cell promoter).

[0059] Developmentally regulated promoters include, for example, promoters active only during an embryonic stage of development, or only in an adult cell.

[0060] “Operable linkage” or being “operably linked” includes juxtaposition of two or more components (e.g., a promoter and another sequence element) such that both components function normally and allow the possibility that at least one of the components can mediate a function that is exerted upon at least one of the other components. For example, a promoter can be operably linked to a coding sequence if the promoter 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 being contiguous with each other or acting in trans (e.g., a regulatory sequence can act at a distance to control transcription of the coding sequence).Attorney Docket No. 057766 / 640819

[0061] “Complementarity” of nucleic acids means that a nucleotide sequence in one strand of nucleic acid, due to orientation of its nucleobase groups, forms hydrogen bonds with another sequence on an opposing nucleic acid strand. The complementary bases in DNA are typically A with T and C with G. In RNA, they are typically C with G and U with A. Complementarity can be perfect or substantial / sufficient. Perfect complementarity between two nucleic acids means that the two nucleic acids can form a duplex in which every base in the duplex is bonded to a complementary base by Watson-Crick pairing. “Substantial” or “sufficient” complementary means that a sequence in one strand is not completely and / or perfectly complementary to a sequence in an opposing strand, but that sufficient bonding occurs between bases on the two strands to form a stable hybrid complex in set of hybridization conditions (e.g., salt concentration and temperature). Such conditions can be predicted by using the sequences and standard mathematical calculations to predict the Tm (melting temperature) of hybridized strands, or by empirical determination of Tm by using routine methods. Tm includes the temperature at which a population of hybridization complexes formed between two nucleic acid strands are 50% denatured (i.e., a population of double-stranded nucleic acid molecules becomes half dissociated into single strands). At a temperature below the Tm, formation of a hybridization complex is favored, whereas at a temperature above the Tm, melting or separation of the strands in the hybridization complex is favored. Tm may be estimated for a nucleic acid having a known G+C content in an aqueous 1 M NaCl solution by using, e.g., Tm=81.5+0.41(% G+C), although other known Tm computations consider nucleic acid structural characteristics.

[0062] Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases are possible. The conditions appropriate for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementation, variables which are well known. The greater the degree of complementation between two nucleotide sequences, the greater the value of the melting temperature (Tm) for hybrids of nucleic acids having those sequences. For hybridizations between nucleic acids with short stretches of complementarity (e.g., complementarity over 35 or fewer, 30 or fewer, 25 or fewer, 22 or fewer, 20 or fewer, or 18 or fewer nucleotides) the position of mismatches becomes important (see Sambrook et al., supra, 11.7-11.8). Typically, the length for a hybridizable nucleic acid is at least about 10 nucleotides. Illustrative minimum lengths for a hybridizable nucleic acid include at least about 15 nucleotides, at least about 20 nucleotides, at least about 22 nucleotides,Attorney Docket No. 057766 / 640819 at least about 25 nucleotides, and at least about 30 nucleotides. Furthermore, the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the region of complementation and the degree of complementation.

[0063] The sequence of polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. Moreover, a polynucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure). A polynucleotide (e.g., gRNA) can comprise at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence complementarity to a target region within the target nucleic acid sequence to which they are targeted. For example, a gRNA in which 18 of 20 nucleotides are complementary to a target region, and would therefore specifically hybridize, would represent 90% complementarity. In this example, the remaining noncomplementaiy nucleotides may be clustered or interspersed with complementary nucleotides and need not be contiguous to each other or to complementary nucleotides.

[0064] Percent complementarity between particular stretches of nucleic acid sequences within nucleic acids can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs (Altschul et al. (1990) J. Mol. Biol. 215:403-410; Zhang and Madden (1997) Genome Res. 7:649-656, each of which is herein incorporated by reference in its entirety for all purposes) or by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482-489, herein incorporated by reference in its entirety for all purposes.

[0065] The methods and compositions provided herein employ a variety of different components. Some components throughout the description can have active variants and fragments. Such components include, for example, Cas proteins, CRISPR RNAs, tracrRNAs, and guide RNAs. Biological activity for each of these components is described elsewhere herein. The term “functional” refers to the innate ability of a protein or nucleic acid (or a fragment or variant thereof) to exhibit a biological activity or function. Such biological activities or functions can include, for example, the ability of a Cas protein to bind to a guide RNA and to a target DNA sequence. The biological functions of functional fragments or variants may be the same or mayAttorney Docket No. 057766 / 640819 in fact be changed (e.g., with respect to their specificity, selectivity, or efficacy) in comparison to the original molecule, but with retention of the molecule’s basic biological function.

[0066] The term “variant” refers to a nucleotide sequence differing from the sequence most prevalent in a population (e.g., by one nucleotide) or a protein sequence different from the sequence most prevalent in a population (e.g., by one amino acid).

[0067] The term “fragment,” when referring to a protein, means a protein that is shorter or has fewer amino acids than the full-length protein. The term “fragment,” when referring to a nucleic acid, means a nucleic acid that is shorter or has fewer nucleotides than the full-length nucleic acid. A fragment can be, for example, when referring to a protein fragment, an N- terminal fragment (i.e., removal of a portion of the C-terminal end of the protein), a C-terminal fragment (i.e., removal of a portion of the N-terminal end of the protein), or an internal fragment (i.e., removal of a portion of each of the N-terminal and C-terminal ends of the protein). A fragment can be, for example, when referring to a nucleic acid fragment, a 5’ fragment (i.e., removal of a portion of the 3’ end of the nucleic acid), a 3’ fragment (i.e., removal of a portion of the 5’ end of the nucleic acid), or an internal fragment (i.e., removal of a portion each of the 5’ and 3’ ends of the nucleic acid).

[0068] “Sequence identity” or “identity” in the context of two polynucleotides or polypeptide sequences refers to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins, residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity.” Means for making this adjustment are well known. Typically, this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. TheAttorney Docket No. 057766 / 640819 scoring of conservative substitutions is calculated, e g., as implemented in the program PC / GENE (Intelligenetics, Mountain View, California).

[0069] “Percentage of sequence identity” includes the value determined by comparing two optimally aligned sequences (greatest number of perfectly matched residues) over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. Unless otherwise specified (e.g., the shorter sequence includes a linked heterologous sequence), the comparison window is the full length of the shorter of the two sequences being compared.

[0070] Unless otherwise stated, sequence identity / similarity values include the value obtained using GAP Version 10 using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program thereof. “Equivalent program” includes any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.

[0071] The term “conservative amino acid substitution” refers to the substitution of an amino acid that is normally present in the sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine, or leucine for another non-polar residue. Likewise, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, or between glycine and serine. Additionally, the substitution of a basic residue such as lysine, arginine, or histidine for another, or the substitution of one acidic residue such as aspartic acid or glutamic acid for another acidic residue are additional examples of conservativeAttorney Docket No. 057766 / 640819 substitutions. Examples of non-conservative substitutions include the substitution of a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, or methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine and / or a polar residue for a non-polar residue. Typical amino acid categorizations are summarized in Table 1 below.

[0072] Table 1. Amino Acid Categorizations.Alanine Ala A Nonpolar Neutral 1.8Arginine Arg R Polar Positive -4.5Asparagine Asn N Polar Neutral -3.5Aspartic acid Asp D Polar Negative -3.5Cysteine Cys C Nonpolar Neutral 2.5Glutamic acid Glu E Polar Negative -3.5Glutamine Gin Q Polar Neutral -3.5Glycine Gly G Nonpolar Neutral -0.4Histidine His H Polar Positive -3.2Isolcucinc lie I Nonpolar Neutral 4.5Leucine Leu L Nonpolar Neutral 3.8Lysine Lys K Polar Positive -3.9Methionine Met M Nonpolar Neutral 1.9Phenylalanine Phe F Nonpolar Neutral 2.8Proline Pro P Nonpolar Neutral -1.6Serine Ser S Polar Neutral -0.8Threonine Thr T Polar Neutral -0.7Tryptophan Trp W Nonpolar Neutral -0.9Tyrosine Tyr Y Polar Neutral -1.3Valine Vai V Nonpolar Neutral 4.2

[0073] A “homologous” sequence (e.g., nucleic acid sequence) includes a sequence that is either identical or substantially similar to a known reference sequence, such that it is, for example, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the known reference sequence. Homologous sequences can include, for example, orthologous sequence and paralogous sequences. Homologous genes, for example, typically descend from a common ancestral DNA sequence, either through a speciation event (orthologous genes) or a genetic duplication event (paralogous genes). “Orthologous” genes include genes in different species that evolved from a common ancestral gene by speciation. Orthologs typically retain the same function in the course of evolution. “Paralogous”Attorney Docket No. 057766 / 640819 genes include genes related by duplication within a genome. Paralogs can evolve new functions in the course of evolution.

[0074] The term “ / / / vitro” includes artificial environments and to processes or reactions that occur within an artificial environment (e.g., a test tube or an isolated cell or cell line). The term “zzz vivo” includes natural environments (e.g., a cell, organism, or body) and to processes or reactions that occur within a natural environment. The term “ex vivo” includes cells that have been removed from the body of an individual and processes or reactions that occur within such cells.

[0075] The term “reporter gene” refers to a nucleic acid having a sequence encoding a gene product (typically an enzyme) that is easily and quantifiably assayed when a construct comprising the reporter gene sequence operably linked to a heterologous promoter and / or enhancer element is introduced into cells containing (or which can be made to contain) the factors necessary for the activation of the promoter and / or enhancer elements. Examples of reporter genes include, but are not limited, to genes encoding beta-galactosidase (lacZ), the bacterial chloramphenicol acetyltransferase (cat) genes, firefly luciferase genes, genes encoding beta-glucuronidase (GUS), and genes encoding fluorescent proteins. A “reporter protein” refers to a protein encoded by a reporter gene.

[0076] The term “fluorescent reporter protein” as used herein means a reporter protein that is detectable based on fluorescence wherein the fluorescence may be either from the reporter protein directly, activity of the reporter protein on a fluorogenic substrate, or a protein with affinity for binding to a fluorescent tagged compound. Examples of fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, eGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, and ZsGreenl), yellow fluorescent proteins (e.g., YFP, eYFP, Citrine, Venus, YPet, PhiYFP, and ZsYellowl), blue fluorescent proteins (e.g., BFP, eBFP, eBFP2, Azurite, mKalamal, GFPuv, Sapphire, and T-sapphire), cyan fluorescent proteins (e.g., CFP, eCFP, Cerulean, CyPet, AmCyanl, and Midoriishi-Cyan), red fluorescent proteins (e.g., RFP, mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFPl, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611, mRaspberry, mStrawberry, and Jred), orange fluorescent proteins (e.g., mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, and tdTomato), and any other suitable fluorescent protein whose presence in cells can be detected by flow cytometry methods.Attorney Docket No. 057766 / 640819

[0077] Repair in response to double-strand breaks (DSBs) occurs principally through two conserved DNA repair pathways: homologous recombination (HR) and non-homologous end joining (NHEJ). See Kasparek & Humphrey (2011) Semin. Cell Dev. Biol. 22:886-897, herein incorporated by reference in its entirety for all purposes. Likewise, repair of a target nucleic acid mediated by an exogenous donor nucleic acid can include any process of exchange of genetic information between the two polynucleotides.

[0078] The term “recombination” includes any process of exchange of genetic information between two polynucleotides and can occur by any mechanism. Recombination can occur via homology directed repair (HDR) or homologous recombination (HR). HDR or HR includes a form of nucleic acid repair that can require nucleotide sequence homology, uses a “donor” molecule as a template for repair of a “target” molecule (i.e., the one that experienced the double-strand break), and leads to transfer of genetic information from the donor to target. Without wishing to be bound by any particular theory, such transfer can involve mismatch correction of heteroduplex DNA that forms between the broken target and the donor, and / or synthesis-dependent strand annealing, in which the donor is used to resynthesize genetic information that will become part of the target, and / or related processes. In some cases, the donor polynucleotide, a portion of the donor polynucleotide, a copy of the donor polynucleotide, or a portion of a copy of the donor polynucleotide integrates into the target DNA. See Wang et al. (2013) Cell 153:910-918; Mandalos et al. (2012) FLoS ONE 7:e45768: l-9; and Wang et al. (2013) Nat Biotechnol. 31 :530-532, each of which is herein incorporated by reference in its entirety for all purposes.

[0079] Non-homologous end joining (NHEJ) includes the repair of double-strand breaks in a nucleic acid by direct ligation of the break ends to one another or to an exogenous sequence without the need for a homologous template. Ligation of non-contiguous sequences by NHEJ can often result in deletions, insertions, or translocations near the site of the double-strand break. For example, NHEJ can also result in the targeted integration of an exogenous donor nucleic acid through direct ligation of the break ends with the ends of the exogenous donor nucleic acid (i .e., NHEJ-based capture). Such NHEJ-mediated targeted integration can be preferred for insertion of an exogenous donor nucleic acid when homology directed repair (HDR) pathways are not readily usable (e.g., in non-dividing cells, primary cells, and cells which perform homology-based DNA repair poorly). In addition, in contrast to homology-directed repair, knowledge concerning largeAttorney Docket No. 057766 / 640819 regions of sequence identity flanking the cleavage site is not needed, which can be beneficial when attempting targeted insertion into organisms that have genomes for which there is limited knowledge of the genomic sequence. The integration can proceed via ligation of blunt ends between the exogenous donor nucleic acid and the cleaved genomic sequence, or via ligation of sticky ends (i.e., having 5’ or 3’ overhangs) using an exogenous donor nucleic acid that is flanked by overhangs that are compatible with those generated by a nuclease agent in the cleaved genomic sequence. See, e.g., US 2011 / 020722, WO 2014 / 033644, WO 2014 / 089290, and Maresca et al. (2013) Genome Res. 23(3):539-546, each of which is herein incorporated by reference in its entirety for all purposes. If blunt ends are ligated, target and / or donor resection may be needed to generation regions of microhomology needed for fragment joining, which may create unwanted alterations in the target sequence.

[0080] Compositions or methods “comprising” or “including” one or more 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 transitional phrase “consisting essentially of’ means that the scope of a claim is to be interpreted to encompass the specified elements recited in the claim and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, the term “consisting essentially of’ when used in a claim of this invention is not intended to be interpreted to be equivalent to “comprising.”

[0081] “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur and that the description includes instances in which the event or circumstance occurs and instances in which the event or circumstance does not.

[0082] Designation of a range of values includes all integers within or defining the range, and all subranges defined by integers within the range. For example, 5-10 nucleotides is understood as 5, 6, 7, 8, 9, or 10 nucleotides, whereas 5-10% is understood to contain 5% and all possible values through 10%.

[0083] At least 17 nucleotides of a 20 nucleotide sequence is understood to include 17, 18, 19, or 20 nucleotides of the sequence provided, thereby providing an upper limit even if one is not specifically provided as it would be clearly understood. Similarly, up to 3 nucleotides would be understood to encompass 0, 1, 2, or 3 nucleotides, providing a lower limit even if one is notAttorney Docket No. 057766 / 640819 specifically provided. When “at least,” “up to,” or other similar language modifies a number, it can be understood to modify each number in the series.

[0084] As used herein, “no more than” or “less than” is understood as the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. For example, a duplex region of “no more than 2 nucleotide base pairs” has a 2, 1, or 0 nucleotide base pairs. When “no more than” or “less than” is present before a series of numbers or a range, it is understood that each of the numbers in the series or range is modified.

[0085] As used herein, it is understood that when the maximum amount of a value is represented by 100% (e.g., 100% inhibition) that the value is limited by the method of detection. For example, 100% inhibition is understood as inhibition to a level below the level of detection of the assay.

[0086] Unless otherwise apparent from the context, the term “about” encompasses values ± 5% of a stated value. In certain embodiments, the term “about” is understood to encompass tolerated variation or error within the art, e.g., 2 standard deviations from the mean, or the sensitivity of the method used to take a measurement, or a percent of a value as tolerated in the art, e.g., with age. When “about” is present before the first value of a series, it can be understood to modify each value in the series.

[0087] 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 combinations when interpreted in the alternative (“or”).

[0088] The term “or” refers to any one member of a particular list and also includes any combination of members of that list.

[0089] The singular forms of the articles “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a protein” or “at least one protein” can include a plurality of proteins, including mixtures thereof.

[0090] Statistically significant means p <0.05.

[0091] In the event of a conflict between a sequence in the application and an indicated accession number or position in an accession number, the sequence in the application predominates.Attorney Docket No. 057766 / 640819DETAILED DESCRIPTIONI. Overview

[0092] Disclosed herein are non-human animal genomes, non-human animal cells, and nonhuman animals comprising a humanized STMN2 locus and methods of using such non-human animal cells and non-human animals. Such non-human animal cells and non-human animals can be used to assess delivery or efficacy of agents as regulators of human STMN2 mRNA splicing in vitro in vivo, or ex vivo. Such non-human animal cells and non-human animals can also be used to assess delivery or efficacy of human-stathmin-2-targeting agents in vitro, ex vivo, or in vivo.

[0093] In some of the non-human animal cells and non-human animals disclosed herein, some or most or all of the human S1MN2 genomic DNA is inserted into the corresponding non- human animal Stmn2 locus (corresponding orthologous non-human animal Stmn2 locus). A humanized STMN2 allele resulting from replacing most or all of the non-human animal genomic DNA one-for-one with corresponding human genomic DNA or inserting human STMN2 genomic sequence in the corresponding non-human Stmn2 locus can provide a model of TDP-43 mediated splicing of STMN2 mRNA, for example, where mouse TDP-43 can function like human TDP-43 in repression of cryptic exon splicing in humanized STMN2 mRNA.

