Compositions and methods for screening 4R tau targeting agents

Abstract

Tau reporter compositions, cells, and animals are provided that include a four-repeat (4R) tau isoform linked to a first reporter protein and a three-repeat (3R) tau isoform linked to a second reporter protein that is different from the first reporter protein. Methods are provided for making such cells and animals, and for using such cells and animals to assess the activity of tau-targeting reagents.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application No. 63 / 309,055, filed February 11, 2022, which is incorporated by reference herein in its entirety for all purposes.

[0002] Submitted as an xml file via EFS Web Reference to sequence listing The sequence listing in file 057766-590201.xml is 123 kilobytes, was created on February 3, 2023, and is incorporated herein by reference. [Background technology]

[0003] The human MAPT gene encodes six different isoforms of tau: 2N4R, 1N4R, 0N4R, 2N3R, 1N3R, 0N3R. The difference between transcripts containing 4R (4 repeat) tau versus 3R (3 repeat) tau is based on the inclusion (4R) or exclusion (3R) of exon 10. Humans normally express equal ratios of 3R and 4R tau. In some tauopathies, such as Alzheimer's disease, experimental evidence from postmortem brain tissue suggests that insoluble aggregates of tau are composed of 3R and 4R tau. In rarer tauopathy diseases, such as progressive supranuclear palsy (PSP) and cortical basal degeneration (CBD), 4R tau protein is the aggregating species of tau. The reasons underlying these different types of aggregates in disease are unclear.

[0004] One major challenge to date has been to develop assays for measuring the mRNA of different isoforms of tau. For example, the "R / repeat" domain of tau makes it difficult to design primer pairs and TaqMan probes for measuring 3R tau. With such a paucity of assays for measuring certain isoforms of tau, it is difficult to accurately test the specificity of reagents (e.g., siRNAs) that only reduce 4R tau and do not affect 3R tau versus reagents (e.g., siRNAs) that should reduce both. Summary of the Invention

[0005] Tau reporter compositions, cells, and animals are provided that include a four-repeat (4R) tau isoform linked to a first reporter protein and a three-repeat (3R) tau isoform linked to a second reporter protein that is different from the first reporter protein. Methods are provided for making such cells and animals, and for using such cells and animals to assess the activity of tau-targeting reagents.

[0006] In one aspect, a cell is provided that comprises a 4-repeat (4R) tau isoform linked to a first reporter protein and a 3-repeat (3R) tau isoform linked to a second reporter protein different from the first reporter protein. In some such cells, the cell comprises a first fusion protein comprising the 4R tau isoform fused to the first reporter protein and a second fusion protein comprising the 3R tau isoform fused to the second reporter protein. In some such cells, the cell comprises a first nucleic acid encoding the first fusion protein and a second nucleic acid encoding the second fusion protein, and the cell expresses the first fusion protein and the second fusion protein.

[0007] In some such cells, the cell comprises a first nucleic acid comprising a coding sequence for a 4R tau isoform and a coding sequence for a first reporter protein, and a second nucleic acid comprising a coding sequence for a 3R tau isoform and a coding sequence for a second reporter protein, and the cell expresses the 4R tau isoform, the 3R tau isoform, the first reporter protein, and the second reporter protein. In some such cells, the coding sequence for the 4R tau isoform and the coding sequence for the first reporter protein are separated by a coding sequence for a first 2A peptide, and the coding sequence for the 3R tau isoform and the coding sequence for the second reporter protein are separated by a coding sequence for a second 2A peptide. In some such cells, the first 2A peptide is a first P2A peptide, and the second 2A peptide is a second P2A peptide.

[0008] In some such cells, the first nucleic acid and the second nucleic acid are integrated into the genome of the cell. In some such cells, the cell comprises a viral vector comprising the first nucleic acid and the second nucleic acid. In some such cells, the viral vector is a lentiviral vector or an adeno-associated virus (AAV) vector. In some such cells, the cell comprises a first viral vector comprising the first nucleic acid and a second viral vector comprising the second nucleic acid. In some such cells, the first viral vector and the second viral vector are lentiviral vectors or adeno-associated virus (AAV) vectors.

[0009] In some such cells, the first reporter protein is a first fluorescent reporter protein and the second reporter protein is a second fluorescent reporter protein. In some such cells, the first reporter protein is eYFP and the second reporter protein is mCherry. In some such cells, the 4R tau isoform and the 3R tau isoform are human.

[0010] In some such cells, the 4R tau isoform is a 2N4R tau isoform. In some such cells, the 3R tau isoform is a 2N3R tau isoform. In some such cells, the 4R tau isoform is a 2N4R tau isoform and the 3R tau isoform is a 2N3R tau isoform. In some such cells, the 4R tau isoform comprises the sequence set forth in SEQ ID NO: 13 and the 3R tau isoform comprises the sequence set forth in SEQ ID NO: 14.

[0011] In some such cells, the 4R tau isoform is a 1N4R tau isoform. In some such cells, the 3R tau isoform is a 1N3R tau isoform. In some such cells, the 4R tau isoform is a 1N4R tau isoform and the 3R tau isoform is a 1N3R tau isoform. In some such cells, the 4R tau isoform comprises a sequence set forth in SEQ ID NO: 23, 27, 31, or 47, and the 3R tau isoform comprises a sequence set forth in SEQ ID NO: 24, 28, 32, or 49.

[0012] In some such cells, the cell is a mammalian cell. In some such cells, the cell is a human cell. In some such cells, the cell is an immortalized cell. In some such cells, the cell is a HEK293 cell.

[0013] In another aspect, a cell population comprising a plurality of any of the above cells is provided.

[0014] In another aspect, a non-human animal is provided that comprises a 4-repeat (4R) tau isoform linked to a first reporter protein and a 3-repeat (3R) tau isoform linked to a second reporter protein different from the first reporter protein. In some such non-human animals, the non-human animal comprises a first fusion protein comprising the 4R tau isoform fused to the first reporter protein and a second fusion protein comprising the 3R tau isoform fused to the second reporter protein. In some such non-human animals, the non-human animal comprises a first nucleic acid encoding the first fusion protein and a second nucleic acid encoding the second fusion protein, and the non-human animal expresses the first fusion protein and the second fusion protein.

[0015] In some such non-human animals, the non-human animal comprises a first nucleic acid comprising a coding sequence for a 4R tau isoform and a coding sequence for a first reporter protein, and a second nucleic acid comprising a coding sequence for a 3R tau isoform and a coding sequence for a second reporter protein, and the non-human animal expresses the 4R tau isoform, the 3R tau isoform, the first reporter protein, and the second reporter protein. In some such non-human animals, the coding sequence for the 4R tau isoform and the coding sequence for the first reporter protein are separated by a coding sequence for a first 2A peptide, and the coding sequence for the 3R tau isoform and the coding sequence for the second reporter protein are separated by a coding sequence for a second 2A peptide. In some such non-human animals, the first 2A peptide is a first P2A peptide, and the second 2A peptide is a second P2A peptide.

[0016] In some such non-human animals, the first nucleic acid and the second nucleic acid are integrated into the genome of the non-human animal. In some such non-human animals, the non-human animal comprises a viral vector comprising the first nucleic acid and the second nucleic acid. In some such non-human animals, the viral vector is a lentiviral vector or an adeno-associated viral (AAV) vector. In some such non-human animals, the non-human animal comprises a first viral vector comprising the first nucleic acid and a second viral vector comprising the second nucleic acid. In some such non-human animals, the first viral vector and the second viral vector are lentiviral vectors or adeno-associated viral (AAV) vectors.

[0017] In some such non-human animals, the first reporter protein is a first fluorescent reporter protein and the second reporter protein is a second fluorescent reporter protein. In some such non-human animals, the first reporter protein is eYFP and the second reporter protein is mCherry.

[0018] In some such non-human animals, the 4R tau isoform and the 3R tau isoform are human, and optionally the 4R tau isoform and the 3R tau isoform each include an R5L mutation, an L237V mutation, or a G243V mutation, or optionally the 4R tau isoform and the 3R tau isoform each include an N279K mutation, an L284R mutation, or a S285R mutation.

[0019] In some such non-human animals, the 4R tau isoform is a 2N4R tau isoform. In some such non-human animals, the 3R tau isoform is a 2N3R tau isoform. In some such non-human animals, the 4R tau isoform is a 2N4R tau isoform and the 3R tau isoform is a 2N3R tau isoform. In some such non-human animals, the 4R tau isoform comprises the sequence set forth in SEQ ID NO: 13 and the 3R tau isoform comprises the sequence set forth in SEQ ID NO: 14.

[0020] In some such non-human animals, the 4R tau isoform is a 1N4R tau isoform. In some such non-human animals, the 3R tau isoform is a 1N3R tau isoform. In some such non-human animals, the 4R tau isoform is a 1N4R tau isoform and the 3R tau isoform is a 1N3R tau isoform. In some such non-human animals, the 4R tau isoform comprises the sequence set forth in SEQ ID NO: 23, 27, 31, or 47, and the 3R tau isoform comprises the sequence set forth in SEQ ID NO: 24, 28, 32, or 49.

[0021] In some such non-human animals, the non-human animal is a mammal. In some such non-human animals, the non-human animal is a rodent. In some such non-human animals, the non-human animal is a mouse. In some such non-human animals, the non-human animal is a rat.

[0022] In some such non-human animals, the 4R tau isoform, the first reporter protein, the 3R tau isoform, and the second reporter protein are expressed in neurons of the central nervous system of the non-human animal. In some such non-human animals, the non-human animal comprises filamentous tau inclusions.

[0023] In another aspect, methods of assessing the activity of a tau targeting reagent are provided. Some such methods include (a) administering a tau targeting reagent to any of the cells described above, and (b) assessing the activity of the tau targeting reagent in the cell. In some such methods, the activity of the tau targeting reagent is assessed relative to a control cell that has not been administered the tau targeting reagent, or is assessed relative to before administration of the tau targeting reagent.

[0024] In some such methods, the evaluating comprises measuring one or more of 4R tau messenger RNA expression, a first reporter protein messenger RNA expression, and a second reporter protein messenger RNA expression. In some such methods, the evaluating comprises measuring 4R tau isoform messenger RNA expression and a second reporter protein messenger RNA expression, where a greater relative decrease in 4R tau isoform messenger RNA expression compared to the second reporter protein messenger RNA expression after administration of the tau targeting reagent to the cell indicates that the tau targeting reagent is a 4R-preferential tau targeting reagent, and optionally, a decrease of at least 70% in 4R tau isoform messenger RNA expression and a decrease of no more than 30% in the second reporter protein messenger RNA expression after administration of the tau targeting reagent to the cell indicates that the tau targeting reagent is a 4R-preferential tau targeting reagent. In some such methods, the evaluating includes measuring a first reporter protein messenger RNA expression and a second reporter protein messenger RNA expression, wherein a greater relative decrease in the first reporter protein messenger RNA expression compared to the second reporter protein messenger RNA expression after administration of the tau targeting reagent to the cell indicates that the tau targeting reagent is a 4R-preferential tau targeting reagent, and optionally, a decrease of at least 70% in the first reporter protein messenger RNA expression and a decrease of no more than 30% in the second reporter protein messenger RNA expression after administration of the tau targeting reagent to the cell indicates that the tau targeting reagent is a 4R-preferential tau targeting reagent.

[0025] In some such methods, the evaluating comprises measuring one or more of a first reporter protein expression and a second reporter protein expression. In some such methods, the evaluating comprises measuring a first reporter protein expression and a second reporter protein expression, where a greater relative decrease in the first reporter protein expression compared to the second reporter protein expression after administration of the tau targeting reagent to the cell indicates that the tau targeting reagent is a 4R-preferential tau targeting reagent, and optionally, a decrease of at least 70% in the first reporter protein expression and a decrease of no more than 30% in the second reporter protein expression after administration of the tau targeting reagent to the cell indicates that the tau targeting reagent is a 4R-preferential tau targeting reagent.

[0026] In some such methods, the first reporter protein is a first fluorescent reporter protein and the second reporter protein is a second fluorescent reporter protein, and the assessing in step (b) comprises immunofluorescence staining or flow cytometry. In some such methods, the assessing in step (b) comprises assessing tau hyperphosphorylation or tau aggregation.

[0027] In some such methods, the tau targeting reagent is an RNAi agent or an antisense oligonucleotide. In some such methods, the tau targeting reagent is an intrabody. In some such methods, the tau targeting reagent is a nuclease agent. In some such methods, the nuclease agent comprises a Cas protein and a guide RNA designed to target a guide RNA target sequence within the tau coding sequence.

[0028] In another aspect, a method of evaluating the activity of a tau-targeting reagent in vivo is provided. Some such methods include (a) administering a tau-targeting reagent to any of the non-human animals described above, and (b) evaluating the activity of the tau-targeting reagent in the non-human animal. In some such methods, the activity of the tau-targeting reagent is evaluated relative to a control non-human animal that has not been administered the tau-targeting reagent, or is evaluated relative to before administration of the tau-targeting reagent.

[0029] In some such methods, the evaluating comprises measuring one or more of 4R tau messenger RNA expression, a first reporter protein messenger RNA expression, and a second reporter protein messenger RNA expression. In some such methods, the evaluating comprises measuring 4R tau isoform messenger RNA expression and a second reporter protein messenger RNA expression, where a greater relative decrease in 4R tau isoform messenger RNA expression compared to the second reporter protein messenger RNA expression after administration of the tau targeting reagent to the non-human animal indicates that the tau targeting reagent is a 4R-preferential tau targeting reagent, and optionally, a decrease of at least 70% in 4R tau isoform messenger RNA expression and a decrease of no more than 30% in the second reporter protein messenger RNA expression after administration of the tau targeting reagent to the non-human animal indicates that the tau targeting reagent is a 4R-preferential tau targeting reagent. In some such methods, the evaluating includes measuring a first reporter protein messenger RNA expression and a second reporter protein messenger RNA expression, wherein a greater relative decrease in the first reporter protein messenger RNA expression compared to the second reporter protein messenger RNA expression after administration of the tau targeting reagent to the non-human animal indicates that the tau targeting reagent is a 4R-preferential tau targeting reagent, and optionally, a decrease of at least 70% in the first reporter protein messenger RNA expression and a decrease of 30% or less in the second reporter protein messenger RNA expression after administration of the tau targeting reagent to the non-human animal indicates that the tau targeting reagent is a 4R-preferential tau targeting reagent.

[0030] In some such methods, the evaluating comprises measuring one or more of a first reporter protein expression and a second reporter protein expression. In some such methods, the evaluating comprises measuring a first reporter protein expression and a second reporter protein expression, where a greater relative decrease in the first reporter protein expression compared to the second reporter protein expression after administration of the tau targeting reagent to the non-human animal indicates that the tau targeting reagent is a 4R-preferential tau targeting reagent, and optionally, a decrease of at least 70% in the first reporter protein expression and a decrease of no more than 30% in the second reporter protein expression after administration of the tau targeting reagent to the non-human animal indicates that the tau targeting reagent is a 4R-preferential tau targeting reagent.

[0031] In some such methods, the first reporter protein is a first fluorescent reporter protein and the second reporter protein is a second fluorescent reporter protein, and the assessing in step (b) comprises immunofluorescence staining or flow cytometry. In some such methods, the assessing in step (b) comprises assessing tau hyperphosphorylation or tau aggregation.

[0032] In some such methods, the tau targeting reagent is an RNAi agent or an antisense oligonucleotide. In some such methods, the tau targeting reagent is an intrabody. In some such methods, the tau targeting reagent is a nuclease agent. In some such methods, the nuclease agent comprises a Cas protein and a guide RNA designed to target a guide RNA target sequence within the tau coding sequence.

[0033] In some such methods, the evaluating is in neurons of the central nervous system of a non-human animal.

[0034] In another aspect, compositions are provided, some such compositions comprising (a) a four-repeat (4R) tau isoform linked to a first reporter protein and a three-repeat (3R) tau isoform linked to a second reporter protein different from the first reporter protein, or (b) a first nucleic acid encoding the 4R tau isoform linked to a first reporter protein and a second nucleic acid encoding the 3R tau isoform linked to a second reporter protein.

[0035] In some such compositions, the composition comprises a first fusion protein comprising a 4R tau isoform fused to a first reporter protein and a second fusion protein comprising a 3R tau isoform fused to a second reporter protein, or the first nucleic acid encodes the first fusion protein and the second nucleic acid encodes the second fusion protein. In some such compositions, the first nucleic acid comprises a coding sequence for a 4R tau isoform separated by a coding sequence for a first 2A peptide and a coding sequence for a first reporter protein, and the second nucleic acid comprises a coding sequence for a 3R tau isoform separated by a coding sequence for a second 2A peptide and a coding sequence for a second reporter protein. In some such compositions, the first 2A peptide is a first P2A peptide and the second 2A peptide is a second P2A peptide.

[0036] In some such compositions, the first nucleic acid and the second nucleic acid are in a viral vector. In some such compositions, the viral vector is a lentiviral vector or an adeno-associated viral (AAV) vector. In some such compositions, the first nucleic acid is in a first viral vector and the second nucleic acid is in a second viral vector. In some such compositions, the first viral vector and the second viral vector are a lentiviral vector or an adeno-associated viral (AAV) vector.

[0037] In some such compositions, the first reporter protein is a first fluorescent reporter protein and the second reporter protein is a second fluorescent reporter protein. In some such compositions, the first reporter protein is eYFP and the second reporter protein is mCherry.

[0038] In some such compositions, the 4R tau isoform and the 3R tau isoform are human.

[0039] In some such compositions, the 4R tau isoform is a 2N4R tau isoform. In some such compositions, the 3R tau isoform is a 2N3R tau isoform. In some such compositions, the 4R tau isoform is a 2N4R tau isoform and the 3R tau isoform is a 2N3R tau isoform. In some such compositions, the 4R tau isoform comprises the sequence set forth in SEQ ID NO: 13 and the 3R tau isoform comprises the sequence set forth in SEQ ID NO: 14.

[0040] In some such compositions, the 4R tau isoform is a 1N4R tau isoform. In some such compositions, the 3R tau isoform is a 1N3R tau isoform. In some such compositions, the 4R tau isoform is a 1N4R tau isoform and the 3R tau isoform is a 1N3R tau isoform. In some such compositions, the 4R tau isoform comprises a sequence set forth in SEQ ID NO: 23, 27, 31, or 47, and the 3R tau isoform comprises a sequence set forth in SEQ ID NO: 24, 28, 32, or 49.

[0041] In another aspect, a cell is provided comprising any of the above compositions. In another aspect, a non-human animal is provided comprising any of the above compositions.

[0042] In another aspect, methods of making any of the above cells are provided. Some such methods include introducing into the cell a four-repeat (4R) tau isoform linked to a first reporter protein and a three-repeat (3R) tau isoform linked to a second reporter protein, or introducing into the cell a first nucleic acid encoding a 4R tau isoform linked to a first reporter protein and a second nucleic acid encoding a 3R tau isoform linked to a second reporter protein.

[0043] In another aspect, methods of making any of the above non-human animals are provided. Some such methods include administering to the non-human animal a four-repeat (4R) tau isoform linked to a first reporter protein and a three-repeat (3R) tau isoform linked to a second reporter protein, or administering to the non-human animal a first nucleic acid encoding a 4R tau isoform linked to a first reporter protein and a second nucleic acid encoding a 3R tau isoform linked to a second reporter protein. [Brief description of the drawings]

[0044] [Figure 1] The different 4-repeat (4R) and 3-repeat (3R) tau isoforms are shown. Exon 10 encodes the "R2 domain" that distinguishes 4R tau from 3R tau. [Diagram 2] Relative expression of 2N4R-eYFP and 2N3R-mCherry mRNA in the dual reporter 2N4R-eYFP / 2N3R-mCherry cell line using eYFP-specific and mCherry-specific TaqMan probes is shown. [Diagram 3] Immunofluorescence images from 2N4R-eYFP / 2N3R-mCherry cell line clones C2, C3, and C5 are shown. The top row shows eYFP, the middle row shows mCherry, and the third row shows a merged image of eYFP and mCherry. [Figure 4]Immunofluorescence images from untreated 2N4R-eYFP / 2N3R-mCherry cell lines (first column) or cell lines treated with 10 nM total tau siRNA (targeting both 3R and 4R tau) or 10 nM mCherry siRNA (HAIJE-000013) are shown. [Diagram 5] Immunofluorescence images from untreated 2N4R-eYFP / 2N3R-mCherry cell lines (first column) or cell lines treated with 10 nM total YFP siRNA (P-002048-01) or 10 nM mCherry siRNA (HAIJE-000015 or HAIJE-000017) are shown. [Figure 6] Possible criteria for selecting "4R-specific" and "4R-preferential" siRNAs are presented. [Figure 7] Percent RNA knockdown as measured by 4R tau and mCherry TaqMan assays for 65 candidate 4R tau siRNAs is shown. [Figure 8] 1 shows the correlation between percent 4R tau knockdown and percent eYFP knockdown as measured by TaqMan. [Figure 9A] TaqMan qPCR data of expression levels of 4R tau and mCherry (3R ​​tau surrogate) after treatment of HEK293-2N4R-YFP+2N3R-mCherry dual reporter cells with 1 nM of 65 different GalNAc-conjugated siRNAs produced by walking through exon 10 of the MAPT gene. Lipofectamine was used as a negative control. Total tau siRNA was also used as a control. [Figure 9B] Relative 4R tau and mCherry expression for 13 4R-preferring siRNAs (1 nM and 0.1 nM) is shown. Lipofectamine was used as a negative control. Two total tau siRNAs were also used as controls. [Figure 10A] Shown is an IC50 graph for two candidate 4R siRNAs using a 3R / 4R dual reporter. siRNAs were tested at eight different concentrations. [Figure 10B] Shown is an IC50 graph for two candidate 4R siRNAs using a 3R-mCherry reporter. siRNAs were tested at eight different concentrations. [Figure 10C] Shown is an IC50 graph for two candidate 4R siRNAs using the 4R-YFP reporter. siRNAs were tested at eight different concentrations. [Figure 11A] Shown is an IC50 graph for two candidate 4R siRNAs using a 3R / 4R dual reporter. siRNAs were tested at eight different concentrations. [Figure 11B] Shown is an IC50 graph for two candidate 4R siRNAs using a 3R-mCherry reporter. siRNAs were tested at eight different concentrations. [Figure 11C] Shown is an IC50 graph for two candidate 4R siRNAs using the 4R-YFP reporter. siRNAs were tested at eight different concentrations. [Figure 12] Validation of four 4R-preferential siRNAs in 4R tau protein knockdown using dot blotting with 4R- and 3R-specific antibodies. Validation of antibodies with recombinant tau 1N3R or 1N4R proteins to confirm specificity is shown on the left, and testing with lysates from siRNA-treated dual reporter cells is shown on the right. The experimental set-up is shown at the top. [Figure 13] Validation of four 4R-preferential siRNAs in 4R tau protein knockdown using total tau and 3R tau ELISAs is shown. In the left panel, total soluble tau was measured using a commercially available total tau ALPHALISA (Perkin Elmer, Cat. No.: AL271C) according to the manufacturer's instructions. In the right panel, 4R tau protein was measured using a PathScan ELISA kit from Cell Signaling Technologies (Cat. No.: 29443). The experimental setup is shown at the top. [Figure 14]RT-qPCR data of total tau and 4R tau expression levels on RNA extracted from the brains of humanized MAPT mice treated with 4R tau siRNA are shown. Values ​​are graphed as relative expression (½ΔΔCt) to the naive group and normalized to mouse GAPDH. [Figure 15] Schematics of 1N4R Tau(R5L)-P2A-eYFP and 1N3R Tau(R5L)-P2A-mCherry, 1N4R Tau(L237V)-P2A-eYFP, and 1N3R Tau(L237V)-P2A-mCherry, as well as 1N4R Tau(G243V)-P2A-eYFP and 1N3R Tau(G243V)-P2A-mCherry for use in generating mouse models for in vivo testing of 4R Tau targeting reagents. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] definition The terms "protein," "polypeptide," and "peptide," as used interchangeably herein, include polymeric forms of amino acids of any length, including coded and non-coded amino acids, and amino acids that have been chemically or biochemically modified or derivatized. The terms also include modified polymers, such as polypeptides having modified peptide backbones. The term "domain" refers to any portion of a protein or polypeptide having a specific function or structure.

[0046] Proteins are said to have an "N-terminus" (amino terminus) and a "C-terminus" (carboxy or carboxyl terminus). The term "N-terminus" refers to the beginning of a protein or polypeptide, which terminates with an amino acid having a free amine group (-NH2). The term "C-terminus" refers to the end of an amino acid chain (protein or polypeptide) terminated by a free carboxyl group (-COOH).

[0047] 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. These include single-stranded, double-stranded, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers that contain purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural, or derivatized nucleotide bases.

[0048] Nucleic acids are said to have a "5' end" and a "3' end" because mononucleotides react to make oligonucleotides in a manner in which the 5' phosphate of one mononucleotide pentose ring is attached in one direction to the 3' oxygen of its neighbor via a phosphodiester bond. 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 the 5' phosphate of another mononucleotide pentose ring. A nucleic acid sequence, even if it is internal to a larger oligonucleotide, may also be said to have 5' and 3' ends. In either a linear or circular DNA molecule, separate elements are referred to as being "upstream" or "downstream" 5' or 3' elements.

[0049] The term "genomically integrated" refers to a nucleic acid that has been introduced into a cell such that the nucleotide sequence is integrated into the genome of the cell. Any protocol can be used for stable integration of a nucleic acid into the genome of a cell.

[0050] The term "expression vector" or "expression construct" or "expression cassette" refers to a recombinant nucleic acid containing a desired coding sequence operably linked to appropriate nucleic acid sequences necessary for expression of the operably linked coding sequence in a particular host cell or organism. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), a ribosome binding site, and other sequences. Eukaryotic cells are generally well known to utilize promoters, enhancers, termination and polyadenylation signals, and some elements may be deleted and others added without sacrificing the required expression.

[0051] The term "targeting vector" refers to a recombinant nucleic acid that can be introduced into a target location in the genome of a cell by homologous recombination, non-homologous end joining mediated ligation, or any other recombination means.

[0052] 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 bacteria, viruses, cells, or other components that may normally be present in situ, up to and including substantially pure preparations of cells, tissues, proteins, and nucleic acids. The term "isolated" also includes cells, tissues, proteins, and nucleic acids that have no naturally occurring counterpart and have been chemically synthesized and thereby substantially free from contaminating other cells, tissues, proteins, and nucleic acids, or that have been separated or purified from most other components (e.g., cellular or biological components) that naturally accompany them (e.g., other cellular proteins, nucleic acids, or cellular or extracellular components).

[0053] The term "wild-type" includes entities that have a structure and / or activity as found in a normal state or context (as opposed to mutant, diseased, altered, etc.). Wild-type genes and polypeptides often exist in multiple alternative forms (e.g., alleles).

[0054] The term "endogenous sequence" refers to a nucleic acid sequence that is naturally occurring in a rat cell or in a rat. For example, an endogenous MAPT sequence in a human cell refers to the native MAPT sequence that is naturally occurring at the MAPT locus in a human cell.

[0055] An "exogenous" molecule or sequence includes a molecule or sequence that is not normally present in a cell in that form. Normal presence includes presence in relation to a particular developmental stage and environmental conditions of the cell. An exogenous molecule or sequence may include, for example, a mutated version of a corresponding endogenous sequence in a cell, such as a humanized version of an endogenous sequence, or may include a sequence that corresponds to an endogenous sequence within a cell, but in a different form (i.e., not within a chromosome). In contrast, an endogenous molecule or sequence includes a molecule or sequence that is normally present in that form, in a particular cell, at a particular developmental stage, and under particular environmental conditions.

[0056] 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 respect to a segment of a nucleic acid or a segment of a protein indicates that the nucleic acid or protein comprises two or more subsequences that are not found in the same relationship (e.g., linked together) to each other in nature. As an example, a "heterologous" region of a nucleic acid vector is a 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 can include a coding sequence adjacent to a sequence that is not found in association with the coding sequence in nature. Similarly, 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 tagged protein). Similarly, a nucleic acid or protein can include a heterologous label or a heterologous secretion or localization sequence.

[0057] "Codon optimization" includes the process of modifying a nucleic acid sequence for enhanced expression in a particular host cell by taking advantage of the degeneracy of codons, as indicated by the diversity of combinations of three base pairs of codons that specify amino acids, and generally by replacing at least one codon of the native sequence with a codon that is more 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 tau protein can be modified to replace a codon that has a higher frequency of usage in a given prokaryotic or eukaryotic cell, including bacterial cells, yeast cells, human cells, non-human cells, mammalian cells, rodent cells, mouse cells, rat cells, hamster cells, or any other host cell, as compared to the naturally occurring nucleic acid sequence. Codon usage tables are readily available, for example, in the "Codon Usage Database." These tables can be applied in a variety of ways. See Nakamura et al. (2000) Nucleic Acids Res. 28(1):292, which is incorporated herein by reference in its entirety for all purposes. Computer algorithms are also available for codon optimization of a particular sequence for expression in a particular host (see, eg, Gene Forge).

[0058] The term "locus" refers to the specific location of a gene (or sequence of interest), DNA sequence, polypeptide coding sequence, or position on a chromosome of the genome of an organism. For example, "MAPT locus" may refer to the specific location of the MAPT gene, MAPT DNA sequence, tau coding sequence, or MAPT locus on a chromosome of the genome of an organism in which such sequence is identified to reside. "MAPT locus" may include regulatory elements of the MAPT gene, including, for example, enhancers, promoters, 5' and / or 3' untranslated regions (UTRs), or combinations thereof.

[0059] The term "gene" refers to a DNA sequence in a chromosome that, when present in nature, may contain at least one coding region and at least one non-coding region. A DNA sequence in a chromosome that encodes a product (e.g., but not limited to, an RNA product and / or a polypeptide product) may include coding regions interrupted by non-coding introns, as well as sequences located adjacent to the coding region at both the 5' and 3' ends, such that a gene corresponds to a full-length RNA (including 5' and 3' untranslated sequences). In addition, other non-coding sequences, including regulatory sequences (e.g., but not limited to, promoters, enhancers, and transcription factor binding sites), polyadenylation signals, internal ribosome entry sites, silencers, insulating sequences, and matrix attachment regions, may also be present in a gene. These sequences may be proximal (e.g., but not limited to, within 10 kb) or distant from the coding region of the gene, and they affect the level or rate of transcription and translation of the gene.

[0060] The term "allele" refers to variant forms of a gene. Some genes have a variety of different forms located at the same position, or locus, on a chromosome. Diploid organisms have two alleles at each locus. Each pair of alleles represents a genotype at a particular gene locus. A genotype is described as homozygous if there are two identical alleles at a particular locus, and heterozygous if the two alleles are different.

[0061] The "coding region" or "coding sequence" of a gene consists of the portion of the gene's DNA or RNA, composed of exons, that codes for a protein. This region begins with an initiation codon at the 5' end and ends with a stop codon at the 3' end.

[0062] A "promoter" is a regulatory region of DNA that typically includes a TATA box that can direct RNA polymerase II to begin RNA synthesis at the appropriate transcription initiation site for a particular polynucleotide sequence. In some cases, a promoter may additionally include other regions that affect the rate of transcription initiation. The promoter sequences disclosed herein regulate the transcription of an operably linked polynucleotide. The promoter may be active in one or more cell types disclosed herein (e.g., mouse cells, rat cells, pluripotent cells, one-cell stage embryos, differentiated cells, or combinations thereof). The promoter may 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, which is incorporated herein by reference in its entirety for all purposes.

[0063] "Operable linkage" or "operably linked" includes the juxtaposition of two or more components (e.g., a promoter and another sequence element) that allows for both components to function normally and for at least one of the components to mediate a function on at least one of the other components. For example, a promoter may be operably linked to a coding sequence if the promoter controls the level of transcription of the coding sequence depending on the presence or absence of one or more transcriptional regulatory factors. Operable linkage may include such sequences being in close proximity to each other, or acting in trans (e.g., regulatory sequences may act at a distance to control transcription of the coding sequence).

[0064] "Sequence identity" or "identity" in the context of two polynucleotide or polypeptide sequences refers to the residues of the two sequences that are the same when aligned for maximum correspondence over a particular comparison window. When using percentages of sequence identity with respect to proteins, residue positions that are not identical often differ by conservative amino acid substitutions, in which an amino acid residue is replaced with another amino acid residue that has similar chemical properties (e.g., charge or hydrophobicity), and thus does not change the functional properties of the molecule. When sequences differ by 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 conservative substitutions as partial mismatches rather than complete mismatches, thereby increasing the percentage sequence identity. Thus, for example, conservative substitutions are given a score of 0 to 1, where identical amino acids are given a score of 1 and non-conservative substitutions are given a score of 0. Scoring of conservative substitutions is calculated, for example, as implemented in the program PC / GENE (Intelligenetics, Mountain View, Calif.).

[0065] "Percentage of sequence identity" includes values ​​determined by comparing two optimally aligned sequences (maximum number of perfectly matching residues) over a comparison window, where the portion of the polynucleotide sequence in the comparison window may contain additions or deletions (i.e., gaps) when compared to a reference sequence (not including additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Unless otherwise specified (e.g., the shorter sequence includes a concatenated non-homologous sequence), the comparison window is the full length of the shorter of the two sequences being compared.

[0066] Unless otherwise indicated, sequence identity / similarity values ​​include values ​​obtained using GAP version 10 with the following parameters: % identity and % similarity for nucleotide sequences using a GAP weight of 50 and a length weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for amino acid sequences using a GAP weight of 8 and a length weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program thereof. "Equivalent programs" include any sequence comparison program that produces alignments that have identical nucleotide or amino acid residue matches and identical percent sequence identity for any two sequences at issue when compared to corresponding alignments produced by GAP version 10.

[0067] The term "conservative amino acid substitution" refers to the replacement of an amino acid normally present in a sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the replacement of a non-polar (hydrophobic) residue, such as isoleucine, valine, or leucine, with another non-polar residue. Similarly, examples of conservative substitutions include the replacement of one polar (hydrophilic) residue with another, such as between arginine and lysine, between glutamine and asparagine, or between glycine and serine. Additionally, the replacement of a basic residue, such as lysine, arginine, or histidine, with another, or the replacement of one acidic residue, such as aspartic acid or glutamic acid, with another, are additional examples of conservative substitutions. Examples of non-conservative substitutions include the 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 the substitution of a polar residue for a non-polar residue. Exemplary amino acid categories are summarized below.

[0068] [Table 1]

[0069] A "homologous" sequence (e.g., a nucleic acid sequence) includes a sequence that is identical or substantially similar to a known reference sequence, e.g., 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 and paralogous sequences. For example, homologous genes typically derive from a common ancestral DNA sequence either through a speciation event (orthologous genes) or a gene duplication event (paralogous genes). "Orthologous" genes include genes in different species that have evolved from a common ancestral gene by speciation. Orthologs typically retain the same function during evolution. "Paralogous" genes include genes that are related by duplication within a genome. Paralogs can evolve new functions during evolution.

[0070] The term "in vitro" includes an artificial environment and processes or reactions that occur within an artificial environment (e.g., a test tube or an isolated cell or cell line). The term "in vivo" includes a natural environment (e.g., an organism or body, or a cell or tissue within an organism or body) and processes or reactions that occur within a natural environment. The term "ex vivo" includes cells removed from an individual's body and processes or reactions that occur within such cells.

[0071] A composition or method that "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 claims are to be interpreted to include the specific elements recited in the claims, as well as elements that do not materially affect the basic and novel characteristics of the claimed invention. Thus, the term "consisting essentially of" when used in the claims of the present invention is not intended to be interpreted as equivalent to "comprising."

[0072] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes examples where the event or circumstance occurs as well as examples where it does not occur.

[0073] The specification of a range of values ​​includes every integer within or defining that range, and every subrange defined by integers within that range.

[0074] Unless otherwise clear from the context, the term "about" encompasses values ​​that are ±5% of the stated value.

[0075] The term "and / or" refers to and includes 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").

[0076] The term "or" refers to any one member of a particular list, and also includes any combination of members of that list.

[0077] The singular 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.

[0078] Statistically significant means p≦0.05. (Mode for carrying out the invention)

[0079] I. Overview Tau reporter compositions, cells, and animals are provided that include a four-repeat (4R) tau isoform linked to a first reporter protein and a three-repeat (3R) tau isoform linked to a second reporter protein that is different from the first reporter protein. Methods are provided for making such cells and animals, and for using such cells and animals to assess the activity of tau-targeting reagents.

[0080] The human MAPT gene encodes six different isoforms of tau: 2N4R, 1N4R, 0N4R, 2N3R, 1N3R, 0N3R. The difference between transcripts containing 4R (4 repeat) tau versus 3R (3 repeat) tau is based on the inclusion (4R) or exclusion (3R) of exon 10. Humans normally express equal ratios of 3R and 4R tau. In some tauopathies, such as Alzheimer's disease, experimental evidence from postmortem brain tissue suggests that insoluble aggregates of tau are composed of 3R and 4R tau. In rarer tauopathy diseases, such as progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD), 4R tau protein is the aggregating species of tau. The reasons underlying these different types of aggregates in disease are unclear. Therapies targeting total tau (e.g., 3R+4R tau) versus only 4R tau may have beneficial effects in different disease states.

[0081] One major challenge to date has been to develop assays for measuring the mRNA of different isoforms of tau. For example, the "R / repeat" domain of tau makes it difficult to design primer pairs and TaqMan probes for measuring 3R tau. With such a paucity of assays for measuring certain isoforms of tau, it is difficult to accurately test the specificity of reagents (e.g., siRNAs) that only reduce 4R tau and do not affect 3R tau versus reagents (e.g., siRNAs) that should reduce both (e.g., total tau targeting strategies).

[0082] Tau reporters, tau reporter cells, and tau reporter non-human animals can identify compounds (e.g., siRNAs or gRNAs) that target total tau versus compounds that specifically target 4R tau. Until now, screening for 4R-tau specific reagents such as siRNAs has not been possible due to the lack of protein and mRNA assays to specifically measure 3R tau.

[0083] II. Compositions Comprising Tau3R and Tau4R Reporters Provided herein are compositions comprising a tau 3R reporter and a tau 4R reporter that can be used in assays to distinguish between tau targeting reagents that specifically or selectively target 4R tau and reagents that target both 4R tau and 3R tau.

[0084] Tau is an intracellular microtubule-associated protein that binds and stabilizes microtubules. It is primarily expressed in neurons. Tau plays a role in stabilizing neuronal microtubules and thus promoting axonal elongation. In Alzheimer's disease (AD) and a family of related neurodegenerative disorders called tauopathies, tau protein is abnormally hyperphosphorylated and aggregates into bundles of filaments (paired helical filaments) that appear as neurofibrillary tangles. Tauopathies are a heterogeneous group of neurodegenerative conditions characterized by abnormal tau deposition in the brain. Tau tangle burden positively correlates with cognitive decline and dying neurons. In Alzheimer's disease (AD), extracellular amyloid beta induces aggregation of both 3R and 4R isoforms of tau (secondary tauopathy), leading to cognitive decline. In primary tauopathies, tau isoforms differentially aggregate due to unknown causes / triggers. For example, 4R aggregates are associated with progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD), whereas 3R aggregates are associated with Pick's disease.

[0085] Tau is encoded by the MAPT gene, which contains 16 exons. Exons 0 and 1 code for the 5' untranslated region (UTR) of MAPT mRNA, while exon 14 codes for part of the 3'UTR. Exons 4a, 6, and 8 are transcribed only in peripheral tissues. Exons 2 and 3 code for a 29 amino acid residue insert near the amino terminus (N1 and N2), and exons 9-12 code for microtubule-binding domain repeats near the carboxyl terminus (R1-R4). Alternative splicing of MAPT results in six isoforms ranging from 352 to 441 amino acids. Tau isoforms are diverse and named based on the number of amino-terminal inserts (0N, 1N, and 2N) and the number of carboxyl-terminal microtubule-binding domain repeats (3R and 4R). Depending on splicing, the repeat domain of tau protein has either three or four repeat regions that constitute the aggregation-prone core of the protein, often referred to as the repeat domain (RD). The three 4-repeat (4R) tau isoforms are 2N4R, 1N4R, and 0N4R. The three 3-repeat (3R) tau isoforms are 2N3R, 1N3R, and 0N3R.

[0086] The compositions described herein may include a 4R tau isoform linked to a first reporter protein and a 3R tau isoform linked to a second reporter protein different from the first reporter protein. Alternatively, the compositions described herein may include a first nucleic acid encoding a 4R tau isoform linked to a first reporter protein and a second nucleic acid encoding a 3R tau isoform linked to a second reporter protein. By "linked" it is meant that the tau isoform and the linked reporter protein are part of the same protein (i.e., a fusion protein) or expressed from the same messenger RNA. In a fusion protein, the tau isoform and the reporter protein may be fused directly to each other or fused to each other via a linker. In a specific example, the tau isoform and the linked reporter protein are fused to each other via a linker. In a first example, the composition may include a first fusion protein comprising a 4R tau isoform fused to a first reporter protein and a second fusion protein comprising a 3R tau isoform fused to a second reporter protein different from the first reporter protein. For example, the composition may include a first nucleic acid (e.g., an expression cassette) encoding the first fusion protein and a second nucleic acid (e.g., an expression cassette) encoding the second fusion protein. In a second example, a composition may comprise a first nucleic acid (e.g., an expression cassette) comprising a coding sequence for a 4R tau isoform and a coding sequence for a first reporter protein, and a second nucleic acid (e.g., an expression cassette) comprising a coding sequence for a 3R tau isoform and a coding sequence for a second reporter protein different from the first reporter protein, wherein the coding sequence for the 4R tau isoform and the coding sequence for the first reporter protein are separated by the coding sequence for a first 2A peptide, and the coding sequence for the 3R tau isoform and the coding sequence for the second reporter protein are separated by the coding sequence for a second 2A peptide.

[0087] 2A peptides are small "self-cleaving" peptides, generally 18-22 amino acids in length, that generate equimolar levels of multiple genes from the same mRNA. The ribosome skips synthesis of the glycyl-prolyl peptide bond at the C-terminus of the 2A peptide, causing a "cleavage" between the 2A peptide and the peptide immediately downstream. See, for example, Kim et al. (2011) PLoS One 6(4):e18556, incorporated herein by reference in its entirety for all purposes. The "cleavage" occurs between the glycine and proline residues at the C-terminus, the upstream cistron adds a few residues to its end, and the downstream cistron starts with a proline. As a result, the "cleaved" downstream peptide has a proline at its N-terminus. 2A-mediated cleavage is a universal phenomenon in all eukaryotic cells. 2A peptides have been identified from picornaviruses, insect viruses, and type C rotaviruses. See, e.g., Szymczak et al. (2005) Expert Opin. Biol. Ther. 5(5):627-638, which is incorporated by reference in its entirety for all purposes. Examples of 2A peptides that can be used include Thoseaasigna virus 2A (T2A), porcine teschovirus-1 2A (P2A), equine rhinitis A virus (ERAV) 2A (E2A), and FMDV 2A (F2A). Exemplary T2A, P2A, E2A, and F2A sequences include: T2A (EGRGSLLTCGDVEENPGP, SEQ ID NO: 9), P2A (ATNFSLLKQAGDVEENPGP, SEQ ID NO: 10), E2A (QCTNYALLKLAGDVESNPGP, SEQ ID NO: 11), and F2A (VKQTLNFDLLKLAGDVESNPGP, SEQ ID NO: 12). A GSG residue can be added to the 5' end of any of these peptides to improve cleavage efficiency. In a specific example, P2A is used.