[0094] Some non-human animals and non-human animal cells disclosed herein comprise a mutation in the Tardbp gene that causes mislocalization of TDP-43 and results in splicing at a cryptic exon of humanized STMN2 gene in the non-human animals and non-human animal cells. Non-human animals and non-human animal cells expressing a humanized STMN2 gene and a mutation in Tardbp gene causing mislocalization of TDP-43 are useful as a model of human ALS. Such non-human animals and non-human animal cells expressing a humanized STMN2 gene and a mutation in Tardbp gene causing mislocalization of TDP-43 are useful in screening an agent for effectiveness as a splicing effector and / or as a TDP-43 therapeutic and / or as an ALS therapeutic.

[0095] Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that affects motor neurons, causing limb paralysis and eventual death as the result of failure of the diaphragm muscle. A nearly universal pathological finding in postmortem examinations of ALS patient tissue is the accumulation of TDP-43 (transactive response DNA binding protein 43 kDa) in cytoplasmic inclusions. TDP-43 is a predominantly nuclear RNA binding protein similar in structure to members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family that isAttorney Docket No. 057766 / 640819 required for the viability 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.

[0096] While the full scope of TDP-43 ’s biological functions may have yet to be fully elucidated, one clear function of TDP-43 is in the regulation of pre-messenger RNA (pre- mRNA) splicing by preventing the use of cryptic exons in large introns and by influencing alternative splicing of several pre-mRNAs. TDP-43 is also proposed to have functions in the cytoplasm, perhaps in the shuttling of RNAs between the nucleus and cytoplasm and in the transport of mRNAs within the axons of neurons. 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 export signal (NES), and a large domain in the carboxyl-terminal half of the protein that has been described as a low complexity, poorly ordered, or prion-like domain (PLD). Of the mutations in TDP-43 that are associated with familial cases of ALS, most are found in the PLD. A leading hypothesis of the molecular mechanisms underlying motor neuron death in ALS is that the redistribution of TDP-43 from the nucleus to the cytoplasm prevents TDP-43 from carrying out its splicing function leading to aberrant cryptic exon inclusion. Cryptic exon inclusion can lead to several potentially damaging effects on gene expression, either through the generation of truncated mRNAs, the loss of mRNA expression due to the presence of a premature stop codon triggering nonsense-mediated decay (NMD), or through novel in-frame amino acids that may disrupt the encoded protein structure or function. Work in both cellular and animal model systems have demonstrated that TDP-43 directly binds to and regulates hundreds of splicing events across the transcriptome, highlighting the profound effect TDP-43 dysfunction may have on gene expression.

[0097] In support of cryptic exon de-repression being linked mechanistically to disease, recent work analyzing post-mortem CNS tissue from ALS patients identified aberrant inclusion of a cryptic exon in stathmin-2 (STMN2) — a protein highly expressed in motor neurons and critical for proper neuron health. While three TDP-43 binding sites in intron 1 of STMN2 pre- mRNA normally prevent recognition of a cryptic 3’ splice site, loss of TDP-43 leads to recognition of this 3’ splice site and inclusion of a cryptic exon. Due to the presence of both an in-frame stop codon and a poly adenylation signal downstream of the cryptic 3’ splice site, the aberrant inclusion of this cryptic exon creates a highly truncated, stable mRNA (referred toAttorney Docket No. 057766 / 640819 herein as cryptic spliced STMN2 RNA transcript) that is non-functional, leading to loss of STMN2 function. Because STMN2 carries out a critical role in controlling microtubule dynamics and stability, its loss can promote the degeneration of injured axons in vitro.

[0098] Although it remains to be determined the phenotypic contribution of STMN2 loss to motor neuron degeneration in ALS, the TDP-43 -dependent cryptic exon in STMN2 nevertheless offers a unique and valuable molecular readout of disease-associated TDP-43 dysfunction. However, because TDP-43 binding sites are largely within introns, and intronic sequences are poorly conserved between species, the splicing events regulated by TDP-43 are generally not conserved between mouse and human, including STMN2. This presents a major barrier to modeling and characterizing the contribution of STMN2 mis-splicing to motor neuron disease in mouse models of TDP-43 pathology.

[0099] To circumvent these limitations, we developed, in some embodiments, a novel system wherein we fully humanized the endogenous mouse Stmn2 allele to model human TDP-43 - dependent splicing. Analysis of our humanized STMN2 allele demonstrated that the cryptic exon is not recognized by the splicing machinery when wild-type mouse TDP-43 is present, providing a critical finding that mouse TDP-43 can regulate splicing events dependent on the human TDP- 43 protein. Furthermore, when TDP-43 is disrupted by endogenous deletion or mutation of critical domains (e.g., APLD, ANLS, ANES) that lead to mislocalization and loss of splicing activity broadly in mouse embryonic stem cell-derived motor neurons, we see strong inclusion of the cryptic exon in humanized STMN2 transcripts. Cryptic exon inclusion in humanized STMN2 leads to loss of full-length STMN2 mRNA and protein, as predicted by studies in human cells, further validating our system. This collective set of data from our humanized STMN allele demonstrates that (1) mouse TDP-43 can compensate for human TDP-43 splicing function and (2) we have generated a system to monitor for ALS-associated human splicing changes in mouse cellular and animal models systems. Given that this humanized STMN2 system responds robustly to disruption in TDP-43 caused by mislocalization, this system represents a novel and highly relevant platform for testing ALS therapeutics that target, either directly or indirectly, the expression, localization, and function of TDP-43.II. Non-Human Animals Comprising a Humanized STMN2 Locus

[0100] The non-human animal genomes, non-human animal cells, and non-human animalsAttorney Docket No. 057766 / 640819 disclosed herein comprise a humanized STMN2 locus. Cells or non-human animals comprising a humanized STMN2 locus express a human stathmin-2 protein.A. STMN2

[0101] The cells and non-human animals described herein comprise a humanized STMN2 locus. Stathmin-2 (also known as STMN2, superior cervical ganglion-10 protein, and protein SCG10) is encoded by the STMN2 gene (also known as SCG10 or SCGN10). Stathmin-2 is a highly conserved cytosolic protein essential for axonal outgrowth and maintenance. Stathmin-2 appears to act as a regulator of microtubule stability.

[0102] This gene encodes a member of the stathmin family of phosphoproteins. Stathmin proteins function in microtubule dynamics and signal transduction. The encoded protein plays a regulatory role in neuronal growth and is also thought to be involved in osteogenesis. Reductions in the expression of this gene have been associated with Down’s syndrome and Alzheimer’s disease. Alternatively spliced transcript variants have been observed for this gene.

[0103] Human STMN2 maps to 8q21.13 on chromosome 8 (NCBI RefSeq Gene ID 11075; Assembly GRCh38.pl4 (GCF_000001405.40); location NC_000008.11 (79611117..79666158)). The gene has been reported to have 5 exons. The wild type human stathmin-2 protein has been assigned UniProt accession number Q93045. At least 2 isoforms are known. The sequence for the canonical isoform, NCBI Accession No. NP_008960.2 (UniProt Q93045-1), is set forth in SEQ ID NO: 1. An mRNA (cDNA) encoding the canonical isoform is assigned NCBI Accession No. NM_007029.4 and is set forth in SEQ ID NO: 3. An exemplary coding sequence (CDS) is set forth in SEQ ID NO: 2 (CCDS ID CCDS43748.1). Nucleotides 1-78 of SEQ ID NO: 2 encode a membrane attachment domain, nucleotides 112-537 of SEQ ID NO: 2 encode a stathmin-like domain (SLD), nucleotides 115-288 of SEQ ID NO: 2 encode a regulatory / phosphorylation domain, and nucleotides 223-537 of SEQ ID NO: 2 encode a coiled- coil domain. The full-length human stathmin-2 protein set forth in SEQ ID NO: 1 has 179 amino acids, including a membrane attachment region (amino acids 1-26), a stathmin-like domain (SLD) (amino acids 38-179), a regulatory / phosphorylation domain (amino acids 39-96), and a coiled coil domain (amino acids 75-179). Delineations between these domains are as designated in UniProt. Reference to human stathmin-2 includes the canonical (wild type) forms as well as all allelic forms and isoforms. Any other forms of human stathmin-2 have amino acids numberedAttorney Docket No. 057766 / 640819 for maximal alignment with the wild type form, aligned amino acids being designated the same number.

[0104] Alternative splicing may lead to inclusion of STMN2 cryptic exon 2a in a truncated STMN2 CDS (hSTMN2_cryptic (ElE2a), resulting in a coding sequence of 51 nucleotides, including a premature stop codon (SEQ ID NO: 8). Nucleotides 1-19 of SEQ ID NO: 8 encode a membrane attachment domain. A premature stop codon is at nucleotides 49-51 of SEQ ID NO: 8. Truncated STMN2 CDS (SEQ ID NO: 8) encodes a truncated stathmin-2 polypeptide (16 amino acids, SEQ ID NO: 7) (Figure 3).

[0105] Mouse Stmn2 maps to 3 2.15 cM on chromosome 3 (NCBI RefSeq Gene ID 20257; Assembly GRCm39 (GCF_000001635.27); location NC_000069.7 (8574587..8626664)). The gene has been reported to have 5 exons. The wild type mouse stathmin-2 protein has been assigned UniProt accession number P55821. The sequence for the canonical isoform, NP_079561.1 (UniProt P55821-1), is set forth in SEQ ID NO: 4. An exemplary mRNA (cDNA) isoform encoding the canonical isoform is assigned NCBI Accession No. NM_025285.2 and is set forth in SEQ ID NO: 6. An exemplary coding sequence (CDS) (CCDS ID CCDS17231.1) is set forth in SEQ ID NO: 5. Nucleotides 1-78 of SEQ ID NO: 5 encode membrane attachment domain, nucleotides 112-537 of SEQ ID NO: 5 encode a stathmin-like domain (SLD), nucleotides 115-288 of SEQ ID NO: 5 encode regulatory / phosphorylation domain, and nucleotides 223-537 of SEQ ID NO: 5 encode coiled-coil domain. The canonical full-length mouse stathmin-2 protein set forth in SEQ ID NO: 4 has 179 amino acids, including a membrane attachment region (amino acids 1-26), a stathmin-like domain (SLD) (amino acids 38-179), a regulatory / phosphorylation domain (amino acids 39-96), and a coiled coil domain (amino acids 75-179). Delineations between these domains are as designated in UniProt. Reference to mouse stathmin-2 includes the canonical (wild type) forms as well as all allelic forms and isoforms. Any other forms of mouse stathmin-2 have amino acids numbered for maximal alignment with the wild type form, aligned amino acids being designated the same number.

[0106] Rat Stmn2 maps to 2q23 on chromosome 2 (NCBI RefSeq Gene ID 84510; Assembly GRCr8 (GCF_036323735.1); location NC_086020.1 (95112017..95159642, complement)). The gene has been reported to have 4 exons. The wild type rat stathmin-2 protein has been assigned UniProt accession number P21818 and NCBI accession number NP_445892.1. An exemplary mRNA (cDNA) isoform encoding the canonical isoform is assigned NCBI Accession No.Attorney Docket No. 057766 / 640819NM_053440.2.

[0107] The humanized STMN2 CDS has SEQ ID NO: 10 and is identical to human STMN2 CDS (SEQ ID NO: 2). Nucleotides 1-78 of SEQ ID NO: 10 encode membrane attachment domain, nucleotides 115-288 of SEQ ID NO: 10 encode regulatory / phosphorylation domain, and nucleotides 223-537 of SEQ ID NO: 10 encode coiled-coil domain. The humanized stathmin-2 protein is set forth in SEQ ID NO: 9. Mouse stathmin-2 protein (SEQ ID NO: 4), human stathmin-2 protein (SEQ ID NO: 1), and humanized stathmin-2 protein (SEQ ID NO: 9) are 100% identical.B. TDP-43

[0108] The cells and non-human animals described herein may comprise a Tardbp wild type or mutated locus. The human TARDBP locus encodes transactive response DNA-binding protein 43 (TDP-43) (UniProt QI 3148), a highly conserved, ubiquitously expressed RNA binding protein that plays a critical role in essential cell functions such as transcription repression, RNA splicing, and translational regulation. TDP-43 is a predominantly nuclear RNA binding protein, similar in structure and function to the hnRNP family of RBPs. TDP-43 regulates its own mRNA abundance by binding to the 3 -UTR. TDP-43 binds GU rich sequences, particularly in large introns, suppressing the splicing of cryptic exons and is involved in alternative splicing. 97% of ALS cases show a post-mortem pathology of cytoplasmic TDP-43 aggregates. Mutations in TDP-43 associated with ALS are extremely rare (~1% cases). In ALS, TDP-43 mislocalizes from its normal location in the nucleus to the cytoplasm, where it is found in aggregates.Aggregated TDP-43 is ubiquitinated, hyperphosphorylated and truncated. STMN2 undergoes cryptic exon inclusion in ALS patients as a result of TDP-43 pathology.

[0109] Mouse Tardbp maps to 4 78.77 cM on chromosome 4 (NCBI RefSeq Gene ID 230908; Assembly GRCm39 (GCF_000001635.27); location NC_000070.7(148696839..148711672, complement)). The wild type mouse TDP-43 protein has been assigned UniProt accession number Q921F2. Multiple isoforms are known. The sequence for the canonical isoform, NP 663531.1 (UniProt Q921F2-1), is set forth in SEQ ID NO: 50. An exemplary mRNA (cDNA) isoform encoding the canonical isoform 1 is assigned NCBI Accession No. NM_145556.4 and is set forth in SEQ ID NO: 51. An exemplary coding sequence (CDS) (CCDS ID CCDS38971.1) is set forth in SEQ ID NO: 52. The canonical full-lengthAttorney Docket No. 057766 / 640819 mouse TDP-43 protein set forth in SEQ ID NO: 50 has 414 amino acids, including a nuclear localization signal (NLS) (amino acids 82-98), RRM1 (amino acids 106-176), RRM2 (amino acids 191-262), a nuclear export signal (NES) (amino acids 239-250), and glycine-rich prion-like disordered C-terminal domain (PLD) (amino acids 274-414). Reference to mouse TDP-43 includes the canonical (wild type) forms as well as all allelic forms and isoforms. Any other forms of mouse TDP-43 have amino acids numbered for maximal alignment with the wild type form, aligned amino acids being designated the same number. Exemplary mutations in mouse Tcirdbp loci are as described in Figure 4 and WO 2020 / 264339 Al and WO 2023 / 235677 Al, each of which is herein incorporated by reference in its entirety for all purposes.C. Humanized STMN2 Loci

[0110] Disclosed herein are humanized endogenous Stmn2 loci in which a segment of an endogenous Stmn2 locus has been deleted and replaced with a corresponding human STMN2 sequence (e.g., a corresponding human STMN2 genomic sequence), wherein a humanized stathmin-2 protein is expressed from the humanized endogenous STMN2 locus. A humanized STMN2 locus can be a Stmn2 locus in which the entire Stmn2 gene is replaced with the corresponding human STMN2 sequence (e.g., corresponding orthologous human S1MN2 sequence) or a codon-optimized version of the corresponding human STMN2 sequence, or it can be a Stmn2 locus in which only a portion of the Stmn2 gene is replaced with the corresponding human STMN2 sequence (i.e., humanized) or a codon-optimized version of the corresponding human STMN2 sequence, it can be a Stmn2 locus in which a portion of a corresponding human STMN2 locus or a codon-optimized version of the portion of the corresponding human STMN2 locus is inserted, or it can be a Stmn2 locus in which a portion of the Stmn2 gene is deleted and a portion of the corresponding human STMN2 locus or a codon-optimized version of the portion of the corresponding human STMN2 locus is inserted. The portion of the corresponding human STMN2 locus that is inserted can, for example, comprise more of the human STMN2 locus than is deleted from the endogenous Stmn2 locus. A human STMN2 sequence corresponding to a particular segment of endogenous Stmn2 sequence refers to the region of human STMN2 that aligns with the particular segment of endogenous Stmn2 sequence when human STMN2 and the endogenous Stmn2 are optimally aligned (greatest number of perfectly matched residues). The corresponding human sequence can comprise, for example, complementary DNA (cDNA) orAttorney Docket No. 057766 / 640819 genomic DNA. Optionally, a codon-optimized version of the corresponding human STMN2 sequence can be used and is modified to be codon-optimized based on codon usage in the nonhuman animal. Replaced or inserted (i.e., humanized) regions can include coding regions such as an exon, non-coding regions such as an intron, an untranslated region, or a regulatory region (e g., a promoter, an enhancer, or a transcriptional repressor-binding element), or any combination thereof. As one example, exons corresponding to 1, 2, 3, 4, or all 5 exons of the human STMN2 gene can be humanized. For example, exons corresponding to exons 1-5 of the human STMN2 gene can be humanized. Alternatively, a region of STMN2 encoding an epitope recognized by an anti-human-STMN2 antigen-binding protein or a region targeted by human- stathmin-2-targeting reagent (e.g., a small molecule) can be humanized. Likewise, introns corresponding to 1, 2, 3, or all 4 introns of the human STMN2 gene can be humanized or can remain endogenous. For example, introns corresponding to the introns between exons 1 and 5 (i.e., introns 1-4) of the human STMN2 gene can be humanized. In another example, intron 1 can be humanized. In another example, exons 1 and 2 and intron 1 can be humanized.