[0088] In compositions comprising a first nucleic acid and a second nucleic acid, such nucleic acid may be RNA (e.g., messenger RNA (mRNA)) or DNA, may be single-stranded or double-stranded, and may be linear or circular. The DNA may be part of a vector, such as an expression vector or a targeting vector. The vector may also be a viral vector, such as an adenovirus, an adeno-associated virus, a lentivirus, and a retrovirus vector.

[0089] Optionally, the nucleic acid can be codon-optimized for efficient translation into a protein in a particular cell or organism. For example, the nucleic acid can be modified to replace codons that have a higher frequency of usage in human cells, non-human cells, mammalian cells, rodent cells, mouse cells, rat cells, bacterial cells, yeast cells, or any other host cell of interest, as compared to the naturally occurring polynucleotide sequence.

[0090] The compositions described herein may be in vitro, in a cell (e.g., an embryonic stem cell), or in a non-human animal. The cell may be any cell type from any organism, such as an embryonic stem cell (e.g., a mouse or rat embryonic stem cell) or an induced pluripotent stem cell (e.g., a human induced pluripotent stem cell). The non-human animal may be any suitable type of non-human animal, as further described elsewhere herein.

[0091] The nucleic acids or expression cassettes may be stably integrated into the genome of the cell or non-human animal (i.e., within a chromosome), or they may be located outside of a chromosome (e.g., extrachromosomally replicating DNA). Stably integrated expression cassettes or nucleic acids may be randomly integrated into the genome of the cell or non-human animal (i.e., transgenic), or they may be integrated into a predetermined region of the genome of the cell or non-human animal (i.e., knock-in). In one example, the nucleic acid or expression cassette is stably integrated into a safe harbor locus described elsewhere herein. The target genomic locus into which the nucleic acid or expression cassette is stably integrated may be heterozygous for the nucleic acid or expression cassette, or may be homozygous for the nucleic acid or expression cassette.

[0092] The nucleic acid or expression cassette described herein may be operably linked to any promoter suitable for expression in vivo in a non-human animal, or in a cell in vitro or ex vivo. The non-human animal may be any suitable non-human animal described elsewhere herein. As an example, the nucleic acid or expression cassette may be operably linked to an endogenous promoter in the target genomic locus, such as the Rosa26 promoter. Alternatively, the nucleic acid or expression cassette may be operably linked to an exogenous promoter, such as a constitutively active promoter (e.g., a CAG 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). Such promoters are well known and are discussed elsewhere herein. Promoters that can be used in the expression constructs include, for example, promoters active in one or more of eukaryotic cells, human cells, non-human cells, mammalian cells, non-human mammalian cells, rodent cells, mouse cells, rat cells, hamster cells, rabbit cells, pluripotent cells, embryonic stem (ES) cells, or zygotes. Such promoters can be, for example, conditional, inducible, constitutive, or tissue-specific promoters.

[0093] The nucleic acids and expression cassettes described herein can be in any form. For example, the expression cassette can be in a vector, such as a viral vector, or a plasmid. The expression cassette can be operably linked to a promoter in an expression construct that can direct the expression of a protein or RNA. Alternatively, the expression cassette can be in a targeting vector. For example, the targeting vector can include homology arms flanking the expression cassette, which are suitable for directing recombination with a desired target genomic locus to facilitate genomic integration and / or replacement of endogenous sequences.

[0094] When the compositions described herein are in a cell or a non-human animal, the compositions may include a first nucleic acid (e.g., an expression cassette) encoding a first fusion protein and a second nucleic acid (e.g., an expression cassette) encoding a second fusion protein, and the first fusion protein and the second fusion protein may be stably expressed in the cell or non-human animal. For example, the first nucleic acid (e.g., an expression cassette) and the second nucleic acid (e.g., an expression cassette) may be integrated into the genome of the cell or non-human animal and operably linked to a promoter active in the cell or non-human animal. For example, the first nucleic acid and the second nucleic acid may be integrated into the genome of the germline of the non-human animal. Similarly, when the compositions described herein are in a cell or non-human animal, the compositions may include a first nucleic acid (e.g., an expression cassette) comprising a coding sequence for a 4R tau isoform and a coding sequence for a first reporter protein (e.g., separated by a first 2A coding sequence) and a second nucleic acid (e.g., an expression cassette) comprising a coding sequence for a 3R tau isoform and a coding sequence for a second reporter protein (e.g., separated by a second 2A coding sequence), and the 4R tau isoform, the first reporter protein, the 3R tau isoform, and the second reporter protein may be stably expressed in the cell or non-human animal. For example, the first nucleic acid (e.g., an expression cassette) and the second nucleic acid (e.g., an expression cassette) may be integrated into the genome of the cell or non-human animal and operably linked to a promoter active in the cell or non-human animal. For example, the first nucleic acid and the second nucleic acid may be integrated into the genome of the germline of the non-human animal. The nucleic acids (e.g., expression cassettes) can be randomly integrated into the genome of the cell or non-human animal, or they can be integrated into a target genomic locus, such as a safe harbor locus. Any target genomic locus capable of expressing a gene can be used. The promoter can be any suitable promoter. For example, the promoter can be a constitutive promoter, such as the EF1α promoter. Alternatively, the promoter can be a tissue-specific promoter or an inducible promoter. For example, as another example, the promoter can be a neuronal cell-specific promoter.One example of a suitable neuron-specific promoter is the synapsin-1 promoter (eg, the human synapsin-1 promoter).

[0095] An example of a target genomic locus into which the nucleic acid or expression cassette described herein may stably integrate is a safe harbor locus in the genome of a cell or non-human animal. Interactions between the integrated exogenous DNA and the host genome may limit the reliability and safety of the integration, resulting in obvious phenotypic effects not due to targeted genetic modification, but instead due to unintended effects of the integration on surrounding endogenous genes. For example, randomly inserted transgenes may be susceptible to position effects and silencing, making their expression unreliable and unpredictable. Similarly, integration of exogenous DNA into a chromosomal locus may affect the surrounding endogenous genes and chromatin, thereby altering the behavior and phenotype of the cell. Safe harbor loci include chromosomal loci where a transgene or other exogenous nucleic acid insert can be stably and reliably expressed in all tissues of interest without obviously altering the behavior or phenotype of the cell (i.e., without adversely affecting the host cell). See, e.g., Sadelain et al. (2012) Nat. Rev. Cancer 12:51-58, which is incorporated by reference in its entirety for all purposes. For example, a safe harbor locus can be a locus where expression of an inserted genetic sequence is not perturbed by read-through expression from adjacent genes. For example, a safe harbor locus can include a chromosomal locus where exogenous DNA can integrate and function in a predictable manner without adversely affecting the structure or expression of the endogenous gene. A safe harbor locus can include extragenic or intragenic regions, e.g., intragenic loci that are non-essential, unwanted, or that can be disrupted without obvious phenotypic consequences.

[0096] For example, the Rosa26 locus and its equivalent in humans provide an open chromatin configuration in all tissues and is ubiquitously expressed during embryonic development and in adults.See, for example, Zambrowicz et al. (1997) Proc.Natl.Acad.Sci.USA 94:3789-3794, which is incorporated herein by reference in its entirety for all purposes.In addition, the Rosa26 locus can be targeted with high efficiency, and disruption of the Rosa26 gene does not result in an obvious phenotype.Other examples of safe harbor loci include CCR5, HPRT, AAVS1, and albumin. See, for example, U.S. Patent Nos. 7,888,121, 7,972,854, 7,914,796, 7,951,925, 8,110,379, 8,409,861, and 8,586,526, as well as U.S. Patent Application Publication Nos. 2003 / 0232410, 2005 / 0208489, 2005 / 0026157, 2006 / 0063231, and 2008 / 0159. See, for example, US Pat. Nos. 2010 / 00218264, 2012 / 0017290, 2011 / 0265198, 2013 / 0137104, 2013 / 0122591, 2013 / 0177983, 2013 / 0177960, and 2013 / 0122591, each of which is incorporated by reference in its entirety for all purposes. Biallelic targeting of safe harbor loci, such as the Rosa26 locus, does not result in negative consequences, and therefore different genes or reporters can be targeted to the two Rosa26 alleles.

[0097] The nucleic acid (e.g., an expression cassette) integrated into the target genomic locus can be operably linked to an endogenous promoter at the target genomic locus or can be operably linked to an exogenous promoter that is heterologous to the target genomic locus.

[0098] The compositions described herein can include a vector (e.g., a viral vector) that includes a first nucleic acid (e.g., an expression cassette) and a second nucleic acid (e.g., an expression cassette), or a first vector (e.g., a viral vector) that includes a first nucleic acid (e.g., an expression cassette) and a second vector (e.g., a viral vector) that includes a second nucleic acid (e.g., an expression cassette).

[0099] Vectors can include additional sequences, such as, for example, origins of replication, promoters, and genes encoding antibiotic resistance. Some vectors can be circular. Alternatively, vectors can be linear. Non-limiting exemplary vectors include plasmids, phagemids, cosmids, artificial chromosomes, mini-chromosomes, transposons, viral vectors, and expression vectors. 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 permitting packaging into a viral vector particle. The vectors and / or particles can be utilized to transfer DNA, RNA, or other nucleic acids into cells in vitro, ex vivo, or in vivo. Many forms of viral vectors are known. The viral vector can be, for example, an adeno-associated virus (AAV) vector or a lentivirus (LV) vector (i.e., a recombinant AAV vector or a recombinant LV vector). Other exemplary viruses / viral vectors include retroviruses, adenoviruses, vaccinia viruses, poxviruses, and herpes simplex viruses. The virus may infect dividing cells, non-dividing cells, or both dividing and non-dividing cells. The virus may integrate into the host genome, or alternatively, may not integrate into the host genome. Such viruses may also be engineered to have reduced immunity. The virus may be replication competent or replication deficient (e.g., defective in one or more genes required for additional rounds of virion replication and / or packaging). The virus may cause transient expression, long-term expression (e.g., at least 1 week, 2 weeks, 1 month, 2 months, or 3 months), or permanent expression.

[0100] In one example, the nucleic acid or expression construct is present in an AAV vector. AAV can be of any suitable serotype and can be single-stranded AAV (ssAAV) or self-complementary AAV (scAAV). The ssDNA AAV genome consists of two open reading frames, Rep and Cap, flanked by two inverted terminal repeats that allow synthesis of complementary DNA strands. When constructing an AAV transfer plasmid, the transgene is placed between the two ITRs, and Rep and Cap can be supplied in trans. In addition to Rep and Cap, AAV may 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 helper plasmid can be transfected into HEK293 cells containing adenovirus genes E1+ to generate infectious AAV particles. Alternatively, Rep, Cap, and adenovirus helper genes can be combined into a single plasmid. Similar packaging cells and methods can be used for other viruses, such as retroviruses.

[0101] Several serotypes of AAV have been identified. These serotypes differ in the type of cells they infect (i.e., their tropism), allowing preferential transduction of certain cell types. Serotypes for CNS tissues include AAV1, AAV2, AAV4, AAV5, AAV8, and AAV9. The selectivity of AAV serotypes for gene delivery in neural cells is discussed, for example, in Hammond et al. (2017) PLoS One 12(12):e0188830, which is incorporated herein by reference in its entirety for all purposes. In a specific embodiment, the AAV-PHP.eB vector is used. The AAV-PHP.eB vector exhibits a high ability to cross the blood-brain barrier, increasing its CNS transduction efficiency. In another specific embodiment, the AAV9 vector is used.

[0102] Tropism can be further refined by pseudotyping, which is a mixture of capsids and genomes from different viral serotypes. For example, AAV2 / 5 refers to a virus that contains a serotype 2 genome packaged in a serotype 5 capsid. The use of pseudotyped viruses can not only improve transduction efficiency but also alter tropism. Hybrid capsids from different serotypes can also be used to alter the tropism of the virus. For example, AAV-DJ contains hybrid capsids from eight serotypes and shows high infectivity across a wide range of cell types in vivo. AAV-DJ8 is another example that shows the properties of AAV-DJ but with enhanced brain uptake. AAV serotypes can also be modified by 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 pseudotype / modified AAV variants include AAV2 / 1, AAV2 / 6, AAV2 / 7, AAV2 / 8, AAV2 / 9, AAV2.5, AAV8.2, and AAV / SASTG.

[0103] To accelerate transgene expression, self-complementary AAV (scAAV) variants can be used. AAV relies on the cell's DNA replication machinery to synthesize the complementary strand of the AAV's single-stranded DNA genome, which can delay transgene expression. To address this delay, scAAV can be used, which contain complementary sequences that can spontaneously anneal upon infection, eliminating the need for host cell DNA synthesis. However, single-stranded AAV (ssAAV) vectors can also be used.

[0104] The 4R and 3R tau isoforms in the composition can be from any animal or mammal, such as human, mouse, or rat. In a specific example, the 4R and 3R tau isoforms are human. The 4R and 3R tau isoforms can also be any combination of 4R and 3R tau isoforms, respectively. In one example, the 4R tau isoform is a 2N4R isoform. For example, the 4R tau isoform can include the sequence set forth in SEQ ID NO: 13, and optionally, the 4R tau isoform is encoded by the sequence set forth in SEQ ID NO: 17. For example, in a composition in which the 4R tau isoform is fused to a first reporter protein, the fusion protein can include the sequence set forth in SEQ ID NO: 15, and optionally, the fusion protein is encoded by the sequence set forth in SEQ ID NO: 19. In another example, the 4R tau isoform is a 1N4R isoform. For example, the 4R tau isoform may comprise the sequence set forth in SEQ ID NO: 23, 27, 31, or 47, optionally the 4R tau isoform is encoded by the sequence set forth in SEQ ID NO: 25, 29, 33, or 48, respectively. For example, in a composition in which a 4R tau isoform is fused to a first reporter protein, the fusion protein may comprise the sequence set forth in SEQ ID NO: 35, optionally the fusion protein is encoded by the sequence set forth in SEQ ID NO: 36. For example, in a composition in which a 4R tau isoform is fused to a first reporter protein, the fusion protein may comprise the sequence set forth in SEQ ID NO: 37, optionally the fusion protein is encoded by the sequence set forth in SEQ ID NO: 38. For example, in a composition in which a 4R tau isoform is fused to a first reporter protein, the fusion protein may comprise the sequence set forth in SEQ ID NO: 39, optionally the fusion protein is encoded by the sequence set forth in SEQ ID NO: 40.

[0105] In one example, the 3R tau isoform is a 2N3R isoform. For example, the 3R tau isoform may comprise the sequence set forth in SEQ ID NO: 14, and optionally the 3R tau isoform is encoded by the sequence set forth in SEQ ID NO: 18. For example, in a composition in which the 3R tau isoform is fused to a second reporter protein, the fusion protein may comprise the sequence set forth in SEQ ID NO: 16, and optionally the fusion protein is encoded by the sequence set forth in SEQ ID NO: 20. In another example, the 4R tau isoform is a 1N3R isoform. For example, the 3R tau isoform may comprise the sequence set forth in SEQ ID NO: 24, 28, 32, or 49, and optionally the 3R tau isoform is encoded by the sequence set forth in SEQ ID NO: 26, 30, 34, or 50, respectively. For example, in a composition in which the 3R tau isoform is fused to a second reporter protein, the fusion protein may comprise the sequence set forth in SEQ ID NO: 41, and optionally the fusion protein is encoded by the sequence set forth in SEQ ID NO: 42. For example, in a composition in which a 3R tau isoform is fused to a second reporter protein, the fusion protein may comprise the sequence set forth in SEQ ID NO: 43, and optionally, the fusion protein is encoded by the sequence set forth in SEQ ID NO: 44. For example, in a composition in which a 3R tau isoform is fused to a second reporter protein, the fusion protein may comprise the sequence set forth in SEQ ID NO: 45, and optionally, the fusion protein is encoded by the sequence set forth in SEQ ID NO: 46.

[0106] In a specific example, the 4R tau isoform is a 2N4R isoform, and the 3R tau isoform is a 2N3R isoform. For example, the 4R tau isoform may comprise the sequence set forth in SEQ ID NO: 13, and the 3R tau isoform may comprise the sequence set forth in SEQ ID NO: 14 (optionally encoded by SEQ ID NO: 17 and 18, respectively). For example, the 4R tau isoform may be fused to a first reporter protein, and the fusion protein may comprise the sequence set forth in SEQ ID NO: 15, and the 3R tau isoform may be fused to a second reporter protein, and the fusion protein may comprise the sequence set forth in SEQ ID NO: 16 (optionally encoded by SEQ ID NO: 19 and 20, respectively). In another specific example, the 4R tau isoform is a 1N4R isoform, and the 3R tau isoform is a 1N3R isoform. For example, the 4R tau isoform may comprise the sequence set forth in SEQ ID NO:23, and the 3R tau isoform may comprise the sequence set forth in SEQ ID NO:24 (optionally encoded by SEQ ID NOs:25 and 26, respectively). For example, the 4R tau isoform may comprise the sequence set forth in SEQ ID NO:27, and the 3R tau isoform may comprise the sequence set forth in SEQ ID NO:28 (optionally encoded by SEQ ID NOs:29 and 30, respectively). For example, the 4R tau isoform may comprise the sequence set forth in SEQ ID NO:31, and the 3R tau isoform may comprise the sequence set forth in SEQ ID NO:32 (optionally encoded by SEQ ID NOs:33 and 34, respectively). For example, the 4R tau isoform may comprise the sequence set forth in SEQ ID NO:47, and the 3R tau isoform may comprise the sequence set forth in SEQ ID NO:49 (optionally encoded by SEQ ID NOs:48 and 50, respectively). For example, a 4R tau isoform may be fused to a first reporter protein, and the fusion protein may comprise the sequence set forth in SEQ ID NO: 35, and a 3R tau isoform may be fused to a second reporter protein, and the fusion protein may comprise the sequence set forth in SEQ ID NO: 41 (optionally encoded by SEQ ID NOs: 36 and 42, respectively).For example, a 4R tau isoform may be fused to a first reporter protein, the fusion protein may comprise the sequence set forth in SEQ ID NO: 37, and a 3R tau isoform may be fused to a second reporter protein, the fusion protein may comprise the sequence set forth in SEQ ID NO: 43 (optionally encoded by SEQ ID NOs: 38 and 44, respectively). For example, a 4R tau isoform may be fused to a first reporter protein, the fusion protein may comprise the sequence set forth in SEQ ID NO: 39, and a 3R tau isoform may be fused to a second reporter protein, the fusion protein may comprise the sequence set forth in SEQ ID NO: 45 (optionally encoded by SEQ ID NOs: 40 and 46, respectively). In addition to the 2N4R / 2N3R and 1N4R / 1N3R combinations disclosed above, any other combination may be used (e.g., 0N4R / 0N3R, 0N4R / 1N3R, 0N4R / 2N3R, 1N4R / 0N3R, 1N4R / 1N3R, 1N4R / 2N3R, 2N4R / 0N3R, 2N4R / 1N3R, or 2N4R / 2N3R).

[0107] The 4R and / or 3R tau isoforms may be wild type. Alternatively, the 4R and / or 3R tau isoforms may include tau pathogenic mutations, such as aggregation-promoting mutations. Such mutations may be, for example, mutations that are associated with (e.g., segregate with) or cause tauopathy. As an example, the mutation may be an aggregation-sensitizing mutation that sensitizes tau to seeding but does not cause tau to aggregate easily by itself, or a mutation that is known to cause the aggregation of both 3R and 4R tau. For example, the tau mutation may be R5L, L237V, or G243V. R5L is a mutation that has been reported in PSP patients (present in all six isoforms of tau, but in human carriers, selective aggregation of only 4R tau is observed), and G243V and L237V are mutations that have been reported to selectively worsen 4R aggregation. R5L mutation means human tau R5L mutation or the corresponding mutation of another tau protein when optimally aligned with human tau protein. L237V mutation means human tau L237V mutation or the corresponding mutation of another tau protein when optimally aligned with human tau protein. G243V mutation means human tau G243V mutation or the corresponding mutation of another tau protein when optimally aligned with human tau protein. In one example, both 4R and 3R tau isoforms contain R5L mutation. In one example, both 4R and 3R tau isoforms contain L237V mutation. In one example, both 4R and 3R tau isoforms contain G243V mutation. In another example, the tau mutation can be N279K, L284R, or S285R. These are all exon 10 mutations that have been reported in humans with PSP-like symptoms and pathology. By N279K mutation is meant the human tau N279K mutation or the corresponding mutation of another tau protein when optimally aligned with the human tau protein.By L284R mutation is meant the human tau L284R mutation or the corresponding mutation of another tau protein when optimally aligned with the human tau protein.By S285R mutation is meant the human tau S285R mutation or the corresponding mutation of another tau protein when optimally aligned with the human tau protein. In one example, both the 4R and 3R tau isoforms contain the N279K mutation. In one example, both the 4R and 3R tau isoforms contain the L284R mutation. In one example, both the 4R and 3R tau isoforms contain the S285R mutation.

[0108] The first and second reporter proteins can be any suitable reporter proteins encoded by any suitable reporter gene. The term "reporter gene" refers to a nucleic acid having a sequence encoding a gene product (typically an enzyme) that is easily and quantitatively assayed when a construct containing a reporter gene sequence operably linked to a heterologous promoter and / or enhancer element is introduced into a cell that contains (or can be made to contain) factors necessary for activation of the promoter and / or enhancer element. Examples of reporter genes include, but are not limited to, a gene encoding beta-galactosidase (lacZ), a bacterial chloramphenicol acetyltransferase (cat) gene, a firefly luciferase gene, a gene encoding beta-glucuronidase (GUS), and a gene encoding a fluorescent protein. "Reporter protein" refers to a protein encoded by a reporter gene. In a specific example, the first and second reporter proteins are fluorescent reporter proteins.