[0111] Flanking untranslated regions including regulatory sequences can also be humanized or remain endogenous. For example, the 5’ untranslated region (UTR), the 3’UTR, or both the 5’ UTR and the 3’ UTR can be humanized, or the 5’ UTR, the 3’UTR, or both the 5’ UTR and the 3’ UTR can remain endogenous. One or both of the human 5’ and 3’ UTRs can be inserted, and / or one or both of the endogenous 5’ and 3’ UTRs can be deleted. In a specific example, both the 5’ UTR and the 3’ UTR remain endogenous. In another specific example, the human 3’ UTR is inserted. In another specific example, the human 3’ UTR is inserted but the 5’ UTR remains endogenous. Depending on the extent of replacement by corresponding human sequences, regulatory sequences, such as a promoter, can be endogenous or supplied by the replacing corresponding human sequence. For example, the humanized STMN2 locus can include the endogenous non-human animal Stmn2 promoter (i.e., the inserted human STMN2 sequence of the humanized STMN2 coding sequence can be operably linked to the endogenous non-human animal Stmn2 promoter).

[0112] One or more or all of the regions encoding the membrane attachment region, stathmin-like domain (SLD), regulatory / phosphorylation domain, or the coiled coil domain can be humanized, or one or more of such regions can remain endogenous. An exemplary coding sequence for a mouse Stmn2 locus is set forth in SEQ ID NO: 5. An exemplary coding sequenceAttorney Docket No. 057766 / 640819 for a human STMN2 locus is set forth in SEQ ID NO: 2. An exemplary coding sequence for a humanized STMN2 locus is set forth in SEQ ID NO: 10.

[0113] For example, all or part of the region of the Stmn2 locus encoding the membrane attachment region can be humanized, and / or all or part of the region of the Stmn2 locus encoding the stathmin-like domain (SLD) can be humanized, and / or all or part of the region of the Stmn2 locus encoding the regulatory / phosphorylation domain can be humanized, and / or all or part of the region of the Stmn2 locus encoding the coiled coil domain can be humanized. In one example, all of the coding region of the Stmii2 locus is humanized (i.e., from the start codon through the stop codon, wherein the human sequence optionally further includes the human 3’ UTR).

[0114] Domains in a humanized stathmin-2 protein that are from a human stathmin-2 protein can be encoded by a fully humanized sequence (i.e., the entire sequence encoding that domain is replaced with the corresponding human STMN2 sequence) or can be encoded by a partially humanized sequence (i.e., some of the sequence encoding that domain is replaced with the corresponding human STMN2 sequence, and the remaining endogenous (i.e., native) sequence encoding that domain encodes the same amino acids as the corresponding human STMN2 sequence such that the encoded domain is identical to that domain in the human stathmin-2 protein). Likewise, domains in a humanized protein that are from the endogenous stathmin-2 protein cay be encoded by a fully endogenous sequence (i.e., the entire sequence encoding that domain is the endogenous Stmn2 sequence) or can be encoded by a partially humanized sequence (i.e., some of the sequence encoding that domain is replaced with the corresponding human STMN2 sequence, but the corresponding human STMN2 sequence encodes the same amino acids as the replaced endogenous Stmn2 sequence such that the encoded domain is identical to that domain in the endogenous stathmin-2 protein). For example, part of the region of the Stmn2 locus encoding the membrane attachment region can be replaced with corresponding human STMN2 sequence, wherein the amino acid sequence of the region of the membrane attachment region encoded by the corresponding human STMN2 sequence is identical to the corresponding endogenous amino acid sequence. For example, part of the region of the Stmn2 locus encoding the stathmin-like domain (SLD) can be replaced with corresponding human STMN2 sequence, wherein the amino acid sequence of the region of the stathmin-like domain (SLD) encoded by the corresponding human STMN2 sequence is identical to the corresponding endogenous aminoAttorney Docket No. 057766 / 640819 acid sequence. For example, part of the region of the Stmn2 locus encoding the regulatory / phosphorylation domain can be replaced with corresponding human STMN2 sequence, wherein the amino acid sequence of the region of the regulatory / phosphorylation domain encoded by the corresponding human STMN2 sequence is identical to the corresponding endogenous amino acid sequence. For example, part of the region of the Stmn2 locus encoding the coiled coil domain can be replaced with corresponding human STMN2 sequence, wherein the amino acid sequence of the region of the coiled coil domain encoded by the corresponding human STMN2 sequence is identical to the corresponding endogenous amino acid sequence.

[0115] For example, the stathmin-2 protein encoded by the humanized STMN2 locus can comprise a sequence that is at least about 85%, 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 about 100% identical to SEQ ID NO: 9 (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9). As another example, the stathmin-2 protein encoded by the humanized STMN2 locus can consist essentially of a sequence that is at least about 85%, 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 about 100% identical to SEQ ID NO: 9 (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9). As another example, the stathmin-2 protein encoded by the humanized STMN2 locus can consist of a sequence that is at least about 85%, 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 about 100% identical to SEQ ID NO: 9 (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9). Optionally, the STMN2 CDS encoded by the humanized STMN2 locus can comprise a sequence that is at least about 85%, 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 about 100% identical to SEQ ID NO: 10 (or degenerates thereof) (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 10 (or degenerates thereof)). Optionally, the STMN2 CDS encoded by the humanized STMN2 locus can consist essentially of a sequence that is at least about 85%, 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 about 100% identical to SEQ ID NO: 10 (or degenerates thereof) (e.g., at least 85%, at least 90%, at least 95%, at least 96%, atAttorney Docket No. 057766 / 640819 least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 10 (or degenerates thereof)). Optionally, the STMN2 CDS encoded by the humanized STMN2 locus can consist of a sequence that is at least about 85%, 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 about 100% identical to SEQ ID NO: 10 (or degenerates thereof) (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 10 (or degenerates thereof)). In each case, the stathmin-2 protein is expressed and can retain the activity of the native mouse stathmin-2 protein and / or human stathmin-2 protein.

[0116] Optionally, a humanized STMN2 locus can comprise other elements. Examples of such elements can include selection cassettes, reporter genes, recombinase recognition sites, or other elements. Alternatively, the humanized STMN2 locus can lack other elements (e.g., can lack a selection marker or selection cassette). Examples of suitable reporter genes and reporter proteins are disclosed elsewhere herein. Examples of suitable selection markers include neomycin phosphotransferase (neor), hygromycin B phosphotransferase (hygr), puromycin-N- acetyltransferase (puror), blasticidin S deaminase (bsrr), xanthine / guanine phosphoribosyl transferase (gpt), and herpes simplex virus thymidine kinase (HSV-k). Examples of recombinases include Cre, Flp, and Dre recombinases. One example of a Cre recombinase gene is Crei, in which two exons encoding the Cre recombinase are separated by an intron to prevent its expression in a prokaryotic cell. Such recombinases can further comprise a nuclear localization signal to facilitate localization to the nucleus (e.g., NLS-Crei). Recombinase recognition sites include nucleotide sequences that are recognized by a site-specific recombinase and can serve as a substrate for a recombination event. Examples of recombinase recognition sites include FRT, FRT11, FRT71, attp, att, rox, and lox sites such as loxP, lox511, lox2272, lox66, lox71, loxM2, and lox5171.

[0117] Other elements such as reporter genes or selection cassettes can be self-del eting cassettes flanked by recombinase recognition sites. See, e.g., US 8,697,851 and US 2013 / 0312129, each of which is herein incorporated by reference in its entirety for all purposes. As an example, the self-deleting cassette can comprise a Crei gene (comprises two exons encoding a Cre recombinase, which are separated by an intron) operably linked to a mouse Prml promoter and a neomycin resistance gene operably linked to a human ubiquitin promoter. By employing the Prml promoter, the self-deleting cassette can be deleted specifically in male germAttorney Docket No. 057766 / 640819 cells of FO animals. The polynucleotide encoding the selection marker can be operably linked to a promoter active in a cell being targeted. Examples of promoters are described elsewhere herein. As another specific example, a self-deleting selection cassette can comprise a hygromycin resistance gene coding sequence operably linked to one or more promoters (e.g., both human ubiquitin and EM7 promoters) followed by a polyadenylation signal, followed by a Crei coding sequence operably linked to one or more promoters (e.g., an mPrml promoter), followed by another polyadenylation signal, wherein the entire cassette is flanked by loxP sites.

[0118] The humanized STMN2 locus can also be a conditional allele. For example, the conditional allele can be a multifunctional allele, as described in US 2011 / 0104799, herein incorporated by reference in its entirety for all purposes. For example, the conditional allele can comprise: (a) an actuating sequence in sense orientation with respect to transcription of a target gene; (b) a drug selection cassette (DSC) in sense or antisense orientation; (c) a nucleotide sequence of interest (NSI) in antisense orientation; and (d) a conditional by inversion module (COIN, which utilizes an exon-splitting intron and an invertible gene-trap-like module) in reverse orientation. See, e.g., US 2011 / 0104799. The conditional allele can further comprise recombinable units that recombine upon exposure to a first recombinase to form a conditional allele that (i) lacks the actuating sequence and the DSC; and (ii) contains the NSI in sense orientation and the COIN in antisense orientation. See, e.g., US 2011 / 0104799.

[0119] One exemplary humanized STMN2 locus (e.g., a humanized mouse STMN2 locus) is one in which a region from the start codon through the stop codon (i.e., including all introns in between) is deleted from the mouse Stmn2 locus, and replaced with the human STMN2 genomic sequence from the start codon through the stop codon (i.e., with all introns in between), optionally including the 3’ UTR.

[0120] One exemplary humanized STMN2 locus (e.g., a humanized mouse STMN2 locus) is one in which a region starting in exon 1 at the start ATG through the first 51 bp of exon 5, including all introns in between, is deleted from the mouse Stmn2 locus, and replaced with the human genomic sequence, starting in exon 1 with the start ATG, through exon 5 coding sequence and 3’ UTR, with all introns in between. The replaced region encodes the amino acids 1-179 human stathmin-2. See Figure 1 and SEQ ID NOS: 48 and 49. Exemplary sequences for a humanized STMN2 locus are set forth in SED ID NOS: 48 and 49.

[0121] In one specific example, the human STMN2 sequence at the humanized endogenousAttorney Docket No. 057766 / 640819STMN2 locus can comprise a sequence at least about 85%, 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 about 100% identical to the sequence set forth in SEQ ID NO: 53 or 54 (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 53 or 54). In another specific example, the humanized STMN2 locus can encode a protein comprising a sequence at least about 85%, 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 about 100% identical to the sequence set forth in SEQ ID NO: 9 (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9). In another specific example, the humanized STMN2 locus can comprise a coding sequence comprising a sequence at least about 85%, 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 about 100% identical to the sequence set forth in SEQ ID NO: 10 (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 10). In another specific example, the humanized STMN2 locus can comprise a sequence at least about 85%, 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 about 100% identical to the sequence set forth in SEQ ID NO: 48 or 49 (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical to SEQ ID NO: 48 or 49).

[0122] In one specific example, the human STMN2 sequence at the humanized endogenous STMN2 locus can consist essentially of a sequence at least about 85%, 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 about 100% identical to the sequence set forth in SEQ ID NO: 53 or 54 (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 53 or 54). In another specific example, the humanized STMN2 locus can encode a protein consisting essentially of a sequence at least about 85%, 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 about 100% identical to the sequence set forth in SEQ ID NO: 9 (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9). In another specific example, the humanized STMN2 locus can comprise a coding sequence consisting essentially of a sequence at least about 85%, at least about 90%, at leastAttorney Docket No. 057766 / 640819 about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence set forth in SEQ ID NO: 10 (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 10). In another specific example, the humanized STMN2 locus can consist essentially of a sequence at least about 85%, 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 about 100% identical to the sequence set forth in SEQ ID NO: 48 or 49 (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical to SEQ ID NO: 48 or 49).

[0123] In one specific example, the human STMN2 sequence at the humanized endogenous STMN2 locus can consist of a sequence at least about 85%, 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 about 100% identical to the sequence set forth in SEQ ID NO: 53 or 54 (e g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 53 or 54). In another specific example, the humanized STMN2 locus can encode a protein consisting of a sequence at least about 85%, 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 about 100% identical to the sequence set forth in SEQ ID NO: 9 (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9). In another specific example, the humanized STMN2 locus can comprise a coding sequence consisting of a sequence at least about 85%, 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 about 100% identical to the sequence set forth in SEQ ID NO: 10 (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 10). In another specific example, the humanized STMN2 locus can consist of a sequence at least about 85%, 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 about 100% identical to the sequence set forth in SEQ ID NO: 48 or 49 (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical to SEQ ID NO: 48 or 49).

[0124] The humanized STMN2 loci can be heterozygous, homozygous, or compound heterozygous (e.g., hemizygous). A diploid organism has two alleles at each genetic locus. Each pair of alleles represents the genotype of a specific genetic locus. Genotypes are described asAttorney Docket No. 057766 / 640819 homozygous if there are two identical alleles at a particular locus and as heterozygous if the two alleles differ. Genotypes are described as compound heterozygous (e.g., hemizygous) if there is a combination of a humanized allele and an inactivated allele (e.g., a collapsed allele in which a region from near the start codon to near the stop codon is deleted). Some compound heterozygous (hemizygous) loci comprise a humanized STMN2 allele and an inactivated allele (e.g., a collapsed allele).D Non-Human Animal Genomes, Non-Human Animal Cells, and Non-Human Animals Comprising a Humanized STMN2 locus

[0125] Non-human animal genomes, non-human animal cells, and non-human animals comprising a humanized STMN2 locus as described elsewhere herein are provided. The genomes, cells, or non-human animals can express a humanized stathmin-2 protein encoded by the humanized STMN2 locus. The genomes, cells, or non-human animals can be male or female. The genomes, cells, or non-human animals can be heterozygous, homozygous, or compound heterozygous (e.g., hemizygous) for the humanized STMN2 locus. A diploid organism has two alleles at each genetic locus. Each pair of alleles represents the genotype of a specific genetic locus. Genotypes are described as homozygous if there are two identical alleles at a particular locus and as heterozygous if the two alleles differ. Genotypes are described as compound heterozygous (e.g., hemizygous) if there is a combination of a humanized allele and an inactivated allele (e.g., a collapsed allele in which a region from near the start codon to near the stop codon is deleted). Some compound heterozygous (hemizygous) non-human animals or non- human animal cells, comprise a humanized STMN2 allele and an inactivated allele (e.g., a collapsed allele). A non-human animal comprising a humanized STMN2 locus can comprise the humanized STMN2 locus in its germline.

[0126] The non-human animals or non-human animal genomes or cells provided herein can be, for example, any non-human animal genome or cell comprising a Stmn2 locus or a genomic locus homologous or orthologous to the human STMN2 locus. The genomes can be from or the cells can be eukaryotic cells, which include, for example, animal cells, mammalian cells, non- human mammalian cells, and human cells. The term “animal” includes any member of the animal kingdom, including, for example, mammals, fishes, reptiles, amphibians, birds, and worms. A mammalian cell can be, for example, a non-human mammalian cell, a rodent cell, a ratAttorney Docket No. 057766 / 640819 cell, or a mouse cell. Other non-human mammals include, for example, non-human primates. The term “non-human” excludes humans. In a specific example, the non-human animal is a rodent. In another specific example, the non-human animal is a mouse or a rat. In another specific example, the non-human animal is a mouse.

[0127] The cells can also be any type of undifferentiated or differentiated state. For example, a cell can be a totipotent cell, a pluripotent cell (e.g., a human pluripotent cell or a non-human pluripotent cell such as a mouse embryonic stem (ES) cell or a rat ES cell), or a non-pluripotent cell (e.g., a non-ES cell). Totipotent cells include undifferentiated cells that can give rise to any cell type, and pluripotent cells include undifferentiated cells that possess the ability to develop into more than one differentiated cell types. Such pluripotent and / or totipotent cells can be, for example, ES cells or ES-like cells, such as an induced pluripotent stem (iPS) cell. ES cells include embryo-derived totipotent or pluripotent cells that are capable of contributing to any tissue of the developing embryo upon introduction into an embryo. ES cells can be derived from the inner cell mass of a blastocyst and are capable of differentiating into cells of any of the three vertebrate germ layers (endoderm, ectoderm, and mesoderm).

[0128] The cells provided herein can also be germ cells (e.g., sperm or oocytes). The cells can be mitotically competent cells or mitotically-inactive cells, meiotically competent cells or meiotically-inactive cells. Similarly, the cells can also be primary somatic cells or cells that are not a primary somatic cell. Somatic cells include any cell that is not a gamete, germ cell, gametocyte, or undifferentiated stem cell. For example, the cells can be neuronal cells. In a specific example, the cells can be ES-cell-derived motor neurons (e.g., mouse ES-cell-derived motor neurons). In another specific example, the cells can be ES cells (e.g., mouse ES cells).

[0129] Suitable cells provided herein also include primary cells. Primary cells include cells or cultures of cells that have been isolated directly from an organism, organ, or tissue. Primary cells include cells that are neither transformed nor immortal. They include any cell obtained from an organism, organ, or tissue which was not previously passed in tissue culture or has been previously passed in tissue culture but is incapable of being indefinitely passed in tissue culture. For example, the primary cells can be neuronal cells (e.g., mouse neuronal cells).

[0130] Other suitable cells provided herein include immortalized cells. Immortalized cells include cells from a multicellular organism that would normally not proliferate indefinitely but, due to mutation or alteration, have evaded normal cellular senescence and instead can keepAttorney Docket No. 057766 / 640819 undergoing division. Such mutations or alterations can occur naturally or be intentionally induced. Numerous types of immortalized cells are well known. Immortalized or primary cells include cells that are typically used for culturing or for expressing recombinant genes or proteins.

[0131] The cells provided herein also include one-cell stage embryos (i.e., fertilized oocytes or zygotes). Such one-cell stage embryos can be from any genetic background (e.g., BALB / c, C57BL / 6, 129, or a combination thereof for mice), can be fresh or frozen, and can be derived from natural breeding or in vitro fertilization.

[0132] The cells provided herein can be normal, healthy cells, or can be diseased or mutantbearing cells. For example, the cells can comprise a mutation associated with amyotrophic lateral sclerosis (ALS). In another example, the cells can comprise a mutation in a Tardbp locus (i.e., encoding a mutant TDP-43 protein such as those disclosed elsewhere herein).

[0133] Non-human animals comprising a humanized STMN2 locus as described herein can be made by the methods described elsewhere herein. The term “animal” includes any member of the animal kingdom, including, for example, mammals, fishes, reptiles, amphibians, birds, and worms. In a specific example, the non-human animal is a non-human mammal. Non-human mammals include, for example, non-human primates and rodents (e g., mice and rats). The term “non-human animal” excludes humans. Preferred non-human animals include, for example, rodents, such as mice and rats (e.g., mice).

[0134] The non-human animals can comprise mutations in other genes. For example, the non-human animals can comprise a mutation associated with amyotrophic lateral sclerosis (ALS). In a specific example, the non-human animal can comprise a mutation in a Tardbp locus (i.e., encoding a mutant TDP-43 protein such as those disclosed elsewhere herein).

[0135] The non-human animals can be from any genetic background. For example, suitable mice can be from a 129 strain, a C57BL / 6 strain, a mix of 129 and C57BL / 6, a BALB / c strain, or a Swiss Webster strain. Examples of 129 strains include 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129Sl / Svlm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, and 129T2. See, e.g., Festing et al. (1999) Mamm. Genome 10(8):836, herein incorporated by reference in its entirety for all purposes. Examples of C57BL strains include C57BL / A, C57BL / An, C57BL / GrFa, C57BL / Kal_wN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr, and C57BL / 01a. Suitable mice can also be from a mix of an aforementioned 129 strain and an aforementioned C57BL / 6Attorney Docket No. 057766 / 640819 strain (e.g., 50% 129 and 50% C57BL / 6). Likewise, suitable mice can be from a mix of aforementioned 129 strains or a mix of aforementioned BL / 6 strains (e.g., the 129S6 (129 / SvEvTac) strain).

[0136] Similarly, rats can be from any rat strain, including, for example, an ACI rat strain, a Dark Agouti (DA) rat strain, a Wistar rat strain, a LEA rat strain, a Sprague Dawley (SD) rat strain, or a Fischer rat strain such as Fisher F344 or Fisher F6. Rats can also be obtained from a strain derived from a mix of two or more strains recited above. For example, a suitable rat can be from a DA strain or an ACI strain. The ACI rat strain is characterized as having black agouti, with white belly and feet and an RTlavIhaplotype. Such strains are available from a variety of sources including Harlan Laboratories. The Dark Agouti (DA) rat strain is characterized as having an agouti coat and an RTlavIhaplotype. Such rats are available from a variety of sources including Charles River and Harlan Laboratories. Some suitable rats can be from an inbred rat strain. See, e.g., US 2014 / 0235933, herein incorporated by reference in its entirety for all purposes.

[0137] The non-human animals, non-human animal cells, and non-human animal genomes described herein model human TDP -43 -dependent splicing. In such non-human animals and non- human animal cells, the cryptic exon is not recognized by the splicing machinery when wild-type mouse TDP -43 is present. When TDP -43 is disrupted by endogenous deletion or mutation of critical domains (e.g., APLD, ANLS, ANES) that lead to mislocalization and loss of splicing activity broadly in mouse embryonic stem cell-derived motor neurons, the cryptic exon is included in humanized STMN2 transcripts. Cryptic exon inclusion in humanized STMN2 leads to reduction or loss of full-length STMN2 mRNA and protein. In some embodiments, RNA transcripts from the humanized STMN2 locus include a cryptic exon when TDP-43 is disrupted by mutations that lead to mislocalization and loss of TDP-43 splicing activity. In some embodiments, RNA transcripts from the humanized STMN2 locus do not include the cryptic exon when wild type mouse TDP-43 is present. In some embodiments, RNA transcripts from the humanized STMN2 locus include a cryptic exon when TDP-43 is disrupted by mutations that lead to mislocalization and loss of TDP-43 splicing activity, and RNA transcripts from the humanized STMN2 locus do not include the cryptic exon when wild type mouse TDP-43 is present.

[0138] Non-human animals comprising a humanized STMN2 locus in a wild-type TardbpAttorney Docket No. 057766 / 640819 background show expression of full-length human STMN2 mRNA by RT-qPCR analysis of transcripts (panel E of Figure 6). Non-human animals comprising a humanized STMN2 locus in a mutant Tardbp background (e.g., ANLS, ANES, and APLD) show expression of humanized STMN2 transcripts including cryptic exon 2A (panel D of Figure 6) by RT-qPCR analysis of transcripts and reduced levels of full-length STMN2 mRNA (by RT-qPCR analysis of transcripts) and full-length protein (by automated capillary-based western blotting analysis) (panels E, F, and G of Figure 6).III. Methods of Making Non-Human Animals and Cells Comprising a Humanized STMN2 Locus

[0139] Various methods are provided for making a non-human animal genome, non-human animal cell, or non-human animal comprising a humanized STMN2 locus as disclosed elsewhere herein. Likewise, various methods are provided for making a humanized STMN2 gene or locus or for making a non-human animal genome or non-human animal cell comprising a humanized STMN2 locus as disclosed elsewhere herein. Any convenient method or protocol for producing a genetically modified organism is suitable for producing such a genetically modified non-human animal. See, e.g., Poueymirou et al. (2007) Nat. Biotechnol. 25(l):91-99; US 7,294,754; US 7,576,259; US 7,659,442; US 8,816,150; US 9,414,575; US 9,730,434; and US 10,039,269, each of which is herein incorporated by reference in its entirety for all purposes (describing mouse ES cells and the VELOCIMOUSE® method for making a genetically modified mouse). See also US 2014 / 0235933 Al, US 2014 / 0310828 Al, each of which is herein incorporated by reference in its entirety for all purposes (describing rat ES cells and methods for making a genetically modified rat). See also Cho et al. (2009) Curr. Protoc. Cell. Biol. 42: 19.11.1-19.11.22 (doi: 10.1002 / 0471143030. cbl911s42) and Gama Sosa et al. (2010) Brain Struct. Funct. 214(2-3):91- 109, each of which is herein incorporated by reference in its entirety for all purposes. Such genetically modified non-human animals can be generated, for example, through gene knock-in at a targeted Stmn2 locus.

[0140] For example, the method of producing a non-human animal comprising a humanized STMN2 locus can comprise: (1) providing a pluripotent cell (e.g., an embryonic stem (ES) cell such as a mouse ES cell or a rat ES cell) comprising the humanized STMN2 locus; (2) introducing the genetically modified pluripotent cell into a non-human animal host embryo; andAttorney Docket No. 057766 / 640819(3) gestating the host embryo in a surrogate mother.

[0141] As another example, the method of producing a non-human animal comprising a humanized STMN2 locus can comprise: (1) modifying the genome of a pluripotent cell (e.g., an embryonic stem (ES) cell such as a mouse ES cell) to comprise the humanized STMN2 locus; (2) identifying or selecting the genetically modified pluripotent cell comprising the humanized STMN2 locus; (3) introducing the genetically modified pluripotent cell into a non-human animal host embryo; and (4) gestating the host embryo in a surrogate mother. The donor cell can be introduced into a host embryo at any stage, such as the blastocyst stage or the pre-morula stage (i.e., the 4 cell stage or the 8 cell stage). Optionally, the host embryo comprising modified pluripotent cell (e.g., a non-human ES cell) can be incubated until the blastocyst stage before being implanted into and gestated in the surrogate mother to produce an F0 non-human animal. The surrogate mother can then produce an F0 generation non-human animal comprising the humanized STMN2 locus (and capable of transmitting the genetic modification through the germline).

[0142] Alternatively, the method of producing the non-human animals described elsewhere herein can comprise: (1) modifying the genome of a one-cell stage embryo to comprise the humanized STMN2 locus; (2) selecting the genetically modified embryo; and (3) gestating the genetically modified embryo in a surrogate mother. Progeny that are capable of transmitting the genetic modification though the germline are generated.

[0143] Nuclear transfer techniques can also be used to generate the non-human mammalian animals. Briefly, methods for nuclear transfer can include the steps of: (1) enucleating an oocyte or providing an enucleated oocyte; (2) isolating or providing a donor cell or nucleus to be combined with the enucleated oocyte; (3) inserting the cell or nucleus into the enucleated oocyte to form a reconstituted cell; (4) implanting the reconstituted cell into the womb of an animal to form an embryo; and (5) allowing the embryo to develop. In such methods, oocytes are generally retrieved from deceased animals, although they may be isolated also from either oviducts and / or ovaries of live animals. Oocytes can be matured in a variety of well-known media prior to enucleation. Enucleation of the oocyte can be performed in a number of well-known manners. Insertion of the donor cell or nucleus into the enucleated oocyte to form a reconstituted cell can be by microinjection of a donor cell under the zona pellucida prior to fusion. Fusion may be induced by application of a DC electrical pulse across the contact / fusion plane (electrofusion), byAttorney Docket No. 057766 / 640819 exposure of the cells to fusion-promoting chemicals, such as polyethylene glycol, or by way of an inactivated virus, such as the Sendai virus. A reconstituted cell can be activated by electrical and / or non-electrical means before, during, and / or after fusion of the nuclear donor and recipient oocyte. Activation methods include electric pulses, chemically induced shock, penetration by sperm, increasing levels of divalent cations in the oocyte, and reducing phosphorylation of cellular proteins (as by way of kinase inhibitors) in the oocyte. The activated reconstituted cells, or embryos, can be cultured in well-known media and then transferred to the womb of an animal. See, e.g., US 2008 / 0092249, WO 1999 / 005266, US 2004 / 0177390, WO 2008 / 017234, and US 7,612,250, each of which is herein incorporated by reference in its entirety for all purposes.

[0144] The modified cell or one-cell stage embryo can be generated, for example, through recombination by (a) introducing into the cell one or more exogenous donor nucleic acids (e.g., targeting vectors) comprising an insert nucleic acid flanked, for example, by 5’ and 3’ homology arms corresponding to 5’ and 3’ target sites (e.g., target sites flanking the endogenous sequences intended for deletion and replacement with the insert nucleic acid), wherein the insert nucleic acid comprises a human STMN2 sequence to generate a humanized STMN2 locus; and (b) identifying at least one cell comprising in its genome the insert nucleic acid integrated at the endogenous Stmn2 locus (i. e. , identifying at least one cell comprising the humanized STMN2 locus). Likewise, a modified non-human animal genome or humanized non-human animal STMN2 gene can be generated, for example, through recombination by (a) contacting the genome or gene with one or more exogenous donor nucleic acids (e.g., targeting vectors) comprising 5’ and 3’ homology arms corresponding to 5’ and 3’ target sites (e.g., target sites flanking the endogenous sequences intended for deletion and replacement with an insert nucleic acid (e.g., comprising a human STMN2 sequence to generate a humanized STMN2 locus) flanked by the 5’ and 3’ homology arms), wherein the exogenous donor nucleic acids are designed for humanization of the endogenous non-human animal Stmn2 locus.

[0145] Alternatively, the modified pluripotent cell or one-cell stage embryo can be generated by (a) introducing into the cell: (i) one or more nuclease agents, wherein each nuclease agent induces a nick or double-strand break at a target site within the endogenous Stmn2 locus; and (ii) one or more exogenous donor nucleic acids (e.g., targeting vectors) comprising an insert nucleic acid flanked by, for example, 5’ and 3’ homology arms corresponding to 5’ and 3’ target sites (e.g., target sites flanking the endogenous sequences intended for deletion and replacement withAttorney Docket No. 057766 / 640819 the insert nucleic acid), wherein the insert nucleic acid comprises a human STMN2 sequence to generate a humanized STMN2 locus; and (c) identifying at least one cell comprising in its genome the insert nucleic acid integrated at the endogenous Stmn2 locus (i.e., identifying at least one cell comprising the humanized STMN2 locus). Likewise, a modified non-human animal genome or humanized non-human animal STMN2 gene can be generated by contacting the genome or gene with: (i) one or more nuclease agents, wherein each nuclease agent induces a nick or double-strand break at a target site within the endogenous Stmn2 locus or gene; and (ii) one or more exogenous donor nucleic acids (e.g., targeting vectors) comprising an insert nucleic acid (e.g., comprising a human STMN2 sequence to generate a humanized STMN2 locus) flanked by, for example, 5’ and 3’ homology arms corresponding to 5’ and 3’ target sites (e.g., target sites flanking the endogenous sequences intended for deletion and replacement with the insert nucleic acid), wherein the exogenous donor nucleic acids are designed for humanization of the endogenous Stmn2 locus. When multiple nuclease agents (e.g., multiple gRNAs) are used, they can target two different locations, such as near the 5’ and 3’ homology arm target sites, or such as near the start codon and near the stop codon of the non-human animal Stmn2 gene. In some embodiments, the non-human animal or cell or genome may be compound heterozygous or hemizygous and comprise one Stmn2 allele that is humanized and one allele that is inactivated or collapsed (e.g., has a deletion between the nuclease agent target sites). Any nuclease agent that induces a nick or double-strand break into a desired recognition site can be used. Examples of suitable nucleases include a Transcription Activator-Like Effector Nuclease (TALEN), a zinc- finger nuclease (ZFN), a meganuclease, and Clustered Regularly Interspersed 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., US 2013 / 0309670 and US 2015 / 0159175, each of which is herein incorporated by reference in its entirety for all purposes. In one example, the nuclease agent comprises a Cas9 protein and a guide RNA. In another example, the nuclease agents comprise a Cas9 protein and two, three, or four guide RNAs.

[0146] The step of modifying the genome can, for example, utilize exogenous repair templates (e.g., targeting vectors) to modify a Stnm2 locus to comprise a humanized STMN2 locus disclosed herein. As one example, the targeting vector can be for generating a humanized STMN2 gene at an endogenous Stmn2 locus (e.g., endogenous non-human animal Stmn2 locus), wherein the targeting vector comprises a nucleic acid insert comprising human STMN2 sequenceAttorney Docket No. 057766 / 640819 to be integrated in the Stmn2 locus flanked by a 5’ homology arm targeting a 5’ target sequence at the endogenous Stmn2 locus and a 3’ homology arm targeting a 3’ target sequence at the endogenous Stmn2 locus. Integration of a nucleic acid insert in the Stmn2 locus can result in addition of a nucleic acid sequence of interest in the Stmn2 locus, deletion of a nucleic acid sequence of interest in the Stmn2 locus, or replacement of a nucleic acid sequence of interest in the Stmn2 locus (i.e., deleting a segment of the endogenous Stmn2 locus and replacing with a corresponding human STMN2 sequence).

[0147] The exogenous repair templates can be for non-homologous-end-joining-mediated insertion or homologous recombination. Exogenous repair templates can comprise deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), they can be single-stranded or doublestranded, and they can be in linear or circular form. For example, a repair template can be a single-stranded oligodeoxynucleotide (ssODN). Exogenous repair templates can also comprise a heterologous sequence that is not present at an untargeted endogenous Stmn2 locus. For example, an exogenous repair template can comprise a selection cassette, such as a selection cassette flanked by recombinase recognition sites.

[0148] In cells other than one-cell stage embryos, the exogenous repair template can be a “large targeting vector” or “LTVEC,” which includes targeting vectors that comprise homology arms that correspond to and are derived from nucleic acid sequences larger than those typically used by other approaches intended to perform homologous recombination in cells. See, e.g., US 2004 / 0018626; WO 2013 / 163394; US 9,834,786; US 10,301,646; WO 2015 / 088643; US 9,228,208; US 9,546,384; US 10,208,317; and US 2019-0112619, each of which is herein incorporated by reference in its entirety for all purposes. LTVECs also include targeting vectors comprising nucleic acid inserts having nucleic acid sequences larger than those typically used by other approaches intended to perform homologous recombination in cells. For example, LTVECs make possible the modification of large loci that cannot be accommodated by traditional plasmid-based targeting vectors because of their size limitations. For example, the targeted locus can be (i.e., the 5’ and 3’ homology arms can correspond to) a locus of the cell that is not targetable using a conventional method or that can be targeted only incorrectly or only with significantly low efficiency in the absence of a nick or double-strand break induced by a nuclease agent (e.g., a Cas protein). LTVECs can be of any length and are typically at least 10 kb in length. The sum total of the 5’ homology arm and the 3’ homology arm in an LTVEC isAttorney Docket No. 057766 / 640819 typically at least 10 kb. Generation and use of large targeting vectors (LTVECs) derived from bacterial artificial chromosome (BAC) DNA through bacterial homologous recombination (BHR) reactions using VELOCIGENE® genetic engineering technology is described, e.g., in US 6,586,251 and Valenzuela et al. (2003) Nat. Biotechnol. 21 (6):652-659, each of which is herein incorporated by reference in its entirety for all purposes. Generation of LTVECs through in vitro assembly methods is described, e.g., in US 2015 / 0376628 and WO 2015 / 200334, each of which is herein incorporated by reference in its entirety for all purposes.