[0109] As used herein, the term "fluorescent reporter protein" refers to a reporter protein that is detectable based on fluorescence, where the fluorescence can be from either the reporter protein directly, the activity of the reporter protein on a fluorogenic substrate, or from a protein that has an affinity for binding to a fluorescently 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, mRFP1, DsRed-Express, DsRed2, DsRed monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611, mRaspberry, mStrawberry, and JRed), orange fluorescent protein (e.g., mOrange, mKO, Kusabira-Orange, monomeric Kusabira-Orange, mTangerine, and tdTomato), and other suitable fluorescent protein methods that can detect the presence in cells by flow cytometry. In a specific example, the first and second reporter proteins are yellow fluorescent protein (e.g., eYFP) and red fluorescent protein (e.g., mCherry), respectively, or vice versa.

[0110] Also provided herein are cells or cell populations comprising the compositions described herein. The cells can be of any cell type and can be in vitro, ex vivo, or in vivo. The cells or cell populations can be monoclonal cell lines or cell populations. The cells can be from any source. For example, the cells can be eukaryotic cells, animal cells, plant cells, or fungal (e.g., yeast) cells. Such cells can be fish or bird cells, or such cells can be mammalian cells, such as human cells, non-human mammalian cells, rodent cells, mouse cells, or rat cells. Mammals include, for example, humans, non-human primates, monkeys, apes, cats, dogs, horses, bulls, deer, bison, sheep, rodents (e.g., mice, rats, hamsters, guinea pigs), livestock (e.g., bovine species such as cows and steers, ovine species such as sheep and goats, porcine species such as pigs and wild boars). Birds include, for example, chickens, turkeys, ostriches, geese, and ducks. Domestic and agricultural animals are also included. The term "non-human animals" excludes humans. In a specific example, the cell is a human cell (e.g., a HEK293 cell).

[0111] The cells can be, for example, totipotent or pluripotent cells (e.g., embryonic stem (ES) cells, such as rodent ES cells, mouse ES cells, or rat ES cells). Totipotent cells include undifferentiated cells that can give rise to any cell type, and pluripotent cells include undifferentiated cells that have the ability to develop into two or more differentiated cell types. Such pluripotent and / or totipotent cells can be, for example, ES cells or ES-like cells, such as induced pluripotent stem (iPS) cells. ES cells include embryo-derived totipotent or pluripotent cells that can contribute to any tissue of the developing embryo upon introduction into the embryo. ES cells can be derived from the inner cell mass of a blastocyst and can differentiate into cells of any of the three vertebrate germ layers (endoderm, ectoderm, and mesoderm).

[0112] The cells may also be primary somatic cells or cells that are not primary somatic cells. Somatic cells may include any cell that is not a gamete, germ cell, gamete cell, or undifferentiated stem cell. The cells may also be primary cells. Primary cells include cells or cultures of cells that are directly isolated from an organism, organ, or tissue. Primary cells include cells that are not transformed or immortalized. They include any cells obtained from an organism, organ, or tissue that have not previously been passaged in tissue culture or that have previously been passaged in tissue culture but cannot be passaged indefinitely in tissue culture. Such cells may be isolated by conventional techniques and include, for example, somatic cells, hematopoietic cells, endothelial cells, epithelial cells, fibroblasts, mesenchymal cells, keratinocytes, melanocytes, monocytes, mononuclear cells, adipocytes, preadipocytes, neurons, glial cells, hepatocytes, skeletal myoblasts, and smooth muscle cells. For example, primary cells may be derived from connective tissue, muscle tissue, nervous system tissue, or epithelial tissue.

[0113] Such cells include those that do not normally proliferate indefinitely, but due to mutations or modifications, can avoid normal cellular senescence and continue to divide instead. Such mutations or modifications can occur naturally or can be induced intentionally. Examples of immortalized cells include Chinese hamster ovary (CHO) cells, human embryonic kidney cells (e.g., HEK293 cells), and mouse embryonic fibroblast cells (e.g., 3T3 cells). Many types of immortalized cells are known. Immortalized or primary cells typically include cells used for culture or recombinant gene or protein expression.

[0114] The cell may also be a differentiated cell, such as a neuronal cell (e.g., a human neuronal cell). The cell may be an isolated cell.

[0115] Also provided herein is a non-human animal comprising the composition described herein. The non-human animal may be any suitable non-human animal. The term "animal" includes any member of the animal kingdom, including, for example, mammals, fish, reptiles, amphibians, birds, and insects. 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.

[0116] The non-human animals may be from any genetic background. For example, suitable mice may be from the 129 strain, the C57BL / 6 strain, a hybrid of 129 and C57BL / 6, the BALB / c strain, or the Swiss Webster strain. Examples of 129 strains include 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / Svlm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, and 129T2. See, for example, Festing et al. (1999) Mamm. Genome 10(8):836, incorporated herein by reference in its entirety for all purposes. Examples of C57BL strains include C57BL / A, C57BL / An, C57BL / GrFa, C57BL / Kal_wN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr, and C57BL / Ola. Suitable mice can also be from a hybrid of the aforementioned 129 strain and the aforementioned C57BL / 6 strain (e.g., 50% 129 and 50% C57BL / 6). Similarly, suitable mice can be from a hybrid of the aforementioned 129 strain or a hybrid of the aforementioned BL / 6 strain (e.g., 129S6 (129 / SvEvTac) strain).

[0117] Similarly, the rats may be from, for example, the ACI rat strain, the Dark Agouti (DA) rat strain, the Wistar rat strain, the LEA rat strain, the Sprague Dawley (SD) rat strain, or the Fischer rat strain, such as Fischer F344 or Fischer F6. Rats may also be obtained from strains derived from crosses of two or more of the above-mentioned strains. For example, suitable rats may be from the DA strain or the ACI strain. The ACI rat strain has white belly and feet, as well as RT1 av1 The Dark Agouti (DA) rat strain is characterized by the presence of the black agouti haplotype. Such strains are available from a variety of sources, including Harlan Laboratories. The Dark Agouti (DA) rat strain has the agouti coat and the RT1 haplotype. av1 The rat is characterized by having a haplotype. Such rats can be obtained from various sources, including Charles River and Harlan Laboratories. Some suitable rats may be derived from inbred rat strains. See, for example, U.S. Patent Application Publication No. 2014 / 0235933, the entirety of which is incorporated herein by reference for all purposes.

[0118] III. Tau Reporter Cells and Tau Reporter Non-Human Animals Provided herein are tau reporter cells or tau reporter cell populations that can be used in cell screening assays to distinguish between tau targeting reagents that specifically or selectively target 4R tau and reagents that target both 4R tau and 3R tau.Similarly, provided herein are tau reporter non-human animals that can be used in in vivo screening assays to distinguish between tau targeting reagents that specifically or selectively target 4R tau and reagents that target both 4R tau and 3R tau.

[0119] The tau reporter cell or tau reporter non-human animal may comprise a 4R tau isoform linked to a first reporter protein and a 3R tau isoform linked to a second reporter protein different from the first reporter protein. Alternatively, the tau reporter cell or tau reporter non-human animal may comprise a first nucleic acid encoding a 4R tau isoform linked to a first reporter protein and a second nucleic acid encoding a 3R tau isoform linked to a second reporter protein. By "linked" it is meant that the tau isoform and the linked reporter protein are part of the same protein (i.e., a fusion protein) or expressed from the same messenger RNA. In a fusion protein, the tau isoform and the reporter protein may be fused directly to each other or fused to each other via a linker. In a specific example, the tau isoform and the linked reporter protein are fused to each other via a linker. In a first example, a tau reporter cell or a tau reporter non-human animal may comprise a first fusion protein comprising a 4R tau isoform fused to a first reporter protein and a second fusion protein comprising a 3R tau isoform fused to a second reporter protein different from the first reporter protein. For example, the cell or non-human animal may comprise a first nucleic acid (e.g., an expression cassette) encoding the first fusion protein and a second nucleic acid (e.g., an expression cassette) encoding the second fusion protein, and the cell or non-human animal expresses the first fusion protein and the second fusion protein.In a second example, a tau reporter cell or tau reporter non-human animal may comprise a first nucleic acid (e.g., an expression cassette) comprising a coding sequence for a 4R tau isoform and a coding sequence for a first reporter protein, and a second nucleic acid (e.g., an expression cassette) comprising a coding sequence for a 3R tau isoform and a coding sequence for a second reporter protein different from the first reporter protein, wherein the coding sequence for the 4R tau isoform and the coding sequence for the first reporter protein are separated by the coding sequence for a first 2A peptide, and the coding sequence for the 3R tau isoform and the coding sequence for the second reporter protein are separated by the coding sequence for a second 2A peptide, and the cell or non-human animal expresses the 4R tau isoform, the 3R tau isoform, the first reporter protein, and the second reporter protein.

[0120] As mentioned above, examples of 2A peptides that can be used include Thoseaasigna virus 2A (T2A), Porcine Teschovirus-1 2A (P2A), Equine Rhinitis A Virus (ERAV) 2A (E2A), and FMDV 2A (F2A). Exemplary T2A, P2A, E2A, and F2A sequences include: T2A (EGRGSLLTCGDVEENPGP, SEQ ID NO: 9), P2A (ATNFSLLKQAGDVEENPGP, SEQ ID NO: 10), E2A (QCTNYALLKLAGDVESNPGP, SEQ ID NO: 11), and F2A (VKQTLNFDLLKLAGDVESNPGP, SEQ ID NO: 12). A GSG residue can be added to the 5' end of any of these peptides. In a specific example, P2A is used.

[0121] In the tau reporter cell or tau reporter non-human animal comprising the first and second nucleic acids, such nucleic acids may be RNA (e.g., messenger RNA (mRNA)) or DNA, may be single-stranded or double-stranded, and may be linear or circular. The DNA may be part of a vector, such as an expression vector or a targeting vector. The vector may also be a viral vector, such as an adenovirus, an adeno-associated virus, a lentivirus, and a retrovirus vector.

[0122] Optionally, the nucleic acid can be codon-optimized for efficient translation into a protein in a particular cell or organism. For example, the nucleic acid can be modified to replace codons that have a higher frequency of usage in human cells, non-human cells, mammalian cells, rodent cells, mouse cells, rat cells, bacterial cells, yeast cells, or any other host cell of interest, as compared to the naturally occurring polynucleotide sequence.

[0123] The nucleic acids or expression cassettes may be stably integrated into the genome of the cell or non-human animal (i.e., within a chromosome), or they may be located outside of a chromosome (e.g., extrachromosomally replicating DNA). Stably integrated expression cassettes or nucleic acids may be randomly integrated into the genome of the cell or non-human animal (i.e., transgenic), or they may be integrated into a predetermined region of the genome of the cell or non-human animal (i.e., knock-in). In one example, the nucleic acid or expression cassette is stably integrated into a safe harbor locus described elsewhere herein. The target genomic locus into which the nucleic acid or expression cassette is stably integrated may be heterozygous for the nucleic acid or expression cassette, or may be homozygous for the nucleic acid or expression cassette.

[0124] The nucleic acid or expression cassette described herein may be operably linked to any promoter suitable for expression in vivo in a non-human animal, or in a cell in vitro or ex vivo. The non-human animal may be any suitable non-human animal described elsewhere herein. As an example, the nucleic acid or expression cassette may be operably linked to an endogenous promoter in the target genomic locus, such as the Rosa26 promoter. Alternatively, the nucleic acid or expression cassette may be operably linked to an exogenous promoter, such as a constitutively active promoter (e.g., a CAG 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). Such promoters are well known and are discussed elsewhere herein. Promoters that can be used in the expression constructs include, for example, promoters active in one or more of eukaryotic cells, human cells, non-human cells, mammalian cells, non-human mammalian cells, rodent cells, mouse cells, rat cells, hamster cells, rabbit cells, pluripotent cells, embryonic stem (ES) cells, or zygotes. Such promoters can be, for example, conditional, inducible, constitutive, or tissue-specific promoters.

[0125] The expression cassette described herein can be in any form. For example, the expression cassette can be in a vector, such as a viral vector, or a plasmid. The expression cassette can be operably linked to a promoter in an expression construct that can direct the expression of a protein or RNA. Alternatively, the expression cassette can be in a targeting vector. For example, the targeting vector can include homology arms flanking the expression cassette, which are suitable for directing recombination with a desired target genomic locus to facilitate genomic integration and / or replacement of endogenous sequences.

[0126] The expression cassettes described herein may be in vitro, they may be ex vivo (e.g., genomically integrated or extrachromosomal) in a cell (e.g., an embryonic stem cell), or they may be in vivo (e.g., genomically integrated or extrachromosomal) in an organism (e.g., a non-human animal). If ex vivo, the expression cassette may be any cell type from any organism, such as a totipotent cell, such as an embryonic stem cell (e.g., a mouse or rat embryonic stem cell) or an induced pluripotent stem cell (e.g., a human induced pluripotent stem cell). If in vivo, the expression cassette may be in any kind of organism (e.g., a non-human animal, as further described elsewhere herein).

[0127] In a tau reporter cell or a tau reporter non-human animal comprising a first nucleic acid (e.g., an expression cassette) encoding a first fusion protein and a second nucleic acid (e.g., an expression cassette) encoding a second fusion protein, the first fusion protein and the second fusion protein can be stably expressed in the cell or non-human animal. For example, the first nucleic acid (e.g., an expression cassette) and the second nucleic acid (e.g., an expression cassette) can be integrated into the genome of the cell or non-human animal and operably linked to a promoter active in the cell or non-human animal. For example, the first nucleic acid and the second nucleic acid can be integrated into the genome of the germline of the non-human animal. Similarly, in a tau reporter cell or non-human animal comprising a first nucleic acid (e.g., an expression cassette) comprising a coding sequence for a 4R tau isoform and a coding sequence for a first reporter protein (e.g., separated by a first 2A coding sequence) and a second nucleic acid (e.g., an expression cassette) comprising a coding sequence for a 3R tau isoform and a coding sequence for a second reporter protein (e.g., separated by a second 2A coding sequence), the 4R tau isoform, the first reporter protein, the 3R tau isoform, and the second reporter protein can be stably expressed in the cell or non-human animal. For example, the first nucleic acid (e.g., an expression cassette) and the second nucleic acid (e.g., an expression cassette) can be integrated into the genome of the cell or non-human animal and operably linked to a promoter active in the cell or non-human animal. For example, the first nucleic acid and the second nucleic acid can be integrated into the genome of the germline of the non-human animal. The nucleic acids (e.g., expression cassettes) can be randomly integrated into the genome of the cell or non-human animal, or they can be integrated into a target genomic locus, such as a safe harbor locus. Any target genomic locus capable of expressing a gene can be used. The promoter can be any suitable promoter. For example, the promoter can be a constitutive promoter, such as the EF1α promoter. Alternatively, the promoter can be a tissue-specific promoter or an inducible promoter. For example, as another example, the promoter can be a neuronal cell-specific promoter.One example of a suitable neuron-specific promoter is the synapsin-1 promoter (eg, the human synapsin-1 promoter).

[0128] An example of a target genomic locus into which the nucleic acid or expression cassette described herein can be stably integrated is a safe harbor locus in the genome of a cell or a non-human animal. Safe harbor loci are described above. For example, the Rosa26 locus and its equivalent in humans provide an open chromatin configuration in all tissues and are ubiquitously expressed during embryonic development and in adults. In addition, the Rosa26 locus can be targeted with high efficiency, and disruption of the Rosa26 gene does not result in obvious phenotypes. Other examples of safe harbor loci include CCR5, HPRT, AAVS1, and albumin. Biallelic targeting of safe harbor loci such as the Rosa26 locus does not result in negative consequences, and therefore different genes or reporters can be targeted to the two Rosa26 alleles.

[0129] The nucleic acid (e.g., an expression cassette) integrated into the target genomic locus can be operably linked to an endogenous promoter at the target genomic locus or can be operably linked to an exogenous promoter that is heterologous to the target genomic locus.

[0130] Alternatively, the tau reporter cell or non-human animal may comprise a vector (e.g., a viral vector) comprising a first nucleic acid (e.g., an expression cassette) and a second nucleic acid (e.g., an expression cassette), or a first vector (e.g., a viral vector) comprising a first nucleic acid (e.g., an expression cassette) and a second vector (e.g., a viral vector) comprising a second nucleic acid (e.g., an expression cassette).

[0131] Vectors are described in more detail above. Some vectors may be circular. Alternatively, vectors may be linear. Non-limiting exemplary vectors include plasmids, phagemids, cosmids, artificial chromosomes, mini-chromosomes, transposons, viral vectors, and expression vectors. Viral vectors are described in more detail above. Many forms of viral vectors are known. Viral vectors may be, for example, adeno-associated viral (AAV) vectors or lentiviral (LV) vectors (i.e., recombinant AAV vectors or recombinant LV vectors). Other exemplary viruses / viral vectors include retroviruses, adenoviruses, vaccinia viruses, poxviruses, and herpes simplex viruses. Viruses may infect dividing cells, non-dividing cells, or both dividing and non-dividing cells. Viruses may integrate into the host genome, or alternatively, do not integrate into the host genome. Such viruses may also be engineered to reduce immunity. Viruses may be replication competent or replication deficient (e.g., defective in one or more genes required for additional rounds of virion replication and / or packaging). The virus may cause a transient expression, a long-term expression (eg, at least 1 week, 2 weeks, 1 month, 2 months, or 3 months), or a permanent expression.

[0132] In one example, the nucleic acid or expression construct is present in an AAV vector. The AAV can be of any suitable serotype and can be single-stranded AAV (ssAAV) or self-complementary AAV (scAAV). Such AAV vectors are described in more detail above.

[0133] The 4R and 3R tau isoforms in the tau reporter cells or non-human animals can be expressed at similar levels. For example, the 4R and 3R tau isoform mRNAs can have similar relative expression, and / or the 4R and 3R tau isoform proteins can have similar relative expression. In one example, the mRNA levels of the 4R and 3R tau isoforms are within one cycle threshold (Ct) of each other when measured by quantitative polymerase chain reaction (qPCR).

[0134] The 4R and 3R tau isoforms in the tau reporter cell or non-human animal can be from any animal or mammal, such as human, mouse, or rat. In a specific example, the 4R and 3R tau isoforms are human. The 4R and 3R tau isoforms can also be any combination of 4R and 3R tau isoforms, respectively. In one example, the 4R tau isoform is a 2N4R isoform. For example, the 4R tau isoform can include the sequence set forth in SEQ ID NO: 13, and optionally, the 4R tau isoform is encoded by the sequence set forth in SEQ ID NO: 17. For example, in a composition in which the 4R tau isoform is fused to a first reporter protein, the fusion protein can include the sequence set forth in SEQ ID NO: 15, and optionally, the fusion protein is encoded by the sequence set forth in SEQ ID NO: 19. In another example, the 4R tau isoform is a 1N4R isoform. For example, the 4R tau isoform may comprise the sequence set forth in SEQ ID NO: 23, 27, 31, or 47, optionally the 4R tau isoform is encoded by the sequence set forth in SEQ ID NO: 25, 29, 33, or 48, respectively. For example, in a composition in which a 4R tau isoform is fused to a first reporter protein, the fusion protein may comprise the sequence set forth in SEQ ID NO: 35, optionally the fusion protein is encoded by the sequence set forth in SEQ ID NO: 36. For example, in a composition in which a 4R tau isoform is fused to a first reporter protein, the fusion protein may comprise the sequence set forth in SEQ ID NO: 37, optionally the fusion protein is encoded by the sequence set forth in SEQ ID NO: 38. For example, in a composition in which a 4R tau isoform is fused to a first reporter protein, the fusion protein may comprise the sequence set forth in SEQ ID NO: 39, optionally the fusion protein is encoded by the sequence set forth in SEQ ID NO: 40.

[0135] In one example, the 3R tau isoform is a 2N3R isoform. For example, the 3R tau isoform may comprise the sequence set forth in SEQ ID NO: 14, and optionally the 3R tau isoform is encoded by the sequence set forth in SEQ ID NO: 18. For example, in a composition in which the 3R tau isoform is fused to a second reporter protein, the fusion protein may comprise the sequence set forth in SEQ ID NO: 16, and optionally the fusion protein is encoded by the sequence set forth in SEQ ID NO: 20. In another example, the 4R tau isoform is a 1N3R isoform. For example, the 3R tau isoform may comprise the sequence set forth in SEQ ID NO: 24, 28, 32, or 49, and optionally the 3R tau isoform is encoded by the sequence set forth in SEQ ID NO: 26, 30, 34, or 50, respectively. For example, in a composition in which the 3R tau isoform is fused to a second reporter protein, the fusion protein may comprise the sequence set forth in SEQ ID NO: 41, and optionally the fusion protein is encoded by the sequence set forth in SEQ ID NO: 42. For example, in a composition in which a 3R tau isoform is fused to a second reporter protein, the fusion protein may comprise the sequence set forth in SEQ ID NO: 43, and optionally, the fusion protein is encoded by the sequence set forth in SEQ ID NO: 44. For example, in a composition in which a 3R tau isoform is fused to a second reporter protein, the fusion protein may comprise the sequence set forth in SEQ ID NO: 45, and optionally, the fusion protein is encoded by the sequence set forth in SEQ ID NO: 46.