[0149] The methods can further comprise identifying a cell or animal having a modified target genomic locus. Various methods can be used to identify cells and animals having a targeted genetic modification. The screening step can comprise, for example, a quantitative assay for assessing modification-of-allele (MO A) of a parental chromosome. See, e.g., US 2004 / 0018626; US 2014 / 0178879; US 2016 / 0145646; WO 2016 / 081923; and Frendewey et al. (2010) Methods Enzymol. 476:295-307, each of which is herein incorporated by reference in its entirety for all purposes. For example, the quantitative assay can be carried out via a quantitative PCR, such as a real-time PCR (qPCR). The real-time PCR can utilize a first primer set that recognizes the target locus and a second primer set that recognizes a non-targeted reference locus. The primer set can comprise a fluorescent probe that recognizes the amplified sequence. Other examples of suitable quantitative assays include fluorescence-mediated in situ hybridization (FISH), comparative genomic hybridization, isothermic DNA amplification, quantitative hybridization to an immobilized probe(s), INVADER® Probes, TAQMAN® Molecular Beacon probes, or ECLIPSE™ probe technology (see, e.g., US 2005 / 0144655, incorporated herein by reference in its entirety for all purposes).

[0150] The various methods provided herein allow for the generation of a genetically modified non-human F0 animal wherein the cells of the genetically modified F0 animal comprise the humanized STMN2 locus. It is recognized that depending on the method used to generate the F0 animal, the number of cells within the F0 animal that have the humanized STMN2 locus will vary. With mice, for example, the introduction of the donor ES cells into a pre-morula stage embryo from the mouse (e.g., an 8-cell stage mouse embryo) via, for example, the VELOCIMOUSE® method allows for a greater percentage of the cell population of the F0 mouse to comprise cells having the targeted genetic modification. For example, at least 50%, 60%, 65%, 70%, 75%, 85%, 86%, 87%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,Attorney Docket No. 057766 / 64081997%, 98%, 99% or 100% of the cellular contribution of the non-human F0 animal can comprise a cell population having the targeted modification. The cells of the genetically modified F0 animal can be heterozygous for the humanized STMN2 locus or can be homozygous for the humanized STMN2 locus.IV. Methods of Using Non-Human Animals and Cells Comprising a Humanized STMN2 Locus for Assessing Effectiveness of Agents as Regulators of Human STMN2 Splicing In Vivo or Ex Vivo

[0151] Various methods are provided for using the non-human animals and cells comprising a humanized STMN2 locus and optionally a mutant Tardbp locus as described elsewhere herein for assessing effectiveness of agents as regulators (directly or indirectly) of human STMN2 mRNA splicing in vivo or ex vivo. Because the non-human animals and cells comprise a humanized STMN2 locus, the non-human animals and cells will more accurately reflect the efficacy of agents as regulators of human STMN2 mRNA splicing. Although it remains to be determined the phenotypic contribution of STMN2 loss to motor neuron degeneration in ALS, the TDP-43 -dependent cryptic exon in STMN2 nevertheless offers a unique and valuable molecular readout of disease-associated TDP-43 dysfunction. However, because TDP-43 binding sites are largely within introns, and intronic sequences are poorly conserved between species, the splicing events regulated by TDP-43 are generally not conserved between mouse and human, including STMN2. The non-human animals and cells disclosed herein circumvent these limitations to accurately model human TDP-43 -dependent splicing: the cryptic exon is not recognized by the splicing machinery when wild-type mouse TDP-43 is present, but when TDP-43 is disrupted by endogenous deletion or mutation of critical domains (e.g., APLD, ANLS, ANES) that lead to mislocalization and loss of splicing activity broadly in mouse embryonic stem cell-derived motor neurons, there is strong inclusion of the cryptic exon in humanized STMN2 transcripts. Given that this humanized STMN2 system responds robustly to disruption in TDP-43 caused by mislocalization, this system represents a novel and highly relevant platform for testing ALS therapeutics that target, either directly or indirectly, the expression, localization, and function of TDP-43.Attorney Docket No. 057766 / 640819A. Methods of Assessing Effectiveness of Agents as Regulators of Human STMN2 mRNA Splicing In Vivo or Ex Vivo

[0152] Various methods are provided for assessing effectiveness of agents as regulators of human S1MN2 mRNA splicing in vivo using non-human animals or cells comprising a humanized STMN2 locus (and optionally a mutant Tardbp locus) as described elsewhere herein. Such methods can comprise: (a) introducing into the non-human animal or cell a putative regulator of STMN2 mRNA splicing; and (b) assessing the splicing of STM 2 transcripts in the non-human animal or cell. In some methods, the non-human animal or cell further comprises a mutant Tardbp locus, such as a deletion or mutation (e.g., of a critical domain, such as APLD, ANLS, or ANES) that leads to mislocalization and loss of splicing activity. The method can then assess the effectiveness of an agent to rescue STMN2 mRNA splicing.

[0153] The agent can be a therapeutic variant of TDP-43, a nucleic acid encoding a therapeutic variant of TDP-43, an RNAi agent, ASO, or shRNA targeting TDP-43, or any other large molecule or small molecule that directly or indirectly targets STMN2 transcripts or directly or indirectly regulates or affects STMN2 mRNA splicing, TDP-43 aggregation, or TDP-43 localization. Some agents can be an aggregation-resistant variants of TDP-43. Some agents can be an shRNA that silences endogenous TDP-43. Alternatively, the agent can be any biological or chemical agent that targets the human STMN2 locus (the human STMN2 gene), targets human STMN2 mRNA (e.g., human STMN2 pre-mRNA), regulates or affects TDP-43 aggregation, or regulates or affects TDP-43 localization. The agent can be, for example, a known amyotrophic lateral sclerosis (ALS) therapeutic agent or candidate ALS therapeutic agent. Examples of ALS therapeutic agents include rapamycin, monepantel, tamoxifen, bosutinib, trehalose (SLS-005), ATXN2 ASO (BIIB105), tideglusib, ibudilast (MN116), colchicine, Withania somnifera, 3K3A- APC, SOD1 ASO (BIIB067), C9orf72 ASO BIIB078 (tofersen, Qalsody), C9orj72 ASO WVE- 004, C9orf72 ASO ASO5-2 (afmeresen), FUS ASO ION363C (jacifusen), stathmin-2 ASO QRL- 201, UNC13A ASO, SYF2 ASO, PIKFYVE ASO AS-202, CRISPR-Cas ribonuclease programming RfxCasl3d (also named CasRx, from Ruminococcus flavefaciens), modified uridine-rich small nuclear RNA (snRNA) gene therapy, such as via U7 snRNA delivery), Risdiplam, riluzole (trade name: Rilutek), edaravone (trade name: Radi cava), AMX0035, which is a combination of sodium phenylbutyrate and taurursodiol (trade name: Relyvrio). See, e.g., Hayes and Kalab (2022) Neurotherapeutics 19: 1061-1084, Mehta et al. (2023) MolecularAttorney Docket No. 057766 / 640819Degeneration 18: 16; and Van Daele et al. (2024) Trends in Molecular Medicine 30(3):252-262), each of which is herein incorporated by reference in its entirety for all purposes.

[0154] Such agents can be administered by any delivery method and by any route of administration. Some agents are administered by AAV-mediated delivery. Some agents are administered by AAV9-mediated delivery.

[0155] Methods for assessing activity of the agent are well-known and are provided elsewhere herein. In some methods, assessing activity of the agent comprises measuring the amount of full-length STMN2 RNA transcript and / or the amount of cryptic spliced STMN2 RNA transcript (i.e., including a cryptic exon as shown in the working examples), for example by semi-quantitative RT-PCR or by qRT-PCR. Assessment of amount of full-length STMN2 RNA transcript and amount of cryptic spliced STMN2 RNA transcript can be in any cell type, any tissue type, or any organ type. In some methods, assessment of activity is in motor neurons.

[0156] Such methods can also comprise measuring expression levels of the mRNA produced by the humanized STMN2 locus, including measuring amount of full-length STMN2 RNA transcript and / or amount of cryptic spliced STMN2 RNA transcript. Some methods can comprise measuring expression levels of the protein encoded by the humanized STMN2 locus including measuring amount of full-length stathmin-2 protein and / or amount of cryptic spliced stathmin-2 protein. For example, protein levels can be measured in a particular cell, tissue, or organ type (e.g., motor neurons). Methods for assessing expression of STMN2 mRNA or protein expressed from the humanized STMN2 locus are provided elsewhere herein and are well-known. As one specific example, levels of full-length and cryptic spliced STMN2 RNA transcripts are measured by semi-quantitative RT-PCR or by qRT-PCR analysis (see, e.g., Example 4 and panels D and E of Figure 6). As one specific example, levels of full-length and cryptic spliced STMN2 RNA transcripts are measured by semi-quantitative RT-PCR analysis or by qRT-PCR analysis (see, e.g., Example 4 and panels D and E of Figure 6). As one specific example, levels of full-length and cryptic spliced stathmin-2 protein are measured by automated capillary-based western blotting analysis (see, e.g., Example 4 and panels F and G of Figure 6).

[0157] STMN2 mRNA splicing in a non-human animal or non-human animal cell treated with an agent can be compared to STMN2 mRNA splicing in a control non-human animal or non-human animal cell not treated with the agent. An increased amount of full-length STMN2 RNA transcript or full-length STMN2 stathmin-2 protein in the treated non-human animal orAttorney Docket No. 057766 / 640819 non-human animal cell compared to that in the control non-human animal or non-human animal cell is an indication of the effectiveness of the agent as a regulator of STMN2 mRNA splicing.

[0158] Some methods for assessing activity of the agent measure cell health and viability, repression of cryptic exons, TDP-43 localization to the nucleus, TDP-43 aggregation resistance, and normal RNA binding (Figure 8).

[0159] The various methods provided above for assessing activity in vivo can also be used to assess the activity of agents that regulate human STMN2 mRNA splicing s ex vivo as described elsewhere herein.B. Putative Agents that Regulate Splicing of Human STMN2 mRNA

[0160] A putative agent that regulates human STMN2 mRNA splicing can be any reagent that targets a human SIMN2 gene or a human STMN2 mRNA or that targets another protein that regulates human STMN2 mRNA splicing (e.g., a protein that targets TDP-43). A regulator of human STMN2 mRNA splicing can be, for example, a known regulator of human STMN2 mRNA splicing, a putative regulator of human STMN2 mRNA splicing (e.g., candidate reagents designed to target human STMN2 gene or a human STMN2 mRNA (e.g., human STMN2 pre- mRNA)), or can be a reagent being screened for activity as a regulator of human STMN2 mRNA splicing. Some agents that regulate splicing of human STMN2 mRNA can be useful as ALS therapeutics.

[0161] The agent can be a therapeutic variant of TDP-43, a nucleic acid encoding a therapeutic variant of TDP-43, or any other large molecule or small molecule that targets STMN2 transcripts or directly or indirectly regulates or affects STMN2 mRNA splicing, TDP-43 aggregation, or TDP-43 localization. Some agents can be an aggregation-resistant TDP-43. Some agents can be an shRNA that silences endogenous TDP-43. Alternatively, the agent can be any biological or chemical agent that targets the human STMN2 locus (the human STMN2 gene), targets the human STMN2 mRNA (e.g., human STMN2 pre-mRNA), regulates or affects TDP-43 aggregation, or regulates or affects TDP-43 localization. For example, an agent that regulates splicing of human STMN2 mRNA can be an antisense oligonucleotide (ASO), a small molecule, or an agent that targets pre-mRNA, but is stably expressed (for example, CRISPR-Cas ribonuclease programming, such as via RfxCasl3d (also named CasRx, from Ruminococcus flavefaciens), and using modified uridine-rich small nuclear RNA (snRNA) gene therapy, such asAttorney Docket No. 057766 / 640819 via U7 snRNA delivery. The agent can be, for example, a known amyotrophic lateral sclerosis (ALS) therapeutic agent or candidate ALS therapeutic agent. Examples of ALS therapeutic agents include rapamycin, monepantel, tamoxifen, bosutinib, trehalose (SLS-005), ATXN2 ASO (BIIB105), tideglusib, ibudilast (MN116), colchicine, Withania somnifera, 3K3A-APC, S0D1 ASO (BIIB067), C9orf72 ASO BIIB078 (tofersen, Qalsody), C9orf72 ASO WVE-004, C9orf72 ASO ASO 5-2 (afineresen), FUS ASO ION363C (jacifusen), stathmin-2 ASO QRL-201, UNC13A ASO, SYF2 ASO, PIKFYVE ASO AS-202, CRISPR-Cas ribonuclease programming RfxCasl3d (also named CasRx, from Ruminococcus flavefaciens)', modified uridine-rich small nuclear RNA (snRNA) gene therapy, such as via U7 snRNA delivery), Risdiplam, riluzole (trade name: Rilutek), edaravone (trade name: Radicava), AMX0035, which is a combination of sodium phenylbutyrate and taurursodiol (trade name: Relyvrio). See, e.g, Hayes and Kalab (2022) Neurotherapeutics 19:1061-1084, Mehta et al. (2023) Molecular Degeneration 18: 16; and Van Daele et al. (2024) Trends in Molecular Medicine 30(3):252-262), each of which is herein incorporated by reference in its entirety for all purposes.

[0162] Other agents that regulate human STMN2 mRNA splicing can include antisense oligonucleotides (ASOs). Single-stranded ASOs and RNA interference (RNAi) share a fundamental principle in that an oligonucleotide binds a target RNA through Watson-Crick base pairing. Without wishing to be bound by theory, during RNAi, a small RNA duplex (RNAi agent) associates with the RNA-induced silencing complex (RISC), one strand (the passenger strand) is lost, and the remaining strand (the guide strand) cooperates with RISC to bind complementary RNA. Argonaute 2 (Ago2), the catalytic component of the RISC, then cleaves the target RNA. The guide strand is always associated with either the complementary sense strand or a protein (RISC). In contrast, an ASO must survive and function as a single strand. ASOs bind to the target RNA and block ribosomes or other factors, such as splicing factors, from binding the RNA or recruit proteins such as nucleases. Different modifications and target regions are chosen for ASOs based on the desired mechanism of action. A gapmer is an ASO oligonucleotide containing 2-5 chemically modified nucleotides (e.g., LNA or 2’-M0E) on each terminus flanking a central 8-10 base gap of DNA. After binding the target RNA, the DNA- RNA hybrid acts substrate for RNase H.Attorney Docket No. 057766 / 640819V. Methods of Using Non-Human Animals or Cells Comprising a Humanized STMN2 Locus for Assessing Delivery or Efficacy of Human SAMNl-Targeting Reagents In Vivo or Ex Vivo

[0163] Various methods are provided for using the non-human animals or cells comprising a humanized STMN2 locus as described elsewhere herein for assessing delivery or efficacy of human-stathmin-2-targeting reagents in vivo or ex vivo. Because the non-human animals or cells comprise a humanized STMN2 locus, the non-human animals or cells will more accurately reflect the efficacy of a human-stathmin-2-targeting reagent.A. Methods of Testing Efficacy of Human-Stathmin-2-Targeting Reagents In Vivo or Ex Vivo

[0164] Various methods are provided for assessing delivery or efficacy of human-stathmin- 2-targeting reagents in vivo using non-human animals or cells comprising a humanized STMN2 locus as described elsewhere herein. Such methods can comprise: (a) introducing into the non- human animal or cell a human-stathmin-2-targeting reagent; and (b) assessing the activity of the human-stathmin-2-targeting reagent.

[0165] The human-stathmin-2-targeting reagent can be a human-stathmin-2-targeting antibody or antigen-binding protein or any other large molecule or small molecule that targets human stathmin-2. Alternatively, the human-stathmin-2-targeting reagent can be any biological or chemical agent that targets the human STMN2 locus (the human STMN2 gene), the human STMN2 mRNA (e.g., human STMN2 pre-mRNA), or the human stathmin-2 protein. Examples of human-stathmin-2-targeting reagents are disclosed elsewhere herein. In a specific example, the human-stathmin-2-targeting reagent is a biological or chemical agent that that targets the human STMN2 locus (the human STMN2 gene) or the human STMN2 mRNA (e.g., human STMN2 pre- mRNA), as these are different from the mouse Stmn2 locus and the mouse Stmn2 mRNA.

[0166] Such human-stathmin-2-targeting reagents can be administered by any delivery method and by any route of administration. Some agents are administered by AAV-mediated delivery. Some agents are administered by AAV9-mediated delivery.

[0167] Methods for assessing activity of the human-stathmin-2-targeting reagent are well- known and are provided elsewhere herein. Any method for assessing activity of the human stathmin-2 targeting reagent can be used. In some methods, assessing activity of the human- stathmin-2-targeting reagent comprises measuring the amount of full-length STMN2 RNAAttorney Docket No. 057766 / 640819 transcript and / or the amount of cryptic spliced STMN2 RNA transcript (i . e ., including a cryptic exon as shown in the working examples), for example by semi-quantitative RT-PCR or by qRT- PCR. In some methods, assessing activity of the human-stathmin-2-targeting reagent comprises measuring expression levels of the protein encoded by the humanized STMN2 locus including measuring amount of full-length stathmin-2 protein and / or amount of cryptic spliced stathmin-2 protein. Assessment of activity can be in any cell type, any tissue type, or any organ type. In some methods, assessment of activity is in motor neuron cells or in neural tissue.