[0136] In a specific example, the 4R tau isoform is a 2N4R isoform, and the 3R tau isoform is a 2N3R isoform. For example, the 4R tau isoform may comprise the sequence set forth in SEQ ID NO: 13, and the 3R tau isoform may comprise the sequence set forth in SEQ ID NO: 14 (optionally encoded by SEQ ID NO: 17 and 18, respectively). For example, the 4R tau isoform may be fused to a first reporter protein, and the fusion protein may comprise the sequence set forth in SEQ ID NO: 15, and the 3R tau isoform may be fused to a second reporter protein, and the fusion protein may comprise the sequence set forth in SEQ ID NO: 16 (optionally encoded by SEQ ID NO: 19 and 20, respectively). In another specific example, the 4R tau isoform is a 1N4R isoform, and the 3R tau isoform is a 1N3R isoform. For example, the 4R tau isoform may comprise the sequence set forth in SEQ ID NO:23, and the 3R tau isoform may comprise the sequence set forth in SEQ ID NO:24 (optionally encoded by SEQ ID NOs:25 and 26, respectively). For example, the 4R tau isoform may comprise the sequence set forth in SEQ ID NO:27, and the 3R tau isoform may comprise the sequence set forth in SEQ ID NO:28 (optionally encoded by SEQ ID NOs:29 and 30, respectively). For example, the 4R tau isoform may comprise the sequence set forth in SEQ ID NO:31, and the 3R tau isoform may comprise the sequence set forth in SEQ ID NO:32 (optionally encoded by SEQ ID NOs:33 and 34, respectively). For example, the 4R tau isoform may comprise the sequence set forth in SEQ ID NO:47, and the 3R tau isoform may comprise the sequence set forth in SEQ ID NO:49 (optionally encoded by SEQ ID NOs:48 and 50, respectively). For example, a 4R tau isoform may be fused to a first reporter protein, and the fusion protein may comprise the sequence set forth in SEQ ID NO: 35, and a 3R tau isoform may be fused to a second reporter protein, and the fusion protein may comprise the sequence set forth in SEQ ID NO: 41 (optionally encoded by SEQ ID NOs: 36 and 42, respectively).For example, a 4R tau isoform may be fused to a first reporter protein, the fusion protein may comprise the sequence set forth in SEQ ID NO: 37, and a 3R tau isoform may be fused to a second reporter protein, the fusion protein may comprise the sequence set forth in SEQ ID NO: 43 (optionally encoded by SEQ ID NOs: 38 and 44, respectively). For example, a 4R tau isoform may be fused to a first reporter protein, the fusion protein may comprise the sequence set forth in SEQ ID NO: 39, and a 3R tau isoform may be fused to a second reporter protein, the fusion protein may comprise the sequence set forth in SEQ ID NO: 45 (optionally encoded by SEQ ID NOs: 40 and 46, respectively). In addition to the 2N4R / 2N3R and 1N4R / 1N3R combinations disclosed above, any other combination may be used (e.g., 0N4R / 0N3R, 0N4R / 1N3R, 0N4R / 2N3R, 1N4R / 0N3R, 1N4R / 1N3R, 1N4R / 2N3R, 2N4R / 0N3R, 2N4R / 1N3R, or 2N4R / 2N3R).

[0137] The 4R and / or 3R tau isoforms may be wild type. Alternatively, the 4R and / or 3R tau isoforms may include tau pathogenic mutations, such as aggregation-promoting mutations. Such mutations may be, for example, mutations that are associated with (e.g., segregate with) or cause tauopathy. As an example, the mutation may be an aggregation-sensitizing mutation that sensitizes tau to seeding but does not cause tau to aggregate easily by itself, or a mutation that is known to cause the aggregation of both 3R and 4R tau. For example, the tau mutation may be R5L, L237V, or G243V. By R5L mutation, we mean the human tau R5L mutation, or the corresponding mutation of another tau protein when optimally aligned with the human tau protein. By L237V mutation, we mean the human tau L237V mutation, or the corresponding mutation of another tau protein when optimally aligned with the human tau protein. By G243V mutation is meant the human tau G243V mutation or the corresponding mutation of another tau protein when optimally aligned with the human tau protein. In one example, both the 4R and 3R tau isoforms contain the R5L mutation. In one example, both the 4R and 3R tau isoforms contain the L237V mutation. In one example, both the 4R and 3R tau isoforms contain the G243V mutation.

[0138] The first and second reporter proteins can be any suitable reporter proteins encoded by any suitable reporter gene. Reporter genes and reporter proteins are described in more detail above. Examples of reporter genes include, but are not limited to, a gene encoding beta-galactosidase (lacZ), a bacterial chloramphenicol acetyltransferase (cat) gene, a firefly luciferase gene, a gene encoding beta-glucuronidase (GUS), and a gene encoding a fluorescent protein. In a specific example, the first and second reporter proteins are fluorescent reporter proteins. Examples of fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, eGFP, Emerald, Azami Green, monomeric Azami), and the like. 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, mRFP1, 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 other suitable fluorescent protein methods that can detect their presence within a cell by flow cytometry.In a specific example, the first and second reporter proteins are yellow fluorescent protein (eg, eYFP) and red fluorescent protein (eg, mCherry), respectively, or vice versa.

[0139] The tau reporter cells disclosed herein can be of any cell type and can be in vitro, ex vivo, or in vivo. The tau reporter cell line or cell population can be a monoclonal cell line or cell population. The cells can be from any source. For example, the cells can be eukaryotic, animal, plant, or fungal (e.g., yeast) cells. Such cells can be fish or bird cells, or such cells can be mammalian cells, such as human cells, non-human mammalian cells, rodent cells, mouse cells, or rat cells. Mammals include, for example, humans, non-human primates, monkeys, apes, cats, dogs, horses, bulls, deer, bison, sheep, rodents (e.g., mice, rats, hamsters, guinea pigs), livestock (e.g., bovine species such as cows and steers, ovine species such as sheep and goats, porcine species such as pigs and wild boars). Birds include, for example, chickens, turkeys, ostriches, geese, and ducks. Domestic animals and agricultural animals are also included. The term "non-human animals" excludes humans. In a specific example, the tau reporter cells are human cells (e.g., HEK293 cells).

[0140] The cells can be, for example, totipotent or pluripotent cells (e.g., embryonic stem (ES) cells, such as rodent ES cells, mouse ES cells, or rat ES cells). Totipotent cells include undifferentiated cells that can give rise to any cell type, and pluripotent cells include undifferentiated cells that have the ability to develop into two or more differentiated cell types. Such pluripotent and / or totipotent cells can be, for example, ES cells or ES-like cells, such as induced pluripotent stem (iPS) cells. ES cells include embryo-derived totipotent or pluripotent cells that can contribute to any tissue of the developing embryo upon introduction into the embryo. ES cells can be derived from the inner cell mass of a blastocyst and can differentiate into cells of any of the three vertebrate germ layers (endoderm, ectoderm, and mesoderm).

[0141] The cells may also be primary somatic cells or cells that are not primary somatic cells. Somatic cells may include any cell that is not a gamete, germ cell, gamete cell, or undifferentiated stem cell. The cells may also be primary cells. Primary cells include cells or cultures of cells that are directly isolated from an organism, organ, or tissue. Primary cells include cells that are not transformed or immortalized. They include any cells obtained from an organism, organ, or tissue that have not previously been passaged in tissue culture or that have previously been passaged in tissue culture but cannot be passaged indefinitely in tissue culture. Such cells may be isolated by conventional techniques and include, for example, somatic cells, hematopoietic cells, endothelial cells, epithelial cells, fibroblasts, mesenchymal cells, keratinocytes, melanocytes, monocytes, mononuclear cells, adipocytes, preadipocytes, neurons, glial cells, hepatocytes, skeletal myoblasts, and smooth muscle cells. For example, primary cells may be derived from connective tissue, muscle tissue, nervous system tissue, or epithelial tissue.

[0142] Such cells include those that do not normally proliferate indefinitely, but due to mutations or modifications, can avoid normal cellular senescence and continue to divide instead. Such mutations or modifications can occur naturally or can be induced intentionally. Examples of immortalized cells include Chinese hamster ovary (CHO) cells, human embryonic kidney cells (e.g., HEK293 cells), and mouse embryonic fibroblast cells (e.g., 3T3 cells). Many types of immortalized cells are known. Immortalized or primary cells typically include cells used for culture or recombinant gene or protein expression.

[0143] The cell may also be a differentiated cell, such as a neuronal cell (e.g., a human neuronal cell). The cell may be an isolated cell.

[0144] The non-human animals described herein can be any suitable non-human animals. The term "animal" includes any member of the animal kingdom, including, for example, mammals, fish, reptiles, amphibians, birds, and insects. 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.

[0145] The non-human animals may be from any genetic background. For example, suitable mice may be from the 129 strain, the C57BL / 6 strain, a hybrid of 129 and C57BL / 6, the BALB / c strain, or the Swiss Webster strain. Examples of 129 strains include 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / Svlm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, and 129T2. See, for example, Festing et al. (1999) Mamm. Genome 10(8):836, incorporated herein by reference in its entirety for all purposes. Examples of C57BL strains include C57BL / A, C57BL / An, C57BL / GrFa, C57BL / Kal_wN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr, and C57BL / Ola. Suitable mice can also be from a hybrid of the aforementioned 129 strain and the aforementioned C57BL / 6 strain (e.g., 50% 129 and 50% C57BL / 6). Similarly, suitable mice can be from a hybrid of the aforementioned 129 strain or a hybrid of the aforementioned BL / 6 strain (e.g., 129S6 (129 / SvEvTac) strain).

[0146] Similarly, the rats may be from, for example, the ACI rat strain, the Dark Agouti (DA) rat strain, the Wistar rat strain, the LEA rat strain, the Sprague Dawley (SD) rat strain, or the Fischer rat strain, such as Fischer F344 or Fischer F6. Rats may also be obtained from strains derived from crosses of two or more of the above-mentioned strains. For example, suitable rats may be from the DA strain or the ACI strain. The ACI rat strain has white belly and feet, as well as RT1 av1The Dark Agouti (DA) rat strain is characterized by the presence of the black agouti haplotype. Such strains are available from a variety of sources, including Harlan Laboratories. The Dark Agouti (DA) rat strain has the agouti coat and the RT1 haplotype. av1 The rat is characterized by having a haplotype. Such rats can be obtained from various sources, including Charles River and Harlan Laboratories. Some suitable rats may be derived from inbred rat strains. See, for example, U.S. Patent Application Publication No. 2014 / 0235933, the entirety of which is incorporated herein by reference for all purposes.

[0147] IV. METHODS OF USING TAU REPORTER CELLS AND TAU REPORTER NON-HUMAN ANIMALS Various methods are provided for using the tau reporter cells and tau reporter non-human animals described elsewhere herein. Such methods can be, for example, for evaluating the activity of tau targeting reagents (e.g., 4R tau targeting reagents). Such tau reporter cells and tau reporter non-human animals are particularly useful for distinguishing between tau targeting reagents that specifically or selectively target 4R tau and those that target both 4R tau and 3R tau.

[0148] A. Methods for assessing the activity of tau-targeting reagents in tau reporter cells Various methods are provided for evaluating the activity of tau targeting reagents in tau reporter cells described herein. Such methods may include (a) administering a tau targeting reagent to the cells, and (b) evaluating the activity of the tau targeting reagent in the cells. The evaluating may be compared, for example, to a control tau reporter cell that has not been administered the tau targeting reagent, or to the tau reporter cell before administration of the tau targeting reagent.

[0149] Methods for evaluating the activity of tau targeting reagents are well known and provided elsewhere herein. In some methods, evaluating can include measuring one or more of 4R tau messenger RNA expression, first reporter protein messenger RNA expression, and third reporter protein messenger RNA expression. Methods for evaluating messenger RNA (mRNA) expression are well known and provided elsewhere herein.

[0150] In one example, the evaluating includes measuring 4R tau isoform mRNA expression and second reporter protein mRNA expression, and a greater relative decrease in 4R tau isoform mRNA expression compared to the second reporter protein mRNA expression after administration of the tau targeting reagent indicates that the tau targeting reagent is a 4R preferential tau targeting reagent. For example, the decrease in 4R tau isoform mRNA expression after administration of the tau targeting reagent can be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, and the decrease in the second reporter protein mRNA expression after administration of the tau targeting reagent can be 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less. In one example, the reduction in 4R tau isoform mRNA expression after administration of tau targeting reagents can be at least 60%, and the reduction in second reporter protein mRNA expression after administration of tau targeting reagents can be 30% or less. In another example, the reduction in 4R tau isoform mRNA expression after administration of tau targeting reagents can be at least 70%, and the reduction in second reporter protein mRNA expression after administration of tau targeting reagents can be 30% or less. In one example, the reduction in 4R tau isoform mRNA expression after administration of tau targeting reagents can be at least 75%, and the reduction in second reporter protein mRNA expression after administration of tau targeting reagents can be 10% or less.

[0151] In another example, the evaluating includes measuring a first reporter protein mRNA expression and a second reporter protein mRNA expression, and a greater relative decrease in the first reporter protein mRNA expression compared to the second reporter protein mRNA expression after administration of the tau targeting reagent indicates that the tau targeting reagent is a 4R-preferential tau targeting reagent. For example, the decrease in the first reporter protein mRNA expression after administration of the tau targeting reagent can be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, and the decrease in the second reporter protein mRNA expression after administration of the tau targeting reagent can be 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less. In one example, the reduction in the first reporter protein mRNA expression after administration of the tau targeting reagent can be at least 60%, and the reduction in the second reporter protein mRNA expression after administration of the tau targeting reagent can be 30% or less. In another example, the reduction in the first reporter protein mRNA expression after administration of the tau targeting reagent can be at least 70%, and the reduction in the second reporter protein mRNA expression after administration of the tau targeting reagent can be 30% or less. In one example, the reduction in the first reporter protein mRNA expression after administration of the tau targeting reagent can be at least 75%, and the reduction in the second reporter protein mRNA expression after administration of the tau targeting reagent can be 10% or less.

[0152] In some methods, the evaluating comprises measuring one or more of the first reporter protein expression and the second reporter protein expression. Methods for evaluating protein expression are provided elsewhere herein and are well known. For example, the evaluating can comprise measuring the first reporter protein expression and the second reporter protein expression, and a greater relative decrease in the first reporter protein expression compared to the second reporter protein expression after administration of the tau targeting reagent indicates that the tau targeting reagent is a 4R preferential tau targeting reagent. For example, the decrease in the first reporter protein expression after administration of the tau targeting reagent can be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, and the decrease in the second reporter protein expression after administration of the tau targeting reagent can be 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less. In one example, the reduction in the expression of the first reporter protein after administration of the tau targeting reagent can be at least 60%, and the reduction in the expression of the second reporter protein after administration of the tau targeting reagent can be 30% or less. In another example, the reduction in the expression of the first reporter protein after administration of the tau targeting reagent can be at least 70%, and the reduction in the expression of the second reporter protein after administration of the tau targeting reagent can be 30% or less. In one example, the reduction in the expression of the first reporter protein after administration of the tau targeting reagent can be at least 75%, and the reduction in the expression of the second reporter protein after administration of the tau targeting reagent can be 10% or less.

[0153] In specific examples, the first reporter protein is a first fluorescent reporter protein and the second reporter protein is a second fluorescent reporter protein, and the evaluating in step (b) comprises immunofluorescence staining or flow cytometry. In some methods, the evaluating comprises assessing tau hyperphosphorylation or tau aggregation.

[0154] Where the tau-targeting reagent is a genome editing reagent (e.g., a nuclease agent), such methods can include evaluating the modification of a nucleic acid encoding a tau4R or tau3R isoform. As an example, evaluating can include measuring non-homologous end joining (NHEJ) activity. This can include, for example, measuring the frequency of insertions or deletions within the target region. For example, evaluating can include sequencing (e.g., next generation sequencing) the nucleic acid encoding the tau4R or tau3R isoform.

[0155] As a specific example, where the tau-targeting reagent is a genome editing reagent (e.g., a nuclease agent), the percent edits of the target nucleic acid (e.g., the total number of insertions or deletions observed relative to the total number of sequences read in a PCR reaction from a pool of lysed cells) can be assessed.

[0156] The tau targeting reagent may be a tau targeting antibody or antigen binding protein (e.g., an intrabody), or any other large or small molecule that targets tau protein. Alternatively, the tau targeting reagent may be any biological or chemical agent that targets the MAPT locus (MAPT gene), MAPT mRNA, or tau protein. Examples of tau targeting reagents are disclosed elsewhere herein.

[0157] Such tau targeting reagents can be administered by any delivery method / vehicle (e.g., adenovirus, lentivirus, AAV, or LNP). Means of delivering the complexes and molecules are disclosed in more detail elsewhere herein. In certain methods, the reagents are delivered by AAV-mediated delivery or lentivirus-mediated delivery. In other certain methods, the reagents are delivered by LNP-mediated delivery. The dose can be any suitable dose.

[0158] B. Methods for assessing the activity of tau-targeting reagents in tau reporter non-human animals Various methods are provided for evaluating the activity of tau targeting reagents in the tau reporter non-human animals described herein. Such methods may include (a) administering a tau targeting reagent to the non-human animal, and (b) evaluating the activity of the tau targeting reagent in the non-human animal. The evaluation may be compared, for example, to a control tau reporter non-human animal that has not been administered the tau targeting reagent, or to the tau reporter non-human animal before administration of the tau targeting reagent.

[0159] Methods for evaluating the activity of tau targeting reagents are well known and provided elsewhere herein. The evaluation of activity can be in any cell type, any tissue type, or any organ type, as disclosed elsewhere herein. In some methods, the evaluation of activity is performed in neurons. In some methods, the evaluating can include measuring one or more of 4R tau mRNA expression, first reporter protein mRNA expression, and third reporter protein mRNA expression. For example, mRNA levels can be measured in a particular cell, tissue, or organ type (e.g., neurons). Methods for evaluating mRNA expression are provided elsewhere herein and are well known.

[0160] In one example, the evaluating includes measuring 4R tau isoform mRNA expression and second reporter protein mRNA expression, and a greater relative decrease in 4R tau isoform mRNA expression compared to the second reporter protein mRNA expression after administration of the tau targeting reagent indicates that the tau targeting reagent is a 4R preferential tau targeting reagent. For example, the decrease in 4R tau isoform mRNA expression after administration of the tau targeting reagent can be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, and the decrease in the second reporter protein mRNA expression after administration of the tau targeting reagent can be 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less. In one example, the reduction in 4R tau isoform mRNA expression after administration of tau targeting reagents can be at least 60%, and the reduction in second reporter protein mRNA expression after administration of tau targeting reagents can be 30% or less. In another example, the reduction in 4R tau isoform mRNA expression after administration of tau targeting reagents can be at least 70%, and the reduction in second reporter protein mRNA expression after administration of tau targeting reagents can be 30% or less. In one example, the reduction in 4R tau isoform mRNA expression after administration of tau targeting reagents can be at least 75%, and the reduction in second reporter protein mRNA expression after administration of tau targeting reagents can be 10% or less.

[0161] In one example, the evaluating includes measuring a first reporter protein mRNA expression and a second reporter protein mRNA expression, and a greater relative decrease in the first reporter protein mRNA expression compared to the second reporter protein mRNA expression after administration of the tau targeting reagent indicates that the tau targeting reagent is a 4R preferential tau targeting reagent. For example, the decrease in the first reporter protein mRNA expression after administration of the tau targeting reagent can be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, and the decrease in the second reporter protein mRNA expression after administration of the tau targeting reagent can be 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less. In one example, the reduction in the first reporter protein mRNA expression after administration of the tau targeting reagent can be at least 60%, and the reduction in the second reporter protein mRNA expression after administration of the tau targeting reagent can be 30% or less. In another example, the reduction in the first reporter protein mRNA expression after administration of the tau targeting reagent can be at least 70%, and the reduction in the second reporter protein mRNA expression after administration of the tau targeting reagent can be 30% or less. In one example, the reduction in the first reporter protein mRNA expression after administration of the tau targeting reagent can be at least 75%, and the reduction in the second reporter protein mRNA expression after administration of the tau targeting reagent can be 10% or less.

[0162] In some methods, the evaluating comprises measuring one or more of the first reporter protein expression and the second reporter protein expression. Methods for evaluating protein expression are provided elsewhere herein and are well known. For example, the evaluating can comprise measuring the first reporter protein expression and the second reporter protein expression, and a greater relative decrease in the first reporter protein expression compared to the second reporter protein expression after administration of the tau targeting reagent indicates that the tau targeting reagent is a 4R preferential tau targeting reagent. For example, the decrease in the first reporter protein expression after administration of the tau targeting reagent can be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, and the decrease in the second reporter protein expression after administration of the tau targeting reagent can be 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less. In one example, the reduction in the expression of the first reporter protein after administration of the tau targeting reagent can be at least 60%, and the reduction in the expression of the second reporter protein after administration of the tau targeting reagent can be 30% or less. In another example, the reduction in the expression of the first reporter protein after administration of the tau targeting reagent can be at least 70%, and the reduction in the expression of the second reporter protein after administration of the tau targeting reagent can be 30% or less. In one example, the reduction in the expression of the first reporter protein after administration of the tau targeting reagent can be at least 75%, and the reduction in the expression of the second reporter protein after administration of the tau targeting reagent can be 10% or less.

[0163] In specific examples, the first reporter protein is a first fluorescent reporter protein and the second reporter protein is a second fluorescent reporter protein, and the evaluating in step (b) comprises immunofluorescence staining or flow cytometry. In some methods, the evaluating comprises assessing tau hyperphosphorylation or tau aggregation.

[0164] When the tau targeting reagent is a genome editing reagent (e.g., a nuclease agent), such a method may include evaluating the modification of a nucleic acid encoding a tau 4R or tau 3R isoform. As an example, evaluating may include measuring non-homologous end joining (NHEJ) activity. This may include, for example, measuring the frequency of insertions or deletions in the target region. For example, evaluating may include sequencing (e.g., next generation sequencing) a nucleic acid encoding a tau 4R or tau 3R isoform. The evaluation may include isolating a target organ or tissue (e.g., a neuron) from the non-human animal and evaluating the modification of a nucleic acid encoding a tau 3R or tau 4R isoform in the target organ or tissue. Similarly, the evaluation may include isolating a non-target organ or tissue (e.g., two or more non-target organs or tissues) from the non-human animal and evaluating the modification of a nucleic acid encoding a tau 3R or tau 4R isoform in the non-target organ or tissue.

[0165] As a specific example, where the tau-targeting reagent is a genome editing reagent (e.g., a nuclease agent), the percent edits of the target nucleic acid (e.g., the total number of insertions or deletions observed relative to the total number of sequences read in a PCR reaction from a pool of lysed cells) can be assessed.

[0166] The tau targeting reagent may be a tau targeting antibody or antigen binding protein (e.g., an intrabody), or any other large or small molecule that targets tau protein. Alternatively, the tau targeting reagent may be any biological or chemical agent that targets the MAPT locus (MAPT gene), MAPT mRNA, or tau protein. Examples of tau targeting reagents are disclosed elsewhere herein.

[0167] Such tau targeting reagents can be administered by any delivery method / vehicle (e.g., adenovirus, lentivirus, AAV, LNP, or injection) and by any route of administration (e.g., intravitreal or intracameral injection). Means of delivering the complexes and molecules and routes of administration are disclosed in more detail elsewhere herein. In certain methods, the reagents are delivered by AAV-mediated delivery or lentivirus-mediated delivery. In other certain methods, the reagents are delivered by LNP-mediated delivery. The dose can be any suitable dose.

[0168] C. Tau-targeting Reagents The tau targeting reagent can be any reagent that targets tau protein, MAPT gene, or MAPT mRNA (e.g., human tau protein, human MAPT gene, or human MAPT mRNA). The tau targeting reagent can be, for example, a known tau targeting reagent, a putative tau targeting reagent (e.g., a candidate reagent designed to target tau protein, MAPT gene, or MAPT mRNA), or a reagent that is being screened for tau targeting activity.

[0169] For example, the tau targeting reagent can be an antigen binding protein that targets an epitope of the tau protein. The term "antigen binding protein" includes any protein that binds to an antigen. Examples of antigen binding proteins include antibodies, antigen-binding fragments of antibodies, multispecific antibodies (e.g., bispecific antibodies), scFVs, bis-scFVs, diabodies, triabodies, tetrabodies, V-NARs, VHHs, VLs, F(ab), F(ab)2, DVDs (dual variable domain antigen binding proteins), SVDs (single variable domain antigen binding proteins), bispecific T-cell engagers (BiTEs), or Davis bodies (U.S. Patent No. 8,586,713, which is incorporated herein by reference in its entirety for all purposes). In a specific example, the antigen binding protein is an intrabody. An intrabody is an antibody that is designed to be expressed intracellularly. Other tau targeting reagents include small molecules that target the tau protein, the MAPT gene, or the MAPT mRNA.

[0170] Other tau targeting reagents may include genome editing reagents such as nuclease agents (e.g., Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) / CRISPR-associated (Cas) (CRISPR / Cas) nucleases, zinc finger nucleases (ZFNs), or Transcription Activator-Like Effector Nucleases (TALENs)) that cleave recognition sites within the MAPT gene. Similarly, a tau targeting reagent may be an exogenous donor nucleic acid (e.g., a targeting vector or single-stranded oligodeoxynucleotide (ssODN)) designed to recombine with the MAPT gene.

[0171] Other tau targeting reagents may include RNAi agents. An "RNAi agent" is a composition that includes small double-stranded RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecules that can facilitate the degradation or inhibition of translation of a target RNA, such as messenger RNA (mRNA), in a sequence-specific manner. The oligonucleotides of an RNAi agent are polymers of linked nucleosides, each of which may or may not be independently modified. RNAi agents function through an RNA interference mechanism (i.e., induce RNA interference through interaction with the RNA interference pathway machinery (RNA-induced silencing complex or RISC) of mammalian cells). Although RNAi agents, as that term is used herein, are believed to act primarily through an RNA interference mechanism, the disclosed RNAi agents are not bound or limited to any particular pathway or mechanism of action. The RNAi agents disclosed herein include sense and antisense strands, and include, but are not limited to, small interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and dicer substrates. The antisense strand of an RNAi agent described herein is at least partially complementary to the sequence (i.e., the sequence or order of nucleobases or nucleotides described as a series of letters using standard nomenclature) of a target RNA.

[0172] Other tau-targeting reagents may include antisense oligonucleotides (ASOs). Single-stranded ASOs and RNA interference (RNAi) share the basic principle that oligonucleotides bind to target RNA via Watson-Crick base pairing. Without wishing to be bound by theory, during RNAi, a small RNA duplex (the RNAi agent) binds to an RNA-induced silencing complex (RISC) and binds to complementary RNA in cooperation with the RISC, where one strand (the passenger strand) is lost and the remaining strand (the guide strand) is lost. Argonaute 2 (Ago2), a catalytic component of RISC, then cleaves the target RNA. The guide strand is always associated with either a complementary sense strand or a protein (RISC). In contrast, ASOs must survive and function as single strands. ASOs bind to target RNA and either block other factors, such as ribosomes or splicing factors, from binding to the RNA or recruit proteins, such as nucleases. Various modifications and target regions are selected for the ASO based on the desired mechanism of action. Gapmers are ASO oligonucleotides that contain 2-5 chemically modified nucleotides (e.g., LNA or 2'-MOE) at each end flanking a central 8-10 base gap in the DNA. After binding to the target RNA, the DNA-RNA hybrid serves as a substrate for RNase H.

[0173] Other tau-targeting reagents include small molecule reagents.

[0174] D. Administration of Tau-Targeting Reagents to Cells or Non-Human Animals The methods disclosed herein may include introducing various molecules (e.g., tau targeting reagents) into a cell or non-human animal, including nucleic acids, proteins, nucleic acid-protein complexes, protein complexes, or small molecules. "Introducing" includes presenting a molecule (e.g., a nucleic acid or a protein) to a cell or non-human animal in a manner that allows access to the interior of the cell or the interior of a cell in a non-human animal. Introducing can be accomplished by any means, and two or more components (e.g., two components, or all components) can be introduced into a cell or non-human animal simultaneously or sequentially in any combination. In addition, two or more components can be introduced into a cell or non-human animal by the same delivery method / vehicle or different delivery methods / vehicles. Similarly, two or more components can be introduced into a non-human animal by the same route of administration or different routes of administration.

[0175] Molecules (e.g., Cas proteins or guide RNAs or RNAi agents or ASOs) introduced into cells or non-human animals can be provided in compositions that include carriers that increase the stability of the introduced molecule (e.g., extend the period during which degradation products remain below a threshold value, e.g., below 0.5% by weight of the starting nucleic acid or protein, under given storage conditions (e.g., −20° C., 4° C., or ambient temperature), or increase 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, reverse micelles, lipid cochelates, and lipid microtubules.

[0176] A variety of methods and compositions are provided herein to allow the introduction of molecules (e.g., nucleic acids or proteins) into cells or non-human animals. Methods for introducing molecules into various cell types are known and include, for example, stable transfection methods, transient transfection methods, and viral-mediated methods.

[0177] Transfection protocols and protocols for introducing molecules into cells can vary. Non-limiting transfection methods include chemical-based transfection methods using liposomes, nanoparticles, calcium phosphate (Graham et al. (1973) Virology 52(2): 456-67, Bacchetti et al. (1977) Proc. Natl. Acad. Sci. USA 74(4): 1590-4, and Kriegler, M (1991). Transfer and Expression: A Laboratory Manual. New York: WH Freeman and Company. pp. 96-97), dendrimers, or cationic polymers such as DEAE-dextran or polyethyleneimine. Non-chemical methods include electroporation, sonoporation, and phototransfection. Particle-based transfection includes the use of a gene gun or magnet-assisted transfection (Bertram (2006) Current Pharmaceutical Biotechnology 7, 277-28). Viral methods may also be used for transfection.

[0178] The introduction of nucleic acid or protein into cells can also be mediated by electroporation, intracytoplasmic injection, viral infection, adenovirus, adeno-associated virus, lentivirus, retrovirus, transfection, lipid-mediated transfection, or nucleofection. Nucleofection is an improved electroporation technique that allows nucleic acid substrates to be delivered not only to the cytoplasm but also to the nucleus through the nuclear membrane. In addition, the use of nucleofection in the methods disclosed herein typically requires much fewer cells than regular electroporation (e.g., only about 2 million cells compared to 7 million cells for regular electroporation). In one example, nucleofection is performed using the LONZA® NUCLEOFECTOR™ system.

[0179] Introduction of molecules (e.g., nucleic acids or proteins) into cells (e.g., zygotes) can also be achieved by microinjection. In zygotes (i.e., one-cell stage embryos), microinjection can be performed into the maternal and / or paternal pronuclei or into the cytoplasm. If microinjection is performed into only one pronucleus, the paternal pronucleus is suitable due to its larger size. Microinjection of mRNA is preferably into the cytoplasm (e.g., to deliver the mRNA directly to the translation machinery), while microinjection of proteins or polynucleotides encoding proteins or encoding RNA is preferred into the nucleus / pronucleus. Alternatively, microinjection can be performed by injection into both the nucleus / pronucleus and the cytoplasm, where the needle can be first introduced into the nucleus / pronucleus and the first amount can be injected, and the second amount can be injected into the cytoplasm while the needle is removed from the one-cell stage embryo. Methods for performing microinjection are well known. See, e.g., Nagy et al. (Nagy A, Gertsenstein M, Vintersten K, Behringer R., 2003, Manipulating the Mouse Embryo. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); also see Meyer et al. (2010) Proc. Natl. Acad. Sci. USA 107:15022-15026, and Meyer et al. (2012) Proc. Natl. Acad. Sci. USA 109:9354-9359.

[0180] Other methods for introducing molecules (e.g., nucleic acids or proteins) into cells or non-human animals may include, for example, vector delivery, particle-mediated delivery, exosome-mediated delivery, lipid nanoparticle-mediated delivery, cell-penetrating peptide-mediated delivery, or implantable device-mediated delivery. As specific examples, nucleic acids or proteins can be introduced into cells or non-human animals in carriers such as poly(lactic acid) (PLA) microspheres, poly(D,L-lactic-coglycolic acid) (PLGA) microspheres, liposomes, micelles, reverse micelles, lipid cochleates, or lipid microtubules. Some specific examples of delivery to non-human animals include hydrodynamic delivery, virus-mediated delivery (e.g., adeno-associated virus (AAV)-mediated delivery, or lentivirus-mediated delivery), and lipid nanoparticle-mediated delivery.

[0181] Introduction of nucleic acids can also be achieved by virus-mediated delivery, such as adenovirus-mediated delivery, AAV-mediated delivery (e.g., AAV-PhPeB), or lentivirus-mediated delivery. Other exemplary viruses / viral vectors include retroviruses, vaccinia viruses, poxviruses, and herpes simplex viruses. The viruses can infect dividing cells, non-dividing cells, or both dividing and non-dividing cells. The viruses can integrate into the host genome, or alternatively do not integrate into the host genome. Such viruses can also be engineered to reduce immunity. The viruses can be replication competent or replication deficient (e.g., defective in one or more genes required for additional rounds of virion replication and / or packaging). The viruses can cause transient expression, long-term expression (e.g., at least 1 week, 2 weeks, 1 month, 2 months, or 3 months), or permanent expression (e.g., of Cas9 and / or gRNA). Exemplary viral titers (e.g., AAV titers) range from 10 12 , 10 13 , 10 14 , 10 15 , and 10 16 Other exemplary viral titers (e.g., AAV titers) include about 10 12 , about 10 13 , about 1014 , about 10 15 , and about 10 16 Vector genomes (vg) / kg body weight are included.

[0182] The ssDNA AAV genome consists of two open reading frames, Rep and Cap, flanked by two inverted terminal repeats that allow synthesis of a complementary DNA strand. When constructing an AAV transfer plasmid, the transgene is placed between the two ITRs, and Rep and Cap can be supplied in trans. In addition to Rep and Cap, AAV may 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 helper plasmid can be transfected into HEK293 cells containing adenovirus genes E1+ to generate infectious AAV particles. Alternatively, Rep, Cap, and adenovirus helper genes can be combined into a single plasmid. Similar packaging cells and methods can be used for other viruses, such as retroviruses.

[0183] Several serotypes of AAV have been identified. These serotypes differ in the type of cells they infect (i.e., their tropism), allowing preferential transduction of certain cell types. Serotypes for CNS tissue include AAV1, AAV2, AAV4, AAV5, AAV8, and AAV9. Serotypes for cardiac tissue include AAV1, AAV8, and AAV9. Serotypes for kidney tissue include AAV2. Serotypes for lung tissue include AAV4, AAV5, AAV6, and AAV9. Serotypes for pancreatic tissue include AAV8. Serotypes for photoreceptor cells include AAV2, AAV5, and AAV8. Serotypes for retinal pigment epithelium tissue include AAV1, AAV2, AAV4, AAV5, and AAV8. Serotypes for skeletal muscle tissue include AAV1, AAV6, AAV7, AAV8, and AAV9. Serotypes for liver tissue include AAV7, AAV8, and AAV9, and in particular AAV8. In a specific embodiment, the AAV-PhPeB serotype is used.

[0184] Tropism can be further refined by pseudotyping, which is a mixture of capsids and genomes from different viral serotypes. For example, AAV2 / 5 refers to a virus that contains a serotype 2 genome packaged in a serotype 5 capsid. The use of pseudotyped viruses can not only improve transduction efficiency but also alter tropism. Hybrid capsids from different serotypes can also be used to alter the tropism of the virus. For example, AAV-DJ contains hybrid capsids from eight serotypes and shows high infectivity across a wide range of cell types in vivo. AAV-DJ8 is another example that shows the properties of AAV-DJ but with enhanced brain uptake. AAV serotypes can also be modified by 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 pseudotype / modified AAV variants include AAV2 / 1, AAV2 / 6, AAV2 / 7, AAV2 / 8, AAV2 / 9, AAV2.5, AAV8.2, and AAV / SASTG.

[0185] To accelerate transgene expression, self-complementary AAV (scAAV) variants can be used. AAV relies on the cell's DNA replication machinery to synthesize the complementary strand of the AAV's single-stranded DNA genome, which can delay transgene expression. To address this delay, scAAV can be used, which contain complementary sequences that can spontaneously anneal upon infection, eliminating the need for host cell DNA synthesis. However, single-stranded AAV (ssAAV) vectors can also be used.

[0186] To increase packaging capacity, a longer transgene can be split between two AAV transfer plasmids, one being the 3' splice donor and the second being the 5' splice acceptor. Upon co-infection of cells, these viruses can form concatemers and be spliced ​​together to express the full-length transgene. This allows for expression of longer transgenes, but at a reduced efficiency of expression. A similar method to increase capacity utilizes homologous recombination. For example, a transgene can be split between two transfer plasmids, but with substantial sequence overlap, such that co-expression induces homologous recombination and expression of the full-length transgene.

[0187] The introduction of nucleic acids and proteins can also be achieved by lipid nanoparticle (LNP)-mediated delivery. Lipid formulations can protect biomolecules from degradation while at the same time improving cellular uptake. Lipid nanoparticles are particles that contain multiple lipid molecules physically bound to each other by intermolecular forces. These include microspheres (including unilamellar and multilamellar vesicles, e.g., liposomes), the dispersed phase in an emulsion, micelles, or the internal phase in a suspension. Such lipid nanoparticles can be used to encapsulate one or more nucleic acids or proteins for delivery. Formulations that include 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 the 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(A1), which is incorporated by reference in its entirety for all purposes in this application. Exemplary lipid nanoparticles can include cationic lipids and one or more other components. In one example, the other components can include a helper lipid, such as cholesterol. In another example, the other components can include a helper lipid, such as cholesterol, and a neutral lipid, such as DSPC. In another example, the other components can include a helper lipid, such as cholesterol, an optional neutral lipid, such as DSPC, and a stealth lipid, such as S010, S024, S027, S031, or S033.

[0188] LNPs may contain one or more or all of the following: (i) lipids for encapsulation and endosomal escape, (ii) neutral lipids for stabilization, (iii) helper lipids for stabilization, and (iv) stealth lipids. See, e.g., Finn et al. (2018) Cell Reports 22:1-9, and WO 2017 / 173054(A1), each of which is incorporated by reference in its entirety for all purposes.

[0189] Exemplary dosages of LNP include, 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) of total RNA cargo content. In one example, an LNP dose of about 0.01 mg / kg to about 10 mg / kg, about 0.1 to about 10 mg / kg, or about 0.01 to about 0.3 mg / kg can be used. For example, an LNP dose 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.

[0190] In vivo administration can be by any suitable route, including, for example, parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. Systemic 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 injection. Intranasal injection and intravitreal injection are other specific examples. Local modes of administration include, for example, intrathecal, intracerebroventricular, intraparenchymal (e.g., localized intraparenchymal delivery to the striatum (e.g., to the caudate nucleus or putamen), cerebral cortex, precentral gyrus, hippocampus (e.g., dentate gyrus or CA3 region), temporal cortex, amygdala, frontal cortex, thalamus, cerebellum, medulla, thalamus, optic tectum, tegmentum, or substantia nigra), intraocular, intraorbital, subconjunctival, intravitreal, subretinal, and transscleral routes. Significantly smaller amounts of components (compared to systemic approaches) may be effective when administered locally (e.g., intraparenchymal or intravitreal) compared to when administered systemically (e.g., intravenously). Local modes of administration may also reduce or eliminate the incidence of potentially toxic side effects that may occur when therapeutically effective amounts of components are administered systemically.

[0191] In vivo administration can be by any suitable route, including, for example, parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. A specific example is intravenous infusion. The composition to be administered can be formulated using one or more physiologically and pharma- ceutically acceptable carriers, diluents, excipients, or adjuvants. The formulation can depend on the route of administration selected. The term "pharmaceutically acceptable" means that the carrier, diluent, excipient, or adjuvant is compatible with the other ingredients of the formulation and is not substantially deleterious to the recipient thereof.

[0192] The frequency and number of administrations may depend on the half-life and route of administration of the administered molecule, among other factors. Introduction of a nucleic acid or protein into a cell or non-human animal may be performed one or more times over a period of time. For example, introduction may be performed at least 2 times over a period of time, at least 3 times over a period of time, at least 4 times over a period of time, at least 5 times over a period of time, at least 6 times over a period of time, at least 7 times over a period of time, at least 8 times over a period of time, at least 9 times over a period of time, at least 10 times over a period of time, at least 11 times over a period of time, at least 12 times over a period of time, at least 13 times over a period of time, at least 14 times over a period of time, at least 15 times over a period of time, at least 16 times over a period of time, at least 17 times over a period of time, at least 18 times over a period of time, at least 19 times over a period of time, or at least 20 times over a period of time.

[0193] E. Measuring Delivery, Activity, or Efficacy of Tau-Targeting Reagents The methods disclosed herein may further include detecting or measuring the activity of the tau targeting reagent.

[0194] When the tau targeting reagent is a genome editing reagent (e.g., CRISPR / Cas designed to target a nucleic acid encoding a tau 4R isoform), measuring can include evaluating the targeted nucleic acid for modification. A variety of methods can be used to identify cells with targeted genetic modification. Screening can include a quantitative assay to evaluate the modification-of-allele (MOA) of a parent chromosome. See, for example, U.S. Patent Application Publication No. 2004 / 0018626, U.S. Patent Application Publication No. 2014 / 0178879, U.S. Patent Application Publication No. 2016 / 0145646, WO 2016 / 081923, and Frendewey et al. (2010) Methods Enzymol. 476:295-307, each of which is incorporated herein by reference in its entirety for all purposes. For example, the quantitative assay can be performed via quantitative PCR, such as real-time PCR (qPCR). Real-time PCR can utilize a first primer set that recognizes the target locus and a second primer set that recognizes the non-target reference locus. The primer set can include a fluorescent probe that recognizes the amplified sequence. Other examples of suitable quantitative assays include fluorescence-mediated in situ hybridization (FISH), comparative genomic hybridization, isothermal DNA amplification, quantitative hybridization to all immobilized probes, INVADER® probe, TAQMAN® molecular beacon probe, or ECLIPSE™ probe technology (see, for example, U.S. Patent Application Publication No. 2005 / 0144655, which is 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".To define the exact nature of the targeted gene modification and whether it is consistent across cell or tissue or organ types, NGS can be used as a screening tool in addition to the MOA assay.

[0195] The evaluation of the modification in non-human animals can be in any cell type from any tissue or organ. For example, the evaluation can be performed in multiple cell types from the same tissue or organ, or in cells from multiple locations within a tissue or organ. This can provide information about which cell types in the target tissue or organ are targeted, or which sections of the tissue or organ are reached by the tau targeting reagent. As another example, the evaluation can be performed in multiple types of tissues or multiple organs. In methods in which a specific tissue, organ, or cell type is targeted, this can provide information about how effectively that tissue or organ is targeted, and whether there are off-target effects on other tissues or organs.

[0196] Where the reagent is designed to inactivate a nucleic acid encoding a Tau4R or Tau3R isoform, to affect expression of a Tau4R or Tau3R isoform, to prevent translation of a Tau4R or Tau3R isoform mRNA, or to remove a Tau4R or Tau3R isoform protein, measuring can include assessing Tau4R isoform, Tau3R isoform, first reporter protein, or second reporter protein mRNA or protein expression, which can be performed, for example, in a neuron.

[0197] The production of Tau4R or Tau3R isoform protein can be evaluated by any known means.For example, expression can be evaluated by measuring the level of encoded mRNA in a non-human animal or the level of encoded protein in a non-human animal using known assays.For example, measuring can be determining whether a Tau targeting reagent reduces the level of Tau4R or Tau3R isoform in a non-human animal.