[0168] If the stathmin-2-targeting reagent is a genome editing reagent (e.g., a nuclease agent), such methods can comprise assessing modification of the humanized STMN2 locus. As one example, the assessing can comprise measuring non-homologous end joining (NHEJ) activity at the humanized STMN2 locus. This can comprise, for example, measuring the frequency of insertions or deletions within the humanized STMN2 locus. For example, the assessing can comprise sequencing the humanized STMN2 locus in one or more cells isolated from the non-human animal (e.g., next-generation sequencing). Assessment can comprise isolating a target organ or tissue (e.g., neural tissue) from the non-human animal and assessing modification of humanized STMN2 locus in the target organ or tissue. Assessment can also comprise assessing modification of humanized STMN2 locus in two or more different cell types within the target organ or tissue. Similarly, assessment can comprise isolating a non-target organ or tissue (e.g., two or more non-target organs or tissues) from the non-human animal and assessing modification of humanized STMN2 locus in the non-target organ or tissue.

[0169] Such methods can also comprise measuring expression levels of the mRNA produced by the humanized STMN2 locus, or by measuring expression levels of the protein encoded by the humanized STMN2 locus. For example, protein levels can be measured in a particular cell, tissue, or organ type (e.g., neural tissue). Methods for assessing expression of STMN2 mRNA or protein expressed from the humanized STMN2 locus are provided elsewhere herein and are well-known.

[0170] As one specific example, if the human-stathmin-2-targeting reagent is a genome editing reagent (e.g., a nuclease agent), percent editing (e.g., total number of insertions or deletions observed over the total number of sequences read in the PCR reaction from a pool of lysed cells) at the humanized STMN2 locus can be assessed (e.g., in liver cells).

[0171] The various methods provided above for assessing activity in vivo can also be used to assess the activity of human-stathmin— 2-targeting reagents ex vivo as described elsewhereAttorney Docket No. 057766 / 640819 herein.B. Human-Stathmin-2-Targeting Reagents

[0172] A human-stathmin-2 -targeting reagent can be any reagent that targets a human stathmin-2 protein, a human STMN2 gene, or a human STMN2 mRNA (e g., human STMN2 pre- mRNA). A human-stathmin-2-targeting reagent can be, for example, a known human-stathmin- 2-targeting reagent, can be a putative human-stathmin-2 -targeting reagent (e.g., candidate reagents designed to target human stathmin-2 or STMN2 mRNA (e.g., human STMN2 pre- mRNA)), or can be a reagent being screened for human-stathmin-2-targeting activity.

[0173] For example, a human-stathmin-2-targeting reagent can be an antigen-binding protein (e.g., agonist antibody) targeting an epitope of a human stathmin-2 protein. The term “antigenbinding protein” includes any protein that binds to an antigen. Examples of antigen-binding proteins include an antibody, an antigen-binding fragment of an antibody, a multispecific antibody (e.g., a bi-specific antibody), an scFv, a bis-scFv, a diabody, a triabody, a tetrabody, a V-NAR, a VHH, a VL, a F(ab), a F(ab)2, a DVD (dual variable domain antigen-binding protein), an SVD (single variable domain antigen-binding protein), a bispecific T-cell engager (BiTE), or a Davisbody (US Pat. No. 8,586,713, herein incorporated by reference herein in its entirety for all purposes). Other human-stathmin-2-targeting reagents include small molecules targeting a human stathmin-2 protein.

[0174] Other human-stathmin-2-targeting reagents can include genome editing reagents such as a nuclease agent (e.g., a Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) / CRISPR-associated (Cas) (CRISPR / Cas) nuclease, a zinc finger nuclease (ZFN), or a Transcription Activator-Like Effector Nuclease (TALEN)) that cleaves a recognition site within the human STMN2 gene. Likewise, a human-stathmin-2-targeting reagent can be an exogenous donor nucleic acid (e.g., a targeting vector or single-stranded oligodeoxynucleotide (ssODN)) designed to recombine with the human STMN2 gene.

[0175] Other human-stathmin-2 -targeting reagents can include RNAi agents. An “RNAi agent” is a composition that comprises a small double-stranded RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule capable of facilitating degradation or inhibition of translation of a target RNA, such as messenger RNA (mRNA), in a sequencespecific manner. The oligonucleotide in the RNAi agent is a polymer of linked nucleosides, eachAttorney Docket No. 057766 / 640819 of which can be independently modified or unmodified. RNAi agents operate through the RNA interference mechanism (i.e., inducing RNA interference through interaction with the RNA interference pathway machinery (RNA-induced silencing complex or RISC) of mammalian cells). While it is believed that RNAi agents, as that term is used herein, operate primarily through the RNA interference mechanism, the disclosed RNAi agents are not bound by or limited to any particular pathway or mechanism of action. RNAi agents disclosed herein comprise a sense strand and an antisense strand, and include, but are not limited to: short interfering RNAs (siRNAs), double-stranded RNAs (dsRNA), micro RNAs (miRNAs), short hairpin RNAs (shRNA), and dicer substrates. The antisense strand of the RNAi agents described herein is at least partially complementary to a sequence (i.e., a succession or order of nucleobases or nucleotides, described with a succession of letters using standard nomenclature) in the target RNA.

[0176] Other human-stathmin-2 -targeting reagents can include antisense oligonucleotides(ASOs). Single-stranded ASOs and RNA interference (RNAi) share a fundamental principle in that an oligonucleotide binds a target RNA through Watson-Crick base pairing. Without wishing to be bound by theory, during RNAi, a small RNA duplex (RNAi agent) associates with the RNA-induced silencing complex (RISC), one strand (the passenger strand) is lost, and the remaining strand (the guide strand) cooperates with RISC to bind complementary RNA. Argonaute 2 (Ago2), the catalytic component of the RISC, then cleaves the target RNA. The guide strand is always associated with either the complementary sense strand or a protein (RISC). In contrast, an ASO must survive and function as a single strand. ASOs bind to the target RNA and block ribosomes or other factors, such as splicing factors, from binding the RNA or recruit proteins such as nucleases. Different modifications and target regions are chosen for ASOs based on the desired mechanism of action. A gapmer is an ASO oligonucleotide containing 2-5 chemically modified nucleotides (e.g., LNA or 2’-M0E) on each terminus flanking a central 8-10 base gap of DNA. After binding the target RNA, the DNA-RNA hybrid acts substrate for RNase H.VI. Administering Putative Agents that Regulate Human STMN2 mRNA Splicing or Human- Stathmin-2-Targeting Reagents to Non-Human Animals or Cells

[0177] The methods disclosed herein can comprise introducing into a non-human animal orAttorney Docket No. 057766 / 640819 cell various molecules (e.g., putative agents that regulate human STMN2 mRNA splicing or human-S'7MV2 -targeting reagents such as therapeutic molecules or complexes), including nucleic acids, proteins, nucleic-acid-protein complexes, protein complexes, or small molecules. “Introducing” includes presenting to the cell or non-human animal the molecule (e.g., nucleic acid or protein) in such a manner that it gains access to the interior of the cell or to the interior of cells within the non-human animal. The introducing can be accomplished by any means, and two or more of the components (e.g., two of the components, or all of the components) can be introduced into the cell or non-human animal simultaneously or sequentially in any combination. In addition, two or more of the components can be introduced into the cell or non-human animal by the same delivery method or different delivery methods. Similarly, two or more of the components can be introduced into a non-human animal by the same route of administration or different routes of administration.

[0178] Molecules (e.g., proteins, mRNAs, DNAs, guide RNAs, RNAi agents, ASOs, or small molecules) introduced into the non-human animal or cell can be provided in compositions comprising a carrier increasing the stability of the introduced molecules (e.g., prolonging the period under given conditions of storage (e.g., -20°C, 4°C, or ambient temperature) for which degradation products remain below a threshold, such below 0.5% by weight of the starting nucleic acid or protein; or increasing the stability in vivo). Non-limiting examples of such carriers include poly(lactic acid) (PLA) microspheres, poly(D,L-lactic-coglycolic-acid) (PLGA) microspheres, liposomes, micelles, inverse micelles, lipid cochleates, and lipid microtubules.

[0179] Various methods and compositions are provided herein to allow for introduction of molecule (e.g., a nucleic acid, protein, or small molecule) into a cell or non-human animal. Methods for introducing molecules into various cell types are known and include, for example, stable transfection methods, transient transfection methods, and virus-mediated methods.

[0180] Transfection protocols as well as protocols for introducing molecules into cells may 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: W. H. Freeman and Company, pp. 96-97); dendrimers; or cationic polymers such as DEAE-dextran or polyethylenimine. Nonchemical methods include electroporation, sonoporation, and optical transfection. Particle-basedAttorney Docket No. 057766 / 640819 transfection includes the use of a gene gun, or magnet-assisted transfection (Bertram (2006) Current Pharmaceutical Biotechnology’ 7 , 277-28). Viral methods can also be used for transfection.

[0181] Introduction of molecules into a cell can also be mediated by electroporation, by intracytoplasmic injection, by viral infection, by adenovirus, by adeno-associated virus, by lentivirus, by retrovirus, by transfection, by lipid-mediated transfection, or by nucleofection. Nucleofection is an improved electroporation technology that enables nucleic acid substrates to be delivered not only to the cytoplasm but also through the nuclear membrane and into the nucleus. In addition, use of nucleofection in the methods disclosed herein typically requires much fewer cells than regular electroporation (e.g., only about 2 million compared with 7 million by regular electroporation). In one example, nucleofection is performed using the LONZA® NUCLEOFECTOR™ system.

[0182] Introduction of molecules into a cell (e.g., a zygote) can also be accomplished by microinjection. In zygotes (i.e., one-cell stage embryos), microinjection can be into the maternal and / or paternal pronucleus or into the cytoplasm. If the microinjection is into only one pronucleus, the paternal pronucleus is preferable due to its larger size. Microinjection of an mRNA is preferably into the cytoplasm (e.g., to deliver mRNA directly to the translation machinery), while microinjection of a protein or a polynucleotide encoding a protein or encoding an RNA is preferable into the nucleus / pronucleus. Alternatively, microinjection can be carried out by injection into both the nucleus / pronucleus and the cytoplasm: a needle can first be introduced into the nucleus / pronucleus and a first amount can be injected, and while removing the needle from the one-cell stage embryo a second amount can be injected into the cytoplasm. Methods for carrying out 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); see also Meyer et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107: 15022-15026 and Meyer et al. (2012) Proc. Natl. Acad. Sci. U.S.A. 109:9354-9359.

[0183] Other methods for introducing molecules into a cell or non-human animal can include, for example, vector delivery, particle-mediated delivery, exosome-mediated delivery, lipid-nanoparticle-mediated delivery, cell-penetrating-peptide-mediated delivery, or implantabledevice-mediated delivery. As specific examples, a nucleic acid or protein can be introduced intoAttorney Docket No. 057766 / 640819 a cell or non-human animal in a carrier such as a poly(lactic acid) (PLA) microsphere, a poly(D,L-lactic-coglycolic-acid) (PLGA) microsphere, a liposome, a micelle, an inverse micelle, a lipid cochleate, or a lipid microtubule. Some specific examples of delivery to a non-human animal include hydrodynamic delivery, virus-mediated delivery (e.g., adeno-associated virus (AAV)-mediated delivery), and lipid-nanoparticle-mediated delivery.

[0184] Introduction of molecules into cells or non-human animals can be accomplished by hydrodynamic delivery (HDD). For gene delivery to parenchymal cells, only essential DNA sequences need to be injected via a selected blood vessel, eliminating safety concerns associated with current viral and synthetic vectors. When injected into the bloodstream, DNA is capable of reaching cells in the different tissues accessible to the blood. Hydrodynamic delivery employs the force generated by the rapid injection of a large volume of solution into the incompressible blood in the circulation to overcome the physical barriers of endothelium and cell membranes that prevent large and membrane-impermeable compounds from entering parenchymal cells. In addition to the delivery of DNA, this method is useful for the efficient intracellular delivery of RNA, proteins, and other small compounds in vivo. See, e.g., Bonamassa et al. (2011) Pharm. Res. 28(4):694-701, herein incorporated by reference in its entirety for all purposes.

[0185] Introduction of nucleic acids can also be accomplished by virus-mediated delivery, such as AAV-mediated delivery or lentivirus-mediated delivery. Other exemplary viruses / viral vectors include retroviruses, adenoviruses, vaccinia viruses, poxviruses, and herpes simplex viruses. The viruses can infect dividing cells, non-dividing cells, or both dividing and nondividing cells. The viruses can integrate into the host genome or alternatively do not integrate into the host genome. Such viruses can also be engineered to have reduced immunity. The viruses can be replication-competent or can be replication-defective (e.g., defective in one or more genes necessary for additional rounds of virion replication and / or packaging). Viruses can cause transient expression, long-lasting expression (e.g., at least 1 week, 2 weeks, 1 month, 2 months, or 3 months), or permanent expression (e.g., of Cas9 and / or gRNA). Exemplary viral titers (e.g., AAV titers) include about 1012, about 1013, about 1014, about 1015, and about 1016vector genomes / mL. Other exemplary viral titers (e.g., AAV titers) include about 1012, about 1013, about 1014, about 1015, and about 1016vector genomes(vg) / kg of body weight.

[0186] The ssDNA AAV genome consists of two open reading frames, Rep and Cap, flanked by two inverted terminal repeats that allow for synthesis of the complementary DNA strand.Attorney Docket No. 057766 / 640819When constructing an AAV transfer plasmid, the transgene is placed between the two TTRs, and Rep and Cap can be supplied in trans. In addition to Rep and Cap, AAV can require a helper plasmid containing genes from adenovirus. These genes (E4, E2a, and VA) mediate AAV replication. For example, the transfer plasmid, Rep / Cap, and the helper plasmid can be transfected into HEK293 cells containing the adenovirus gene E1+ to produce infectious AAV particles. Alternatively, the Rep, Cap, and adenovirus helper genes may be combined into a single plasmid. Similar packaging cells and methods can be used for other viruses, such as retroviruses.

[0187] Multiple serotypes of AAV have been identified. These serotypes differ in the types of cells they infect (i.e., their tropism), allowing preferential transduction of specific cell types. Serotypes for CNS tissue include AAV1, AAV2, AAV4, AAV5, AAV8, and AAV9. Serotypes for heart tissue include AAV1, AAV8, and AAV9. Serotypes for kidney tissue include AAV2. Serotypes for lung tissue include AAV4, AAV5, AAV6, and AAV9. Serotypes for pancreas tissue include AAV8. Serotypes for photoreceptor cells include AAV2, AAV5, and AAV8. Serotypes for retinal pigment epithelium tissue include AAV1, AAV2, AAV4, AAV5, and AAV8. Serotypes for skeletal muscle tissue include AAV1, AAV6, AAV7, AAV8, and AAV9. Serotypes for liver tissue include AAV7, AAV8, and AAV9, and particularly AAV8.

[0188] Tropism can be further refined through pseudotyping, which is the mixing of a capsid and a genome from different viral serotypes. For example, AAV2 / 5 indicates a virus containing the genome of serotype 2 packaged in the capsid from serotype 5. 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 a hybrid capsid from eight serotypes and displays high infectivity across a broad range of cell types in vivo. AAV-DJ8 is another example that displays the properties of AAV-DJ but with enhanced brain uptake. AAV serotypes can also be modified through mutations. Examples of mutational modifications of AAV2 include Y444F, Y500F, Y730F, and S662V. Examples of mutational modifications of AAV3 include Y705F, Y731F, and T492V. Examples of mutational modifications of AAV6 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.

[0189] To accelerate transgene expression, self-complementary AAV (scAAV) variants can be used. Because AAV depends on the cell’s DNA replication machinery to synthesize theAttorney Docket No. 057766 / 640819 complementary strand of the AAV’s single-stranded DNA genome, transgene expression may be delayed. To address this delay, scAAV containing complementary sequences that are capable of spontaneously annealing upon infection can be used, eliminating the requirement for host cell DNA synthesis. However, single-stranded AAV (ssAAV) vectors can also be used.

[0190] To increase packaging capacity, longer transgenes may 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 a cell, these viruses form concatemers, are spliced together, and the full-length transgene can be expressed. Although this allows for longer transgene expression, expression is less efficient. Similar methods for increasing capacity utilize homologous recombination. For example, a transgene can be divided between two transfer plasmids but with substantial sequence overlap such that co-expression induces homologous recombination and expression of the full- length transgene.

[0191] Introduction of molecules can also be accomplished by lipid nanoparticle (LNP)- mediated delivery. For example, biodegradable lipids improve clearance, improve tolerability, and decrease immunogenicity. Lipid formulations can protect biological molecules from degradation while improving their cellular uptake. Lipid nanoparticles are particles comprising a plurality of lipid molecules physically associated with each other by intermolecular forces. These include microspheres (including unilamellar and multilamellar vesicles, e.g., liposomes), a dispersed phase in an emulsion, micelles, or an internal phase in a suspension. Such lipid nanoparticles can be used to encapsulate one or more nucleic acids or proteins for delivery. Formulations which contain cationic lipids are useful for delivering polyanions such as nucleic acids. Other lipids that can be included are neutral lipids (i.e., uncharged or zwitterionic lipids), anionic lipids, helper lipids that enhance transfection, and stealth lipids that increase the length of time for which nanoparticles can exist in vivo. Examples of suitable cationic lipids, neutral lipids, anionic lipids, helper lipids, and stealth lipids can be found in WO 2016 / 010840 Al, herein incorporated by reference in its entirety for all purposes. An exemplary lipid nanoparticle can comprise a cationic lipid and one or more other components. In one example, the other component can comprise a helper lipid such as cholesterol. In another example, the other components can comprise a helper lipid such as cholesterol and a neutral lipid such as DSPC. In another example, the other components can comprise a helper lipid such as cholesterol, an optional neutral lipid such as DSPC, and a stealth lipid such as SO 10, S024, S027, S031, or S033.Attorney Docket No. 057766 / 640819

[0192] The LNP may contain one or more or all of the following: (i) a lipid for encapsulation and for endosomal escape; (ii) a neutral lipid for stabilization; (iii) a helper lipid for stabilization; and (iv) a stealth lipid. See, e.g., Finn et al. (2018) Cell Reports 22: 1-9 and WO 2017 / 173054 Al, each of which is herein incorporated by reference in its entirety for all purposes.