[0198] The evaluation in non-human animals can be in any cell type from any tissue or organ. For example, the evaluation can be done in multiple cell types from the same tissue or organ (e.g., the central nervous system), or in cells from multiple locations within a tissue or organ. This can provide information about which cell types within the target tissue or organ are being targeted, or which sections of the tissue or organ are being reached by the tau targeting reagent. As another example, the evaluation can be done in multiple types of tissues or multiple organs. In methods in which a specific tissue, organ, or cell type is targeted, this can provide information about how effectively that tissue or organ is targeted, and whether there are off-target effects on other tissues or organs.

[0199] One example of an assay that can be used is the RNASCOPE™ and BASESCOPE™ RNA in situ hybridization (ISH) assays, which are methods that can quantify cell-specific edited transcripts, including single nucleotide changes, in the context of intact fixed tissue. The BASESCOPE™ RNA ISH assay can complement NGS and qPCR in the characterization of gene editing. While NGS / qPCR can provide quantitative averages of wild-type and edited sequences, it does not provide information on the heterogeneity or percentage of edited cells in the tissue. The BASESCOPE™ ISH assay can provide a landscape view of the entire tissue and quantification of wild-type vs. edited transcripts at single-cell resolution that can quantify the actual number of cells in the target tissue that contain the edited mRNA transcript. The BASESCOPE™ assay uses paired oligo ("ZZ") probes to amplify the signal without non-specific background, achieving single molecule RNA detection. However, the BASESCOPE™ probe design and signal amplification system enable detection of single molecule RNA using ZZ probes, allowing differential detection of single nucleotide edits and mutations in intact, fixed tissues.

[0200] Assessment of any of these phenotypes can be compared to a control cell or non-human animal. The control cell or non-human animal can be, for example, the same age and / or the same sex as the test cell or non-human animal. Assessment of any of these phenotypes can also be compared to a control cell or non-human animal that is identical to the test cell non-human animal except that it has not been treated with a tau-targeting reagent. Assessment of any of these phenotypes can also be compared to the test cell or test non-human animal prior to administration of a tau-targeting reagent to the test cell non-human animal.

[0201] Assessment of any of these phenotypes can be performed on a single cell or non-human animal and changes in that cell or non-human animal can be assessed. Alternatively, assessment can be performed on a population of cells or non-human animals, e.g., comparing the percentage of cells or non-human animals having a particular phenotype.

[0202] V. Methods for Producing Tau Reporter Cells and Tau Reporter Non-Human Animals The tau reporter cells and non-human animals disclosed herein can be generated by any known means. For example, the tau reporter cells and non-human animals disclosed herein can be generated by introducing a first nucleic acid encoding a 4R tau isoform linked to a first reporter protein and a 3R tau isoform linked to a second reporter protein, or a second nucleic acid encoding a 4R tau isoform linked to a first reporter protein and a second nucleic acid encoding a 3R tau isoform linked to a second reporter protein into a cell or non-human animal. The 4R tau isoform linked to a first reporter protein and a 3R tau isoform linked to a second reporter protein, or the first nucleic acid encoding a 4R tau isoform linked to a first reporter protein and a second nucleic acid encoding a 3R tau isoform linked to a second reporter protein, can be introduced into a cell or non-human animal in any form (e.g., DNA, RNA, or protein) by any known means. "Introducing" includes presenting a nucleic acid or protein to a cell or non-human animal in such a way that the sequence can be accessed inside the cell or the cell in the non-human animal. The methods provided herein do not depend on a particular method for introducing a nucleic acid or protein into a cell or non-human animal, but only on the nucleic acid or protein being accessible to the inside of at least one cell. Methods for introducing nucleic acids and proteins into various cell types are known, including, for example, stable transfection, transient transfection, and virus-mediated methods. Optionally, a targeting vector can be used.

[0203] Transfection protocols and protocols for introducing nucleic acids or proteins into cells or non-human animals can vary. Non-limiting transfection methods include chemical-based transfection methods using liposomes, nanoparticles, calcium phosphate (Graham et al. (1973) Virology 52(2): 456-67, Bacchetti et al. (1977) Proc. Natl. Acad. Sci. USA 74(4): 1590-4, and Kriegler, M (1991). Transfer and Expression: A Laboratory Manual. New York: WH Freeman and Company. pp. 96-97), dendrimers, or cationic polymers such as DEAE-dextran or polyethyleneimine. Non-chemical methods include electroporation, sonoporation, and phototransfection. Particle-based transfection includes the use of a gene gun or magnet-assisted transfection (Bertram (2006) Current Pharmaceutical Biotechnology 7, 277-28). Viral methods may also be used for transfection.

[0204] The introduction of nucleic acid or protein into cells or non-human animals can also be mediated by electroporation, intracytoplasmic injection, viral infection, adenovirus, adeno-associated virus, lentivirus, retrovirus, transfection, lipid-mediated transfection, or nucleofection. Nucleofection is an improved electroporation technique that allows nucleic acid substrates to be delivered not only to the cytoplasm but also to the nucleus through the nuclear membrane. In addition, the use of nucleofection in the methods disclosed herein typically requires much fewer cells than regular electroporation (e.g., only about 2 million cells compared to 7 million cells for regular electroporation). In one example, nucleofection is performed using the LONZA® NUCLEOFECTOR™ system.

[0205] The introduction of nucleic acids or proteins into cells can also be achieved by microinjection. Microinjection of mRNA is preferably into the cytoplasm (e.g., to deliver mRNA directly to the translation machinery), while microinjection of proteins or DNA encoding proteins is preferably into the nucleus. Alternatively, microinjection can be performed by injection into both the nucleus and the cytoplasm, but the needle can be first introduced into the nucleus and the first amount can be injected, and the second amount can be injected into the cytoplasm while the needle is removed from the cell. Methods for performing microinjection are well known. See, e.g., Nagy et al. (Nagy A, Gertsenstein M, Vintersten K, Behringer R., 2003, Manipulating the Mouse Embryo. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press), Meyer et al. (2010) Proc. Natl. Acad. Sci. USA 107:15022-15026, and Meyer et al. (2012) Proc. Natl. Acad. Sci. USA 109:9354-9359.

[0206] Other methods for introducing a nucleic acid or protein into a cell or non-human animal may include, for example, vector delivery, particle-mediated delivery, exosome-mediated delivery, lipid nanoparticle-mediated delivery, cell-penetrating peptide-mediated delivery, or implantable device-mediated delivery. Methods for administering a nucleic acid or protein to a subject to modify cells in vivo are disclosed elsewhere herein.

[0207] In one example, a first nucleic acid encoding a 4R tau isoform linked to a first reporter protein and a second nucleic acid encoding a 3R tau isoform linked to a second reporter protein can be introduced via viral transduction, such as lentiviral transduction or AAV transduction.

[0208] Screening of cells or non-human animals containing a 4R tau isoform linked to a first reporter protein and a 3R tau isoform linked to a second reporter protein can be carried out by any known means.

[0209] As an example, a reporter gene can be used to screen cells or non-human animals that have a 4R tau isoform linked to a first reporter protein and a 3R tau isoform linked to a second reporter. Exemplary reporter genes include those encoding luciferase, β-galactosidase, green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (eBFP), DsRed, ZsGreen, MmGFP, mPlum, mCherry, tdTomato, mStrawberry, J-Red, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, Cerulean, T-Sapphire, and alkaline phosphatase. For example, if the first and second reporters are fluorescent proteins (e.g., mCherry and eYFP), cells containing these reporters can be selected by flow cytometry to select double-positive cells. The double-positive cells can then be combined to generate polyclonal lines, or monoclonal lines can be generated from a single double-positive cell.

[0210] As another example, a selection marker can be used to screen for cells with a 4R tau isoform linked to a first reporter protein and a 3R tau isoform linked to a second reporter. Exemplary selection markers include neomycin phosphotransferase (neomycin phosphotransferase). r ), hygromycin B phosphotransferase (hyg r ), puromycin N-acetyltransferase (puro r ), blasticidin S deaminase (bsr r ), xanthine / guanine phosphoribosyl transferase (gpt), or herpes simplex virus thymidine kinase (HSV-k). Another exemplary selectable marker is the bleomycin resistance protein encoded by the Sh ble gene (Streptoalloteichus hindustanus bleomycin gene), which confers resistance to Zeocin (phleomycin D1).

[0211] Various other methods are provided for producing non-human animals comprising a 4R tau isoform linked to a first reporter protein and a 3R tau isoform linked to a second reporter protein, or a first nucleic acid encoding a 4R tau isoform linked to a first reporter protein and a second nucleic acid encoding a 3R tau isoform linked to a second reporter protein. Any convenient method or protocol for producing genetically modified organisms is suitable for producing such genetically modified non-human animals. See, for example, Poueymirou et al. (2007) Nat. Biotechnol. 25(1):91-99, U.S. Patent No. 7,294,754, U.S. Patent No. 7,576,259, U.S. Patent No. 7,659,442, U.S. Patent No. 8,816,150, U.S. Patent No. 9,414,575, U.S. Patent No. 9,730,434, and U.S. Patent No. 10,039,269, each of which is incorporated herein by reference in its entirety for all purposes (describing the VELOCIMOUSE® method for producing mouse ES cells and genetically modified mice). See also U.S. Patent Application Publication No. 2014 / 0235933(A1), U.S. Patent Application Publication No. 2014 / 0310828(A1), each of which is incorporated herein by reference in its entirety for all purposes (describing methods for producing rat ES cells and genetically modified rats). See also Cho et al. (2009) Curr. Protoc. Cell. Biol. 42:19.11.1-19.11.22 (doi:10.1002 / 0471143030.cb1911s42) and Gama Sosa et al. (2010) Brain Struct. Funct. 214(2-3):91-109, each of which is incorporated by reference in its entirety for all purposes.

[0212] For example, a method of producing a tau reporter non-human animal described herein may include: (1) providing a pluripotent cell (e.g., an embryonic stem (ES) cell, e.g., a mouse ES cell or a rat ES cell) comprising a first nucleic acid encoding a 4R tau isoform linked to a first reporter protein and a second nucleic acid encoding a 3R tau isoform linked to a second reporter protein; (2) introducing the genetically modified pluripotent cell into a non-human animal host embryo; and (3) gestation of the host embryo in a surrogate mother.

[0213] As another example, a method for producing a tau reporter non-human animal described herein may include: (1) modifying the genome of a pluripotent cell (e.g., an embryonic stem (ES) cell, e.g., a mouse ES cell or a rat ES cell) that comprises a first nucleic acid encoding a 4R tau isoform linked to a first reporter protein and a second nucleic acid encoding a 3R tau isoform linked to a second reporter protein; (2) identifying or selecting a genetically modified pluripotent cell that comprises a first nucleic acid encoding a 4R tau isoform linked to a first reporter protein and a second nucleic acid encoding a 3R tau isoform linked to a second reporter protein; (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 may be introduced into the host embryo at any stage, such as at the blastocyst stage or pre-morula stage (i.e., 4-cell stage or 8-cell stage). Optionally, the host embryo comprising the modified pluripotent cells (e.g., non-human ES cells) may be incubated to the blastocyst stage before being implanted and gestation in a surrogate mother to produce an F0 non-human animal. The surrogate mother may then produce an F0 generation non-human animal comprising a 4R tau isoform linked to a first reporter protein and a 3R tau isoform linked to a second reporter protein, or a first nucleic acid encoding a 4R tau isoform linked to a first reporter protein and a second nucleic acid encoding a 3R tau isoform linked to a second reporter protein. The non-human animal may be capable of transmitting the first nucleic acid encoding a 4R tau isoform linked to a first reporter protein and the second nucleic acid encoding a 3R tau isoform linked to a second reporter protein through the germline.

[0214] Alternatively, the method of producing a tau reporter non-human animal described elsewhere herein may include (1) modifying the genome of a one-cell stage embryo to include a first nucleic acid encoding a 4R tau isoform linked to a first reporter protein and a second nucleic acid encoding a 3R tau isoform linked to a second reporter protein using the methods described above for modifying pluripotent cells, (2) selecting the genetically modified embryo, and (3) gestating the genetically modified embryo in a surrogate mother. Offspring capable of transmitting the genetic modification through the germline are generated.

[0215] Nuclear transfer techniques can also be used to generate non-human mammals. Briefly, methods for nuclear transfer may 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 in the uterus of an animal to form an embryo; and (5) allowing the embryo to develop. In such methods, oocytes are generally retrieved from dead animals, but may also be isolated from the oviducts and / or ovaries of living animals. The oocytes may be matured in a variety of well-known media prior to enucleation. Enucleation of the oocytes may be accomplished by a number of well-known methods. Insertion of the donor cell or nucleus into the enucleated oocyte to form the reconstituted cell may be accomplished by microinjecting the donor cell under the zona pellucida prior to fusion. Fusion can be induced by application of a DC electric pulse across the contact / fusion surface (electrofusion), exposure of the cells to fusion-promoting chemicals such as polyethylene glycol, or by inactivated viruses such as Sendai virus. The reconstituted cells can be activated by electrical and / or non-electrical means before, during, and / or after fusion of the nuclear donor and recipient oocytes. Activation methods include electrical pulses, chemically induced shocks, penetration with sperm, increasing the levels of divalent cations in the oocyte, and reducing phosphorylation of cellular proteins in the oocyte (with kinase inhibitors). The activated reconstituted cells, or embryos, can be cultured in well-known media and then implanted into the uterus of an animal. See, e.g., U.S. Patent Application Publication No. 2008 / 0092249, WO 1999 / 005266, WO 2004 / 0177390, WO 2008 / 017234, and U.S. Patent No. 7,612,250, each of which is incorporated by reference in its entirety for all purposes.

[0216] Modified cells or one-cell stage embryos can be generated, for example, via recombination by: (a) introducing into a cell one or more exogenous donor nucleic acids (e.g., targeting vectors) comprising an insert nucleic acid flanked by 5' and 3' homology arms corresponding to, for example, 5' and 3' target sites (e.g., target sites adjacent to an endogenous sequence desired to be deleted and replaced with an insert nucleic acid), the insert nucleic acid comprising a first nucleic acid encoding a 4R tau isoform linked to a first reporter protein and a second nucleic acid encoding a 3R tau isoform linked to a second reporter protein; and (b) identifying at least one cell comprising in its genome the first nucleic acid encoding the 4R tau isoform linked to a first reporter protein and the second nucleic acid encoding the 3R tau isoform linked to a second reporter protein. Similarly, modified non-human animal genomes or humanized non-human animal target loci can be generated through recombination, for example, by (a) contacting the genome or gene with one or more exogenous donor nucleic acids (e.g., targeting vectors) that include 5' and 3' homology arms corresponding to 5' and 3' target sites (e.g., target sites adjacent to endogenous sequences desired for deletion and replacement with one or more inserted nucleic acids (e.g., a first nucleic acid encoding a 4R tau isoform linked to a first reporter protein and a second nucleic acid encoding a 3R tau isoform linked to a second reporter protein). Optionally, a nuclease agent that targets the endogenous sequence desired for deletion can be introduced along with the exogenous donor nucleic acid. Any nuclease agent that induces a nick or double-stranded break at the desired recognition site can be used. Examples of suitable nucleases include transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), meganucleases, and clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) systems (e.g., CRISPR / Cas9 systems) or components of such systems (e.g., CRISPR / Cas9).See, e.g., U.S. Patent Application Publication Nos. 2013 / 0309670 and 2015 / 0159175, each of which is incorporated by reference in its entirety for all purposes. In one example, the nuclease comprises a Cas9 protein and a guide RNA. In another example, the nuclease comprises a Cas9 protein and two or more, three or more, or four or more guide RNAs.

[0217] The exogenous donor nucleic acid may be for insertion or homologous recombination via non-homologous end joining. The exogenous donor nucleic acid may comprise deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), which may be single-stranded or double-stranded, and which may be in linear or circular form. For example, the donor nucleic acid may be a single-stranded oligodeoxynucleotide (ssODN). The heterologous donor nucleic acid may also comprise a heterologous sequence that is not present in the untargeted endogenous locus. For example, the exogenous donor nucleic acid may comprise a selection cassette, such as a selection cassette flanked by recombinase recognition sites.

[0218] In cells other than one-cell stage embryos, the exogenous donor nucleic acid may be a "targeting vector" or "LTVEC," including targeting vectors that contain homology arms corresponding to and derived from larger nucleic acid sequences than those typically used in other approaches aimed at achieving homologous recombination in cells. See, e.g., U.S. Patent Application Publication No. 2004 / 0018626, WO 2013 / 163394, U.S. Patent No. 9,834,786, U.S. Patent No. 10,301,646, WO 2015 / 088643, U.S. Patent No. 9,228,208, U.S. Patent No. 9,546,384, U.S. Patent No. 10,208,317, and U.S. Patent Application Publication No. 2019-0112619, each of which is incorporated herein by reference in its entirety for all purposes. LTVEC also includes targeting vectors that contain nucleic acid inserts with nucleic acid sequences larger than those typically used in other approaches aimed at performing homologous recombination in cells. For example, LTVEC allows modification of large loci that cannot be accommodated by conventional plasmid-based targeting vectors due to size limitations. For example, the targeted locus can be a cellular locus that cannot be targeted using conventional methods or that can only be targeted imprecisely or with significantly lower efficiency in the absence of nicks or double-strand breaks induced by nuclease agents (e.g., Cas proteins) (i.e., the 5' and 3' homology arms can be accommodated). LTVEC can be of any length, and is usually at least 10 kb long. The sum of the 5' and 3' homology arms of LTVEC is typically at least 10 kb.For the generation and use of large targeting vectors (LTVEC) derived from bacterial artificial chromosome (BAC) DNA by bacterial homologous recombination (BHR) reactions using VELOCIGENE® genetic engineering technology, see, e.g., U.S. Patent No. 6,586,251 and Valenzuela et al. (2003) Nat. Biotechnol. 21(6):652-659, each of which is incorporated by reference in its entirety for all purposes. For the generation of LTVEC by in vitro assembly methods, see, e.g., U.S. Patent Application Publication No. 2015 / 0376628 and WO 2015 / 200334, each of which is incorporated by reference in its entirety for all purposes.

[0219] The method may further include identifying cells or animals that have a modified target genomic locus. A variety of methods can be used to identify cells and animals that have a targeted genetic modification. The screening step can include, for example, a quantitative assay to evaluate the modification of parental chromosome alleles (MOA). See, for example, U.S. Patent Application Publication No. 2004 / 0018626, U.S. Patent Application Publication No. 2014 / 0178879, U.S. Patent Application Publication No. 2016 / 0145646, WO 2016 / 081923, and Frendewey et al. (2010) Methods Enzymol. 476:295-307, each of which is incorporated herein by reference in its entirety for all purposes. For example, the quantitative assay can be performed via quantitative PCR, such as 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-target reference locus. The primer set can include a fluorescent probe that recognizes the amplified sequence. Other examples of suitable quantitative assays include fluorescence-mediated in situ hybridization (FISH), comparative genomic hybridization, isothermal DNA amplification, quantitative hybridization to immobilized probes, INVADER® probes, TAQMAN® molecular beacon probes, or ECLIPSE™ probe technology (see, e.g., U.S. Patent Application Publication No. 2005 / 0144655, which is incorporated by reference in its entirety for all purposes).

[0220] Various methods provided herein allow for the generation of genetically modified non-human F0 animals, where the cells of the genetically modified F0 animals include a first nucleic acid encoding a 4R tau isoform linked to a first reporter protein and a second nucleic acid encoding a 3R tau isoform linked to a second reporter protein. It is recognized that depending on the method used to generate the F0 animal, the number of cells in the F0 animal that have a first nucleic acid encoding a 4R tau isoform linked to a first reporter protein and a second nucleic acid encoding a 3R tau isoform linked to a second reporter protein will vary. In mice, the introduction of donor ES cells into a pre-morula stage embryo (e.g., an 8-cell stage mouse embryo) from a mouse, for example, via the VELOCIMOUSE® method, allows a greater percentage of the cell population of the F0 mouse to contain cells with targeted genetic modifications. For example, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 86%, at least 87%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the cellular contribution of the non-human F0 animal may comprise a cell population having a targeted modification. The cells of the genetically modified F0 animal may be heterozygous for a first nucleic acid encoding a 4R tau isoform linked to a first reporter protein and a second nucleic acid encoding a 3R tau isoform linked to a second reporter protein, or may be homozygous for a first nucleic acid encoding a 4R tau isoform linked to a first reporter protein and a second nucleic acid encoding a 3R tau isoform linked to a second reporter protein.

[0221] All patent applications, websites, other publications, accession numbers, etc. cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item was specifically and individually indicated to be incorporated by reference. Where different versions of a sequence are associated with an accession number at different times, the version associated with the accession number at the effective filing date of this application is meant. The effective filing date means the filing date of the actual application or the priority application prior to the accession number, if applicable. Similarly, where different versions of a publication, website, etc. are published at different times, the version published closest to the effective filing date of this application is meant, unless otherwise specified. Any feature, step, element, embodiment, or aspect of the invention can be used in combination with any other, unless otherwise indicated. The invention has been described in some detail through diagrams and examples for purposes of clarity and understanding, but it will be apparent that certain changes and modifications can be made within the scope of the appended claims.

[0222] A brief description of the sequence 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 starting at the 5'-end of the sequence and proceeding forward (i.e., left to right on 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 strand shown. Where a nucleotide sequence encoding an amino acid sequence is provided, it is understood that codon-degenerate variants thereof which encode the same amino acid sequence are also provided. The amino acid sequences follow the standard convention of starting at the amino terminus of the sequence and proceeding forward (i.e., left to right on each line) to the carboxy terminus.