[0193] Exemplary dosing of LNPs includes, for example, about 0.1, about 0.25, about 0.3, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 8, or about 10 mg / kg (mpk) with respect to total RNA cargo content. In one example, LNP doses between about 0.01 mg / kg and about 10 mg / kg, between about 0.1 and about 10 mg / kg, or between about 0.01 and about 0.3 mg / kg can be used. For example, LNP doses of about 0.01, about 0.03, about 0.1, about 0.3, about 1, about 3, or about 10 mg / kg can be used.

[0194] Administration in vivo can be by any suitable route including, for example, parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. Systemic modes of administration include, for example, oral and parenteral routes. Examples of parenteral routes include intravenous, intraarterial, intraosseous, intramuscular, intradermal, subcutaneous, intranasal, and intraperitoneal routes. A specific example is intravenous infusion. Nasal instillation and intravitreal injection are other specific examples. Local modes of administration include, for example, intrathecal, intracerebroventricular, intraparenchymal (e.g., localized intraparenchymal delivery to the striatum (e.g., into the caudate 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, subconjuctival, intravitreal, subretinal, and transscleral routes. Significantly smaller amounts of the components (compared with systemic approaches) may exert an effect when administered locally (for example, intraparenchymal or intravitreal) compared to when administered systemically (for example, intravenously). Local modes of administration may also reduce or eliminate the incidence of potentially toxic side effects that may occur when therapeutically effective amounts of a component are administered systemically.

[0195] Compositions comprising the administered agents can be formulated using one or more physiologically and pharmaceutically acceptable carriers, diluents, excipients or auxiliaries. The formulation can depend on the route of administration chosen. The term “pharmaceuticallyAttorney Docket No. 057766 / 640819 acceptable” means that the carrier, diluent, excipient, or auxiliary is compatible with the other ingredients of the formulation and not substantially deleterious to the recipient thereof.

[0196] The frequency of administration and the number of dosages can depend on the halflife of the administered agents and the route of administration among other factors. The introduction of nucleic acids or proteins into the cell or non-human animal can be performed one time or multiple times over a period of time. For example, the introduction can be performed at least two times 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 times, at least ten times over a period of time, at least eleven times, 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.VII. Measuring Delivery, Activity, or Efficacy of Putative Agents that Regulate Splicing of Human STMN2 mRNA or Human-Stathmin-2-Targeting Reagents In Vivo or Ex Vivo

[0197] The methods disclosed herein can further comprise detecting or measuring activity of putative agents that regulate splicing of human STMN2 mRNA or human-stathmin-2-targeting reagents. In some methods, the activity of the agents that regulate splicing of human STMN2 mRNA or human-stathmin-2-targeting reagents include measuring a level of full-length STMN2 mRNA and / or a level of cryptic spliced STMN2 mRNA. In some methods, the activity of the agents that regulate splicing of human STMN2 mRNA or human-stathmin-2-targeting reagents include measuring a level of full-length stathmin-2 protein and / or a level of cryptic spliced stathmin-2 protein.

[0198] If the human-stathmin-2 -targeting reagent is a genome editing reagent, the measuring can comprise assessing the humanized STMN2 locus for modifications. Various methods can be used to identify cells having a targeted genetic modification. The screening can comprise a quantitative assay for assessing modification-of-allele (MO A) of a parental chromosome. See, e.g., US 2004 / 0018626; US 2014 / 0178879; US 2016 / 0145646; WO 2016 / 081923; andAttorney Docket No. 057766 / 640819Frendewey et al. (2010) Methods Enzymol. 476:295-307, each of which is herein incorporated by reference in its entirety for all purposes. For example, the quantitative assay can be carried out via a quantitative PCR, such as a real-time PCR (qPCR). The real-time PCR can utilize a first primer set that recognizes the target locus and a second primer set that recognizes a non-targeted reference locus. The primer set can comprise a fluorescent probe that recognizes the amplified sequence. Other examples of suitable quantitative assays include fluorescence-mediated in situ hybridization (FISH), comparative genomic hybridization, isothermic DNA amplification, quantitative hybridization to an immobilized probe(s), INVADER® Probes, TAQMAN® Molecular Beacon probes, or ECLIPSE™ probe technology (see, e.g., US 2005 / 0144655, herein incorporated by reference in its entirety for all purposes). Next-generation sequencing (NGS) can also be used for screening. Next-generation sequencing can also be referred to as “NGS” or “massively parallel sequencing” or “high throughput sequencing.” NGS can be used as a screening tool in addition to the MOA assays to define the exact nature of the targeted genetic modification and whether it is consistent across cell types or tissue types or organ types.

[0199] If the reagent is designed to inactivate the humanized STMN2 locus, affect expression of the humanized STMN2 locus, prevent translation of the humanized STMN2 mRNA, or affect splicing of the humanized STMN2 mRNA, the measuring can comprise assessing humanized STMN2 mRNA or protein expression.

[0200] The assessing in a non-human animal can be in any cell type from any tissue or organ. For example, the assessment can be in multiple cell types from the same tissue or organ (e g., neural tissue) or in cells from multiple locations within the tissue or organ. This can provide information about which cell types within a target tissue or organ are being targeted or which sections of a tissue or organ are being reached by the human-stathmin-2-targeting reagent. As another example, the assessment can be in multiple types of tissue or in multiple organs. In methods in which a particular tissue, organ, or cell type is being targeted, this can provide information about how effectively that tissue or organ is being targeted and whether there are off-target effects in other tissues or organs.

[0201] All patent filings, websites, other publications, accession numbers and the like cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. If different versions of a sequence are associated with an accession number at differentAttorney Docket No. 057766 / 640819 times, the version associated with the accession number at the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or filing date of a priority application referring to the accession number if applicable. Likewise, if different versions of a publication, website or the like are published at different times, the version most recently published at the effective filing date of the application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the invention can be used in combination with any other unless specifically indicated otherwise. Although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.BRIEF DESCRIPTION OF THE SEQUENCES

[0202] The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three-letter code for amino acids. The nucleotide sequences follow the standard convention of beginning at the 5’ end of the sequence and proceeding forward (i.e., from left to right in each line) to the 3’ end. Only one strand of each nucleotide sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand. When a nucleotide sequence encoding an amino acid sequence is provided, it is understood that codon degenerate variants thereof that encode the same amino acid sequence are also provided. The amino acid sequences follow the standard convention of beginning at the amino terminus of the sequence and proceeding forward (i.e., from left to right in each line) to the carboxy terminus.

[0203] Table 2. Description of Sequences.Attorney Docket No. 057766 / 640819EXAMPLESExample 1. Generation of Mice Comprising a Wild Type Humanized STMN2 Locus

[0204] TDP-43 regulates expression of STMN2 by binding sites within STMN2 intron 1 .Lowered TDP-43 reduces binding to STMN2 and uncovers a cryptic poly(A) site in an alternate exon 2a within the STMN2 first intron. Use of this alternative exon 2a causes earlier terminationAttorney Docket No. 057766 / 640819 of the STMN2 protein. As this phenomenon has not been shown in mouse, a humanization of full-length Stmn2 was undertaken.

[0205] A large targeting vector (LTVEC) comprising a 5’ homology arm comprising 26 kb of the mouse Stmn2 locus and 3’ homology arm comprising 22 kb of the mouse Stmn2 locus was generated to replace a region of 50.7 kb from the mouse Stmn2 gene with 55.3 kb of the corresponding sequence of the human STMN2 gene. Information on mouse Stmn2 and human STMN2 genes is provided in Table 3. A description of the generation of the large targeting vector is provided in Table 4. Generation and use of large targeting vectors (LTVECs) derived from bacterial artificial chromosome (BAC) DNA through bacterial homologous recombination (BHR) reactions using VELOCIGENE® genetic engineering technology is described, e.g., in US 6,586,251 and Valenzuela et al. (2003) Nat. Biotechnol. 21(6):652-659, each of which is herein incorporated by reference in its entirety for all purposes. Generation of LTVECs through in vitro assembly methods is described, e.g., in US 2015 / 0376628 and WO 2015 / 200334, each of which is herein incorporated by reference in its entirety for all purposes.

[0206] Table 3. Mouse Stmn2 and Human STMN2.

[0207] Table 4. Mouse Stmn2 Large Targeting Vector for MAID8636.

[0208] Specifically, a region, starting in exon 1 at the start ATG through the first 51 bp of exon 5, including all introns in between, was deleted from the mouse Stmn2 locus, leaving the last 3 codons (TCT GGC TGA, encoding Ser, Gly, stop) and the mouse Stmn2 3’ untranslated region (UTR). A human region, starting in exon 1 with the start ATG, through exon 5 coding sequence and 3’ UTR, with all introns in between, was inserted in place of the deleted mouse region. The humanized region includes a fully human coding sequence, including alternate exon 2a, and introns. A self-deleting, neomycin resistance cassette was placed just 3’ to the human region. This is the MAID 8636 (Stmn2 humanized region with self-deleting cassette) (SEQ IDAttorney Docket No. 057766 / 640819NO: 48). See Figure 1. The MAID 8637 allele (Stmn2 humanized region without self-deleting cassette) (SEQ ID NO: 49) is the allele after cassette deletion. See Figure 1.

[0209] Sequences for targeted allele with self-del eting selection cassette (MAID 8636) are shown in Table 5, with numbering with reference to SEQ ID NO: 48, sequences for cassette- deleted allele (MAID 8637) are shown in Table 6, with numbering with reference to SEQ ID NO: 49.

[0210] Table 5. Targeted Allele with Self-Deleting Selection Cassette, MAID 8636Mouse Sequence 1-402Human ex 1 CDS 403-421Human intron 422-6024Human alternate exon 2a 6025-6251Human intron 6252-26004Human exon 2 26005-26100Human intron 26101-30580Human exon 3 30581-30753Human intron 30754-44073Human exon 4 44074-44265Human intron 44266-54017Human exon 5 CDS 54018-54077Human exon 5. 3’ UTR 54078-55361Human sequence following 3’ UTR 55362-55724Xhol 55725-55731LoxP 55731-55764Protamine promoter 55771-56451Cre (exon 1) 56452-56892Intron 56893-56959Cre (exon 2) 56960-57592Poly(A) 57605-57834 hUb promoter 57889-59101Em7 promoter 59102-59168Neo 59169-59972Poly (A) 59973-60457LoxP 60463-60496I_Ceu-l 60503-60528Nhel 60529-60534Mouse exon 5 60535-60543Mouse sequence 60544-62375Attorney Docket No. 057766 / 640819

[0211] Table 6. Cassette-Deleted Allele, MAID 8637Mouse Sequence 1-402Human ex 1 CDS 403-421Human intron 422-6024Human alternate exon 2a 6025-6251Human intron 6252-26004Human exon 2 26005-26100Human intron 26101-30580Human exon 3 30581-30753Human intron 30754-44073Human exon 4 44074-44265Human intron 44266-54017Human exon 5 CDS 54018-54077Human exon 5, 3’ UTR 54078-55361Human sequence following 3’ UTR 55362-55724Xhol 55725-55731LoxP 55731-55764I_Ceu-l 55771-55796Nhel 55797-55802Mouse exon 5 55803-57090Mouse sequence 57091-57643

[0212] An alignment of the mouse stathmin-2, human stathmin-2, humanized stathmin-2, and humanized stathmin-2 using alternate exon are depicted in Figure 3. The mouse and human stathmin-2 proteins are 100% identical at the protein level. The mouse Stmn2 and human STMN2 coding sequences are set forth in SEQ ID NOS: 5 and 2, respectively. The mouse and human stathmin-2 protein sequences are set forth in SEQ ID NOS: 4 and 1, respectively. The sequences for the expected humanized STMN2 coding sequence and the expected humanized stathmin-2 protein are set forth in SEQ ID NOS: 10 and 9, respectively. The sequences for the expected humanized STMN2 coding sequence and the expected humanized stathmin-2 protein when the alternate exon is used due to cryptic splicing are set forth in SEQ ID NOS: 8 and 7, respectively. Coding sequences and amino acid sequences are set forth in Table 2.

[0213] To generate the mutant allele, CRISPR / Cas9 components including four guide RNAs were introduced into mouse embryonic stem cells together with the large targeting vector. Specifically, 2 x 106mouse ES cells were electroporated with the following: 0.4 ug Stmn2 LTVEC; and 125 pmol each of the gRNAs: gU2, gU3, gD3, and gD4. Antibiotic selection was performed using G418 at a concentration of 75 mg / mL. See, e.g., US 2015-0376651 and WO 2015 / 200805, each of which is herein incorporated by reference in its entirety for all purposes. Following antibiotic selection, colonies were picked, expanded, and screened by TAQMAN®. See Figure 2. Loss-of-allele assays were performed to detect loss of the endogenous mouseAttorney Docket No. 057766 / 640819 allele (8636mTU, 8636m TD, 8636mTD2), gain-of-allele assays (8636hTU, 8636hTD, 8636hTD2) were performed to detect gain of the humanized allele, CRISPR retention assays (90108mretU, 90108mretU2, 90108mretD, 90108mretD2), and CRISPR assays (90108mTM) were performed using the primers and probes set forth in Table 7.

[0214] Table 7. Guide RNAs and Screening Assays.

[0215] Modification-of-allele (MO A) assays including loss-of-allele (LOA) and gain-of- allele (GOA) assays are described, for example, in US 2014 / 0178879; US 2016 / 0145646; WOAttorney Docket No. 057766 / 6408192016 / 081923; and Frendewey et al. (2010) Methods Enzymol. 476:295-307, each of which is herein incorporated by reference in its entirety for all purposes. The loss-of-allele (LOA) assay inverts the conventional screening logic and quantifies the number of copies in a genomic DNA sample of the native locus to which the mutation was directed. In a correctly targeted heterozygous cell clone, the LOA assay detects one of the two native alleles (for genes not on the X or Y chromosome), the other allele being disrupted by the targeted modification. The same principle can be applied in reverse as a gain-of-allele (GOA) assay to quantify the copy number of the inserted targeting vector in a genomic DNA sample.

[0216] Retention assays are described in US 2016 / 0145646 and WO 2016 / 081923, each of which is herein incorporated by reference in its entirety for all purposes. Retention assays distinguish between correct targeted insertions of a nucleic acid insert into a target genomic locus from random transgenic insertions of the nucleic acid insert into genomic locations outside of the target genomic locus by assessing copy numbers of DNA templates from 5’ and 3’ target sequences corresponding to the 5’ and 3’ homology arms of the targeting vector, respectively. Specifically, retention assays determine copy numbers in a genomic DNA sample of a 5’ target sequence DNA template intended to be retained in the modified target genomic locus and / or the 3’ target sequence DNA template intended to be retained in the modified target genomic locus. In diploid cells, correctly targeted clones will retain a copy number of two. Copy numbers greater than two generally indicate transgenic integration of the targeting vector randomly outside of the target genomic locus rather than at the target genomic locus. Copy numbers of less than generally indicate large deletions extending beyond the region targeted for deletion.

[0217] CRISPR assays are TAQMAN® assays designed to cover the region that is disrupted by the CRISPR gRNAs. When a CRISPR gRNA cuts and creates an indel (insertion or deletion), the TAQMAN® assay will fail to amplify and thus reports CRISPR cleavage.

[0218] F0 mice were generated from the modified ES cells using the VELOCIMOUSE® method. Specifically, mouse ES cell clones comprising the humanized STMN2 loci described above that were selected by the MOA assay described above were injected into 8-cell stage embryos using the VELOCIMOUSE® method. See, e.g., US 7,576,259; US 7,659,442; US 7,294,754; US 2008 / 0078000; and Poueymirou et al. (2007) Nat. Biotechnol. 25(l):91-99, each of which is herein incorporated by reference in its entirety for all purposes. In the VELOCIMOUSE® method, targeted mouse ES cells are injected through laser-assisted injectionAttorney Docket No. 057766 / 640819 into pre-morula stage embryos, e g., eight-cell -stage embryos, which efficiently yields FO generation mice that are fully ES-cell-derived. In the VELOCIMOUSE® method, the injected pre-morula stage embryos are cultured to the blastocyst stage, and the blastocyst-stage embryos are introduced into and gestated in surrogate mothers to produce the FO generation mice. When starting with mouse ES cell clones homozygous for the targeted modification, FO mice homozygous for the targeted modification are produced. When starting with mouse ES cell clones heterozygous for the targeted modification, subsequent breeding can be performed to produce mice homozygous for the targeted modification.Example 2. Generation of Embryonic Stem Cells Expressing a Mutated Tardbp Gene

[0219] Since TDP-43 is essential for viability, embryonic stem (ES) cells comprising a conditional knockout on a first endogenous Tardbp allele and a mutation on the other second endogenous Tardbp allele may be generated such that wild type TDP-43 from the first endogenous allele sustains viability of the ES cell until activation of the condition, after which activation the effects of the mutant TDP-43 polypeptide expressed from the second allele may be ascertained.

[0220] To evaluate the biological, biochemical, and / or pathogenic role(s) played by various TDP-43 structural domains, mouse ES cells were modified to comprise: (i) at an endogenous Tardbp locus, a conditional knockout mutation, and (ii) at the other Tardbp locus on a homologous chromosome, a mutated Tardbp gene that encodes a mutant TDP-43 polypeptide in which one of the five structural domains — the nuclear localization signal (NLS), RNA recognition motif 1 (RRM1), RNA recognition motif 2 (RRM2), a putative export signal (E), or the prion like domain (PLD) — was either altered in ways predicted to abolish their functions or deleted. See Figure 4 and WO 2020 / 264339, herein incorporated by reference in its entirety for all purposes.

[0221] The conditional allele was designed based on previously published work that shows deletion of TDP-43 exon 3 produces no functional protein. Chiang et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107: 16320-16324, herein incorporated by reference in its entirety for all purposes. Exon 3 of the endogenous mouse TARDBP gene was floxed with loxp sites. After Cre-mediated recombination, deletion of the genomic coordinates chr4: 147995844-147996841 was effected. ES cells comprising the floxed exon 3 were further modified with a mutated Tardbp gene asAttorney Docket No. 057766 / 640819 described herein. As a control, mouse ES cells modified with the conditional knockout mutation on one allele and a deletion from the start codon of the second exon to the stop codon (genomic coordinates chr4: 147992370-147999471) on the other allele were also created.Example 3. Differentiation of Embryonic Stem (ES) Cells into Motor Neurons

[0222] ES cells were differentiated into motor neurons (embryonic-stem-cell-derived motor neurons, or ESMNs) in culture. See Figure 5 and Example 2 of WO 2020 / 264339, herein incorporated by reference in its entirety for all purposes. ES cells were cultured in embryonic stem cell medium (ESM; DMEM + 15% fetal bovine serum + penicillin / streptomycin + glutamine + non-essential amino acids + nucleosides + b-mercaptoethanol + sodium pyruvate + LIF) for 2 days, during which the medium was changed daily. ES medium was replaced with 7 mb of ADFNK medium (advanced DMEM / F12 + neurobasal medium + 10% knockout serum + penicillin / streptomycin + glutamine + b-mercaptoethanol) 1 hour before trypsinization. ADFNK medium was aspirated, and ESCs were trypsinized with 0.05% trypsin-EDTA. Pelleted cells were resuspended in 12 mL of ADFNK and grown for two days in suspension. Dissociated motor neurons were plated and matured in embryonic-stem-cell-derived motor neuron medium (ESMN; neurobasal medium + 2% horse serum + B27 + glutamine + penicillin / streptomycin + b-mercaptoethanol + 10 ng / mL GDNF, BDNF, CNTF). The conditional knockout allele was activated using Cre recombinase delivered via electroporation at the ES cell stage or seven days after plating.Example 4. Use of Humanized STMN2 ES-Cell-Derived Motor Neurons

[0223] Loss of mTDP-43 function in ES cell-derived motor neurons leads to inclusion of a human cryptic exon in STMN2 and loss of full length STMN2 mRNA and protein. While multiple cryptic exons regulated by TDP-43 have been shown to be aberrantly de-repressed in ALS patient samples, only a select few have been postulated to contribute to disease processes, specifically those in STMN2 and UNC13A. Intronic sequences harboring these cryptic exons and TDP-43 binding sites are generally not conserved between mouse and human. See Figure 7, panel A. Therefore, to study how mis-localized TDP-43 directly affects splicing of disease- associated cryptic exons in our mouse systems, we humanized mouse Stmn2 in the background of TDP-43 WT or domain mutant alleles (Figures 6 (panel C) and 7 (panel B)). Cells wereAttorney Docket No. 057766 / 640819 obtained with one humanized STMN2 allele and one collapsed KO allele (STMN2HumIn / -) caused by cleavage with the gRNAs described in Example 1. RT-qPCR analysis of transcripts encoded by our humanized STMN2 allele (STMN2HumIn / -) in ES-cell-derived motor neurons showed that expression of the cryptic exon is repressed when wild-type mouse TDP-43 is present (Figure 6, panels D and E), supporting the notion that mouse TDP-43 can regulate splicing events dependent on the human TDP-43 protein. Importantly, when TDP-43 is disrupted by endogenous deletion or mutation of critical domains (i.e., ANLS, ANES, and APLD), inclusion of the cryptic exon in humanized STMN2 transcripts is dramatically induced (Figure 6, panels D and E). Furthermore, cryptic exon inclusion in humanized STMN2 led to loss of full-length STMN2 mRNA and protein (Figure 6, panels D, E, F, and G). This collective set of data from our humanized STMN2 allele demonstrates that (1) mouse TDP-43 can compensate for human TDP-43 splicing function and (2) our mouse system of endogenous TDP-43 domain mutant alleles can induce human-specific splicing changes associated with ALS, providing both cellular and animal model systems to test the contribution of these splicing events to motor neuron disease in vivo (Figure 8).

Claims

1. Attorney Docket No. 057766 / 640819We claim:

1. A non-human animal or non-human animal cell comprising in its genome a humanized endogenous STMN2 locus in which a segment of the endogenous Stmn2 locus has been deleted and replaced with a corresponding human STMN2 sequence.

2. The non-human animal or non-human animal cell of claim 1, wherein a region of the endogenous Stmn2 locus comprising both coding sequence and non-coding sequence has been deleted and replaced with the corresponding human STMN2 sequence comprising both coding sequence and non-coding sequence.

3. The non-human animal or non-human animal cell of claim 1 or 2, wherein at least one intron and at least one exon of the endogenous Stmn2 locus have been deleted and replaced with the corresponding human STMN2 sequence.

4. The non-human animal or non-human animal cell of any preceding claim, wherein exon 1, intron 1, and exon 2 of the endogenous Stmn2 locus have been deleted and replaced with the corresponding human STMN2 sequence.

5. The non-human animal or non-human animal cell of any preceding claim, wherein the humanized endogenous STMN2 locus encodes a human stathmin-2.

6. The non-human animal or non-human animal cell of any preceding claim, wherein the deleted segment of the endogenous Stmn2 locus comprises a region of endogenous Stmn2 genomic sequence from the start codon through the stop codon, and wherein the corresponding human STMN2 sequence comprises the human STMN2 genomic sequence from the start codon through the stop codon.

7. The non-human animal or non-human animal cell of any preceding claim, wherein the endogenous Stimi2 5’ untranslated region has not been deleted and replaced with the corresponding human STMN2 sequence and / or wherein the humanized endogenous STMN2 locus comprises a human STMN2 3’ untranslated region.Attorney Docket No. 057766 / 6408198. The non-human animal or non-human animal cell of any preceding claim, wherein the humanized endogenous STMN2 locus comprises the endogenous Stmn2 promoter, wherein the human STMN2 sequence is operably linked to the endogenous Stmn2 promoter.

9. The non-human animal or non-human animal cell of any preceding claim, wherein the deleted segment of the endogenous Stmn2 locus comprises a region of endogenous Stmn2 genomic sequence from the start codon through the stop codon, and wherein the corresponding human STMN2 sequence comprises the human STMN2 genomic sequence from the start codon through the stop codon, wherein the endogenous Stmn2 5’ untranslated region has not been deleted and replaced with the corresponding human STMN2 sequence, wherein the humanized endogenous STMN2 locus comprises a human STMN2 3’ untranslated region, and wherein the humanized endogenous STMN2 locus comprises the endogenous Stmn2 promoter, wherein the human STMN2 sequence is operably linked to the endogenous Stmn2 promoter.

10. The non-human animal or non-human animal cell of any preceding claim, wherein:(i) the humanized endogenous STMN2 locus comprises a human STMN2 sequence comprising, consisting essentially of, or consisting of a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO: 53 or 54; or(ii) the humanized endogenous STMN2 locus encodes a protein comprising, consisting essentially of, or consisting of a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO: 9, optionally wherein the humanized endogenous STMN2 locus encodes a protein comprising, consisting essentially of, or consisting of the sequence set forth in SEQ ID NO: 9; or(iii) the humanized endogenous STMN2 locus comprises a coding sequence comprising, consisting essentially of, or consisting of a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO: 10, optionally wherein the humanized endogenous STMN2 locus comprises aAttorney Docket No. 057766 / 640819 coding sequence comprising, consisting essentially of, or consisting of the sequence set forth in SEQ ID NO: 10; or(iv) the humanized endogenous STMN2 locus comprises a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO: 48 or 49, optionally wherein the humanized endogenous STMN2 locus comprises the sequence set forth in SEQ ID NO: 48 or 49.

11. The non-human animal or non-human animal cell of any preceding claim, wherein the humanized endogenous STMN2 locus does not comprise a selection cassette or a reporter gene.

12. The non-human animal or non-human animal cell of any preceding claim, wherein the non-human animal or non-human animal cell is homozygous for the humanized endogenous STMN2 locus.

13. The non-human animal or non-human animal cell of any one of claims 1- 11, wherein the non-human animal or non-human animal cell is heterozygous for the humanized endogenous STMN2 locus.

14. The non-human animal or non-human animal cell of any one of claims 1- 11, wherein the non-human animal or non-human animal cell is compound heterozygous or hemizygous for the humanized endogenous STMN2 locus.

15. The non-human animal or non-human animal cell of claim 14, wherein:(I) (i) the first allele of the humanized endogenous STMN2 locus comprises a human STMN2 sequence comprising, consisting essentially of, or consisting of a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO: 53 or 54; or(ii) the first allele of the humanized endogenous STMN2 locus encodes a protein comprising, consisting essentially of, or consisting of a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO: 9, optionally wherein the first allele of the humanized endogenous STMN2Attorney Docket No. 057766 / 640819 locus encodes a protein comprising, consisting essentially of, or consisting of the sequence set forth in SEQ ID NO: 9; or(iii) the first allele of the humanized endogenous STMN2 locus comprises a coding sequence comprising, consisting essentially of, or consisting of a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO: 10, optionally wherein the first allele of the humanized endogenous STMN2 locus comprises a coding sequence comprising, consisting essentially of, or consisting of the sequence set forth in SEQ ID NO: 10; or(iv) the first allele of the humanized endogenous STMN2 locus comprises a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO: 48 or 49, optionally wherein the first allele of the humanized endogenous STMN2 locus the sequence set forth in SEQ ID NO: 48 or 49; and(II) (i) the second allele of the humanized endogenous STMN2 locus is an inactivated allele comprising a deletion.

16. The non-human animal or non-human animal cell of any preceding claim, wherein the non-human animal or non-human animal cell further comprises a mutation in the Tardbp locus.

17. The non-human animal or non-human animal cell of claim 16, wherein the mutation in the Tardbp locus causes mislocalization of encoded TAR DNA-binding protein 43 (TDP-43) protein.

18. The non-human animal or non-human animal cell of claim 16 or 17, wherein the encoded TDP-43 protein comprises a mutation in the nuclear localization signal (NLS), nuclear export signal (NES), or prion-like domain (PLD) of TDP-43.

19. The non-human animal or non-human animal cell of claim 18, wherein:(I) the mutation in the NLS comprises point mutations at positions 82-84, 95, 97, and 98, optionally wherein the TDP-43 protein is a mouse TDP-43 protein, and the mutation comprises K82A, R83A, K84A, K95A, K97A, and R98A point mutations;Attorney Docket No. 057766 / 640819(II) the mutation in the NES comprises a deletion of between positions 239 and 250, optionally wherein the TDP-43 protein is a mouse TDP-43 protein, and the mutation comprises a deletion between V239 and 1250; or(III) the mutation in the PLD comprises a deletion between positions 274 and 414, optionally wherein the TDP-43 protein is a mouse TDP-43 protein, and the mutation comprises a deletion between G274 and M414.

20. The non-human animal or non-human animal cell of any preceding claim, wherein the non-human animal is a mammal.

21. The non-human animal or non-human animal cell of claim 20, wherein the mammal is a rodent, optionally wherein the rodent is a rat or a mouse.

22. The non-human animal or non-human animal cell of claim 21, wherein the mammal is the mouse.

23. The non-human animal or non-human animal cell of any preceding claim, wherein RNA transcripts from the humanized endogenous STMN2 locus include a cryptic exon when TDP-43 is disrupted by mutations that lead to mislocalization and loss of TDP-43 splicing activity.

24. The non-human animal or non-human animal cell of claim 23, wherein RNA transcripts from the humanized endogenous STMN2 locus do not include the cryptic exon when wild type mouse TDP-43 is present.

25. The non-human animal of any preceding claim, wherein the non-human animal comprises the humanized endogenous STMN2 locus in its germline.

26. The non-human animal cell of any one of claims 1-24, wherein the non- human animal cell is a neuron, optionally wherein the non-human animal cell is an embryonic- stem-cell -derived motor neuron (ESMN), optionally wherein the ESMN is a mouse ESMN.

27. The non-human animal cell of any one of claims 1-24 and 26, wherein the cell is in vitro.Attorney Docket No. 057766 / 64081928. A non-human animal genome comprising a humanized endogenous STMN2 locus in which a segment of the endogenous Stmn2 locus has been deleted and replaced with a corresponding human STMN2 sequence.

29. A targeting vector for generating a humanized endogenous STMN2 locus in which a segment of the endogenous Stmn2 locus has been deleted and replaced with a corresponding human STMN2 sequence, wherein the targeting vector comprises an insert nucleic acid comprising the corresponding human STMN2 sequence flanked by a 5’ homology arm targeting a 5’ target sequence at the endogenous Stmn2 locus and a 3’ homology arm targeting a 3’ target sequence at the endogenous Stmn2 locus.

30. A nucleic acid comprising humanized non-human animal Stmn2 gene in which a segment of the non-human animal Stmn2 gene has been deleted and replaced with a corresponding human STMN2 sequence.

31. A method of assessing the effectiveness of an agent as a regulator of human S1MN2 messenger RNA (mRNA) splicing, comprising:(a) administering the agent to the non-human animal of any one of claims 1- 25 or the non-human animal cell of any one of claims 1-24, 26, and 27;(b) measuring the splicing of human STMN2 RNA transcripts encoded by the humanized endogenous STMN2 locus in the non-human animal or non-human animal cell; and(c) comparing the measured splicing of human STMN2 RNA transcripts in the non-human animal or non-human animal cell with the splicing of human STMN2 RNA transcripts in a control non-human animal or non-human animal cell, wherein splicing is measured as an amount of full-length STMN2 mRNA or as an amount of full-length stathmin-2 protein, wherein an increase in full-length STMN2 mRNA or an increase in full-length stathmin-2 protein in the non-human animal or non-human animal cell administered the agent relative to that in the control non-human animal or non-human animal cell indicates that the agent is effective as a regulator of human STMN2 mRNA splicing.

32. The method of claim 31, wherein the splicing is measured as the amount of full-length STMN2 mRNA encoded by the humanized endogenous STMN2 locus.Attorney Docket No. 057766 / 64081933. The method of claim 32, wherein the assessing further comprises measuring expression of a cryptic spliced STMN2 mRNA encoded by the humanized endogenous STMN2 locus.

34. The method of claim 32, wherein the assessing comprises measuring expression of a full-length STMN2 mRNA encoded by the humanized endogenous STMN2 locus and a cryptic spliced STMN2 mRNA encoded by the humanized endogenous STMN2 locus.

35. The method of claim 31, wherein the splicing is measured as the amount of full-length stathmin-2 protein.

36. The method of claim 35, wherein the assessing further comprises measuring expression of a truncated human stathmin-2 encoded by the humanized endogenous STMN2 locus.

37. The method of claim 35, wherein the assessing comprises measuring expression of the full-length human stathmin-2 encoded by the humanized endogenous STMN2 locus and the truncated human stathmin-2 encoded by the humanized endogenous STMN2 locus.

38. The method of any one of claims 31-37, wherein the agent is a TDP-43 variant.

39. The method of any one of claims 31-38, wherein the agent is an amyotrophic lateral sclerosis (ALS) therapeutic agent or a candidate ALS therapeutic agent.

40. A method of making the non-human animal of any one of claims 1-25, comprising:(I) (a) modifying the genome of a non-human animal embryonic stem(ES) cell to comprise in its genome the humanized endogenous STMN2 locus;(b) identifying or selecting the genetically modified non-human animal ES cell comprising in its genome the humanized endogenous STMN2 locus;(c) introducing the genetically modified non-human animal ES cell into a non-human animal host embryo; andAttorney Docket No. 057766 / 640819(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 comprise in its genome the humanized endogenous STMN2 locus;(b) selecting the genetically modified non-human animal one-cell stage embryo comprising in its genome the humanized endogenous STMN2 locus; and(c) gestating the genetically modified non-human animal one-cell stage embryo in a surrogate mother.

41. A method of making the non-human animal cell of any one of claims 1-25, comprising modifying the genome of a non-human animal cell to comprise in its genome the humanized endogenous STMN2 locus.

42. The method of claim 41, wherein the non-human animal cell is an embryonic stem (ES) cell.

43. The method of any one of claims 40-42, wherein the non-human animal is a mouse or a rat.

44. The method of claim 43, wherein the non-human animal is the mouse.

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