[0223] [Table 2] EXAMPLES

[0224] Example 1. Development of a Cellular Screening Assay to Distinguish Between Reagents Targeting 4R and 3R Tau The human MAPT gene encodes six different isoforms of tau: 2N4R, 1N4R, 0N4R, 2N3R, 1N3R, 0N3R. See FIG. 1. The difference between transcripts containing 4R (4 repeat) tau versus 3R (3 repeat) tau is based on the inclusion (4R) or exclusion (3R) of exon 10. Humans normally express equal ratios of 3R and 4R tau. In some tauopathies, such as Alzheimer's disease, experimental evidence from postmortem brain tissue suggests that insoluble aggregates of tau are composed of 3R and 4R tau. In rarer tauopathy diseases, such as progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD), 4R tau protein is the aggregating species of tau. The reasons underlying these different types of aggregates in disease are unclear. Therapies targeting total tau (e.g., 3R+4R tau) versus only 4R tau may have beneficial effects in different disease states.

[0225] One major challenge to date has been to develop assays for measuring the mRNA of different isoforms of tau. For example, the "R / repeat" domain of tau makes it difficult to design primer pairs and TaqMan probes for measuring 3R tau. With such a paucity of assays for measuring certain isoforms of tau, it is difficult to accurately test the specificity of reagents (e.g., siRNAs) that only reduce 4R tau and do not affect 3R tau versus reagents (e.g., siRNAs) that should reduce both (e.g., total tau targeting strategies).

[0226] We, for example, sought to identify 4R tau targeting siRNAs, but the lack of an mRNA assay to measure 3R tau made it impossible to perform this type of screening. To circumvent this problem, we generated a cell screening assay to identify compounds (e.g., siRNAs or gRNAs) that target total tau versus compounds that specifically target 4R tau. This involved generating a secondary cell line (e.g., HEK293) that expresses fluorescently tagged fusion proteins of 4R and 3R tau. More specifically, the HEK293 cell line equally expressed both xN4R-tau-eYFP and xN3R-tau-mCherry (where xN=0N, 1N, or 2N), providing a simple and robust system to screen for compounds that specifically target 4R tau. Furthermore, the fusion messenger RNA and protein can be used as surrogate readouts for the reduction of each form of tau. For example, an siRNA targeting 4R tau should reduce four readouts: (1) 4R tau mRNA using a 4R-specific TaqMan probe, (2) eYFP mRNA using an eYFP-specific TaqMan probe, (3) eYFP protein levels (e.g., as measured by any protein detection technique (western blotting, ELISA)), and (4) eYFP fluorescence. Alternatively, an siRNA that also reduces 3R tau should affect (1) mCherry messenger RNA using an mCherry-specific TaqMan probe, (2) mCherry protein levels (e.g., as measured by any protein detection technique (western blotting, ELISA)), and (3) mCherry fluorescence.

[0227] This cell line can be used for screening many different therapeutic modalities, including siRNA, gRNA, and intrabodies (intracellular antibodies). Intrabodies can be useful for intracellular degradation of proteins located in the cytoplasm. For example, intrabodies targeting 4R tau or 3R tau should reduce yellow (eYFP) or red (mCherry) protein signals, respectively.

[0228] To generate the cell lines, full-length human 2N4R tau (NCBI accession number: NM_005910.6) and 2N3R tau (NCBI accession number: NM_001203252.2) were cloned in frame with either eYFP or mCherry into the pLvX vector to generate 2N4R-eYFP and 2N3R-mCherry fusion proteins. The fusion protein coding sequences were operably linked to the EF1A promoter. The plasmid DNA was packaged into lentivirus. The 2N4R-eYFP fusion protein is set forth in SEQ ID NO: 15 and encoded by the sequence set forth in SEQ ID NO: 19. The 2N3R-mCherry fusion protein is set forth in SEQ ID NO: 16 and encoded by the sequence set forth in SEQ ID NO: 20.

[0229] HEK293 cells were grown to 80% confluence in 24-well plates and grown in 10% fetal bovine serum in high glucose DMEM. The cells were then transduced with lentivirus carrying 2N4R-eYFP. After 3 days, eYFP+ cells were sorted into single cells in individual wells to expand the clonal cell lines. Once the eYFP+ cells reached confluence, five cell lines were selected for subsequent cryopreservation and further expanded for transduction with lentivirus carrying 2N3R-mCherry. After another 3 days, eYFP+mCherry+ cells were sorted into single cells in 96-well plates and the cells were grown for approximately 2 weeks to identify surviving clones that continued to grow. 20 distinct eYFP / mCherry double-positive clonal cell lines were generated and used for subsequent screening in the TaqMan assay. The three 2N4R-eYFP / 2N3R-mCherry cell lines (clones C2, C3, and C5) expressed eYFP and mCherry mRNA at approximately a 1:1 ratio. See Figure 2. Clone C3 was selected as having high copy numbers of 2N4R-eYFP and 2N3R mCherry RNA and similar relative expression levels of each. See Figure 3.

[0230] HEK293, 2N4R-eYFP / 2N3R-mCherry cells were maintained in complete medium (DMEM, 10% FBS, penicillin, streptomycin, GlutaMAX, G418, and hygromycin) at 37 °C and 5% CO2. To demonstrate that eYFP and mCherry can serve as surrogates for 4R tau and 3R tau levels, respectively, dual reporter 2N4R-eYFP / 2N3R-mCherry cells were plated at 1.6 × 10 5 Cells / well were seeded. On the day of transfection (approximately 70% confluence), complete medium was aspirated from each well and replaced with 450 μL of OptiMem. siRNA duplex solutions were diluted to 200 nM, 20 nM, and 2 nM in OptiMEM. Lipofectamine RNAMax was diluted in OptiMem according to the manufacturer's protocol (3 uL Lipofectamine / 50 uL of medium). Equal volumes of siRNA duplex solution and diluted Lipofectamine solution were combined (10× siRNA / lipid solution), mixed, and incubated at room temperature for a minimum of 5 minutes. 50 uL of each 10× siRNA / lipid solution was added in triplicate to the appropriate wells of a 24-well plate. Plates were then returned to the incubator for 48 hours before immunofluorescence analysis. Specifically, cells were treated with either 10 nM total tau siRNA (targeting both 3R and 4R isoforms) or 10 nM mCherry siRNA (HAIJE-000013, SEQ ID NOs: 1 and 2). After 48 hours, eYFP and mCherry immunofluorescence were measured. mCherry siRNA reduced the mCherry signal, but eYFP signal was unaffected. In contrast, total tau siRNA reduced both eYFP signal intensity and mCherry intensity. See Figure 4.

[0231] Similar experiments were then performed using 10 nM mCherry siRNA (HAIJE-000015 (SEQ ID NOs: 3 and 4) or HAIJE-000017 (SEQ ID NOs: 5 and 6) or 10 nM YFP siRNA (P-002048-01, SEQ ID NOs: 7 and 8). Both mCherry siRNAs reduced the mCherry signal intensity but not the eYFP signal intensity, whereas YFP siRNA reduced the eYFP signal intensity but not the mCherry signal intensity. See FIG. 5.

[0232] Potential 4R-specific siRNAs were then designed by designing 65 siRNAs along exon 10 of the MAPT gene, which encodes the R2 domain. The R2 domain is set forth in SEQ ID NO: 21, and the coding sequence of the R2 domain is set forth in SEQ ID NO: 22. Two total tau siRNAs were designed as positive controls, a YFP siRNA was used as an eYFP knockdown control, and an mCherry siRNA was used as an mCherry knockdown control. 24 hours after treatment, doses of 0.1 nM and 1 nM were tested in triplicate. TaqMan assays were performed for total tau, 4R tau, mCherry, and eYFP.

[0233] To select 4R-specific or 4R-preferential siRNAs, parameters can be set, for example, as in FIG. 6. Pure 4R-specific siRNAs can be selected, for example, as those with robust knockdown of 4R tau and eYFP (e.g., at least 75% reduction in mRNA) and no effect on mCherry (e.g., about 0% reduction in mCherry mRNA). 4R-preferential siRNAs can be selected, for example, as those with robust knockdown of 4R tau and eYFP (e.g., at least 70% reduction in mRNA) and little to no knockdown effect on mCherry (e.g., 30% or less reduction in mCherry mRNA). Using the "4R-preferential" criteria, 13 of 65 candidate 4R siRNAs met the criteria when administered at 1 nM (less than 30% 4R tau message remaining at 1 nM and less than 30% mCherry knockdown). See FIG. 7. We also found a near perfect correlation in TaqMan assays for 4R tau and eYFP, validating eYFP as a surrogate for 4R tau levels, see Figure 8. Similarly, mCherry was a reliable surrogate for 3R tau levels.

[0234] Relative 4R tau and mCherry expression for 65 candidate 4R siRNA duplexes is shown in Figure 9A. HEK293-2N4R-YFP+2N3R-mCherry cells were treated with 1 nM GalNAc-conjugated siRNA in lipofectamine and harvested 48 hours later for TaqMan assays (mCherry and YFP). For each sample, the left bar shows the results of the 4R assay and the right bar shows the results of the mCherry assay (surrogate for 3R tau). Relative 4R tau and mCherry expression for 13 selected siRNAs is shown in Figure 9B. Solid bars are samples treated with 1 nM siRNA and hatched bars are samples treated with 0.1 nM siRNA. This validated the cell line as a tool for screening 4R-specific reagents. Until now, screening of 4R-specific reagents such as siRNAs has not been possible due to the lack of protein and mRNA assays to measure 3R tau. To address this issue, we generated and validated a dual reporter HEK293 2N4R-eYFP+2N3R-mCherry fusion protein cell line to screen for 4R-specific candidates.

[0235] Using the 3R / 4R dual reporter cells, dose-response analysis was performed on each compound as described above using TaqMan to examine relative 4R tau expression and relative eYFP expression (4R tau surrogate) and relative mCherry expression (3R tau surrogate). Results for two 4R tau-preferring siRNAs are shown in FIG. 10A, and results for two additional candidate 4R tau siRNAs are shown in FIG. 11A. The data show that the 4R-preferring siRNAs are highly specific for 4R tau, with little to no effect on 3R tau. However, based on the sequence homology of the R2 domain to the other R domains, we sought to ensure that the 4R-preferring siRNAs would retain specificity only given the 2N3R-mCherry target. Therefore, we performed an identical series of experiments in HEK293 2N3R-mCherry-only cells, using the same dose ranges tested in the dual reporter cell line. We found no effect of 4R siRNA molecules when tested in this cell line (i.e., no knockdown of 2N3R-mCherry). See Figures 10B and 11B. As a final control, we performed the same experiment in HEK293 2N4R-YFP only cells and the efficacy of 4R-preferential siRNA was still observed. See Figures 10C and 11C.

[0236] The same four 4R tau siRNAs were then validated for 4R tau protein knockdown using protein dot blotting with commercially available total tau antibody (mouse monoclonal, clone tau12), 4R tau antibody (mouse monoclonal, clone 7D12.1), and 3R tau antibody (mouse monoclonal, clone 8E6\C11). The antibodies were first validated to confirm specificity by blotting with recombinant 1N3R or 1N4R protein (left panel of FIG. 12). HEK293 dual reporter cells were then treated with vehicle control or siRNA (1 nM for 48 h) in Lipofectamine RNAiMax transfection reagent (diluted according to manufacturer's instructions) (three 4R-preferential siRNAs, and one mixed-selective siRNA). Lysates were harvested in RIPA buffer supplemented with protease / phosphatase inhibitors, and a total of 7 μg (for total tau dot blots) or 21 μg (for 4R tau dot blots), or 7 μg (for 3R tau dot blots) were spotted onto the dot blot apparatus, and blots were subjected to blotting with total tau, 4R tau, and 3R tau antibodies. The results are shown in Figure 12, and the dual reporter HEK293 2N4R-eYFP+2N3R-mCherry fusion protein cell line is further validated to screen for 4R-specific candidates.

[0237] We also verified 4R preferential knockdown using the same 4R tau siRNA using a protein ELISA type assay. As shown in Figure 13, total soluble tau was measured using a commercially available Total Tau ALPHALISA (Perkin Elmer, Cat. No. AL271C) according to the manufacturer's instructions, and 4R tau protein was measured using a PathScan ELISA kit from Cell Signaling Technologies (Cat. No. 29443). As expected, 4R tau levels were lower than that of total tau, since the total tau assay measures both 4R tau (which should be reduced) and 3R tau (which should be minimally or unaffected) levels. A 3R tau ELISA assay is performed to quantitatively measure 3R tau levels. Flow cytometry analysis of eYFP and mCherry intensity is also performed to assess the impact on protein levels.

[0238] The same 4R tau siRNA was then tested in vivo in MAPT humanized mice to further validate the dual reporter HEK293 2N4R-eYFP+2N3R-mCherry fusion protein cell line. siRNA with identical targeting sequences was synthesized using C16 conjugates to enable CNS cell delivery. We then performed intracerebral injections of 300 μg of each siRNA and allowed the siRNA to incubate in vivo for 30 days. Naive and vehicle (artificial cerebrospinal fluid, aCSF) were used as negative controls. Total tau TaqMan assay (to assess the overall levels of 3R+4R tau) and 4R tau assay were performed and the results are shown in FIG. 14.

[0239] Dot blotting was performed on brain lysates from MAPT humanized mice treated with 4R tau siRNA using total tau, 3R tau, and 4R tau antibodies. Lysates were harvested and a total of 1 μg / well (for total tau dot blot), 1 μg / well (for 4R tau dot blot), or 5 μg / well (for 3R tau dot blot) was spotted onto a dot blot apparatus, and blots were subjected to blotting with total tau, 4R tau, and 3R tau antibodies. Results confirmed 4R-preferential knockdown for the top four siRNAs (data not shown). RNAscope assays to detect 3R tau, 4R tau, and total tau mRNA are performed on sections from neuronal cultures (in vitro) and brains of mice treated with 4R tau-preferential siRNA.

[0240] Example 2. Development of an in vivo screening assay to distinguish between reagents targeting 4R and 3R tau A mouse model for in vivo testing of 4R siRNA is generated. Two constructs: 1N4R tau(R5L)-P2A-eYFP and 1N3R tau(R5L)-P2A-mCherry are virally transduced into tau knockout mice using AAV-PhPeB with a synapsin promoter (driving robust expression in neurons of the central nervous system). The 1N4R tau(R5L)-P2A-eYFP fusion protein is set forth in SEQ ID NO: 35 and encoded by the sequence set forth in SEQ ID NO: 36. The 1N3R tau(R5L)-P2A-mCherry fusion protein is set forth in SEQ ID NO: 41 and encoded by the sequence set forth in SEQ ID NO: 42. Similarly, AAV-PhPeB with the synapsin promoter (driving robust expression in neurons of the central nervous system) is used to virally transduce two constructs: 1N4R tau(L237V)-P2A-eYFP and 1N3R tau(L237V)-P2A-mCherry into tau knockout mice. The 1N4R tau(L237V)-P2A-eYFP fusion protein is set forth in SEQ ID NO: 37 and encoded by the sequence set forth in SEQ ID NO: 38. The 1N3R tau(L237V)-P2A-mCherry fusion protein is set forth in SEQ ID NO: 43 and encoded by the sequence set forth in SEQ ID NO: 44. Similarly, AAV-PhPeB with the synapsin promoter (driving robust expression in neurons of the central nervous system) is used to virally transduce two constructs: 1N4R tau(G243V)-P2A-eYFP and 1N3R tau(G243V)-P2A-mCherry into tau knockout mice. The 1N4R tau(G243V)-P2A-eYFP fusion protein is set forth in SEQ ID NO: 39 and encoded by the sequence set forth in SEQ ID NO: 40. The 1N3R tau(G243V)-P2A-mCherry fusion protein is set forth in SEQ ID NO: 45 and encoded by the sequence set forth in SEQ ID NO: 46. See FIG. 15. Similar to the in vitro screens using the cell lines described above, eYFP and mCherry mRNA levels are used as surrogate readouts for 4R tau and 3R tau mRNA levels, respectively.The difference is that because we are driving neuronal expression of aggregating tau, we do not want eYFP and mCherry to interfere with the proper function / folding and microtubule binding of these aggregating tau forms. Therefore, P2A is used in each construct, which allows two proteins to be produced from a single mRNA molecule due to ribosomal skipping.

[0241] The mouse model is used to test candidate 4R tau targeting reagents using the readout described in Example 1. Behavioral metrics are performed to assess motor and eye defects. Behavioral and disease pathology such as tau aggregates are assessed.

Claims

1. 1. A method for assessing the activity of a tau-targeting reagent, comprising: assessing the activity of the tau-targeting reagent in cells previously administered the tau-targeting reagent, wherein the cells comprise a four-repeat (4R) tau isoform linked to a first reporter protein and a three-repeat (3R) tau isoform linked to a second reporter protein different from the first reporter protein; (I) said evaluating comprises measuring 4R tau isoform messenger RNA expression and second reporter protein messenger RNA expression, and a greater relative decrease in 4R tau isoform messenger RNA expression compared to second reporter protein messenger RNA expression after administering the tau targeting reagent to the cell indicates that the tau targeting reagent is a 4R-preferential tau targeting reagent; (II) said evaluating comprises measuring first reporter protein messenger RNA expression and second reporter protein messenger RNA expression, and a greater relative decrease in first reporter protein messenger RNA expression compared to second reporter protein messenger RNA expression after administering said tau targeting reagent to said cell indicates that said tau targeting reagent is a 4R-preferential tau targeting reagent; or (III) said evaluating comprises measuring a first reporter protein expression and a second reporter protein expression, wherein a greater relative decrease in first reporter protein expression compared to second reporter protein expression after administering the tau targeting reagent to the cell indicates that the tau targeting reagent is a 4R-preferential tau targeting reagent. method.

2. 2. The method of claim 1, wherein the activity of the tau-targeting reagent is assessed relative to control cells that have not been administered the tau-targeting reagent, or is assessed relative to before administration of the tau-targeting reagent.

3. (I) at least a 70% reduction in 4R tau isoform messenger RNA expression and no more than a 30% reduction in second reporter protein messenger RNA expression after administration of the tau targeting reagent to the cells indicates that the tau targeting reagent is a 4R-preferential tau targeting reagent; or (II) at least a 70% decrease in first reporter protein messenger RNA expression and no more than a 30% decrease in second reporter protein messenger RNA expression after administering the tau targeting reagent to the cell, indicates that the tau targeting reagent is a 4R-preferential tau targeting reagent; or (III) a decrease of at least 70% in expression of a first reporter protein and a decrease of no more than 30% in expression of a second reporter protein after administering the tau targeting reagent to the cells indicates that the tau targeting reagent is a 4R-preferential tau targeting reagent.

3. The method according to claim 1 or 2. (I) the 4R tau isoform and the 3R tau isoform are human; and / or (II) (a) the cell comprises a first fusion protein comprising the 4R tau isoform fused to the first reporter protein and a second fusion protein comprising the 3R tau isoform fused to the second reporter protein; Optionally, the cell comprises a first nucleic acid encoding the first fusion protein and a second nucleic acid encoding the second fusion protein, and the cell expresses the first fusion protein and the second fusion protein; or (b) the cell comprises a first nucleic acid comprising a coding sequence for the 4R tau isoform and a coding sequence for the first reporter protein, and a second nucleic acid comprising a coding sequence for the 3R tau isoform and a coding sequence for the second reporter protein, wherein the cell expresses the 4R tau isoform, the 3R tau isoform, the first reporter protein, and the second reporter protein; Optionally, the coding sequence for the 4R tau isoform and the coding sequence for the first reporter protein are separated by a coding sequence for a first 2A peptide, and the coding sequence for the 3R tau isoform and the coding sequence for the second reporter protein are separated by a coding sequence for a second 2A peptide; Optionally, the first 2A peptide is a first P2A peptide and the second 2A peptide is a second P2A peptide.

3. The method according to claim 1 or 2. (I) the first nucleic acid and the second nucleic acid are integrated into the genome of the cell; and / or (II) (a) the cell comprises a viral vector comprising the first nucleic acid and the second nucleic acid; Optionally, the viral vector is a lentiviral vector or an adeno-associated viral (AAV) vector; or (b) the cell comprises a first viral vector comprising the first nucleic acid and a second viral vector comprising the second nucleic acid; Optionally, the first viral vector and the second viral vector are lentiviral vectors or adeno-associated viral (AAV) vectors. The method of claim 4.

6. The cell is a mammalian cell, Optionally, the cell is a human cell, the cell is an immortalized cell, and / or the cell is a HEK293 cell.

3. The method according to claim 1 or 2.

7. 1. A method for assessing the activity of a tau-targeting reagent in vivo, comprising: (a) administering the tau targeting reagent to a non-human animal comprising a four-repeat (4R) tau isoform linked to a first reporter protein and a three-repeat (3R) tau isoform linked to a second reporter protein different from the first reporter protein; (b) assessing the activity of the tau-targeting reagent in the non-human animal; (I) said evaluating comprises measuring 4R tau isoform messenger RNA expression and second reporter protein messenger RNA expression, and a greater relative decrease in 4R tau isoform messenger RNA expression compared to second reporter protein messenger RNA expression after administering the tau-targeting reagent to the non-human animal indicates that the tau-targeting reagent is a 4R-preferential tau-targeting reagent; or (II) said evaluating comprises measuring first reporter protein messenger RNA expression and second reporter protein messenger RNA expression, and a greater relative decrease in first reporter protein messenger RNA expression compared to second reporter protein messenger RNA expression after administering said tau targeting reagent to said non-human animal indicates that said tau targeting reagent is a 4R-preferential tau targeting reagent; or (III) said evaluating comprises measuring a first reporter protein expression and a second reporter protein expression, wherein a greater relative decrease in first reporter protein expression compared to second reporter protein expression after administering the tau targeting reagent to the non-human animal indicates that the tau targeting reagent is a 4R-preferential tau targeting reagent. method.

8. 8. The method of claim 7, wherein the activity of the tau-targeting reagent is assessed relative to a control non-human animal that has not been administered the tau-targeting reagent, or is assessed relative to before administration of the tau-targeting reagent.

9. (I) at least a 70% reduction in 4R tau isoform messenger RNA expression and no more than a 30% reduction in second reporter protein messenger RNA expression after administration of the tau targeting reagent to the non-human animal indicates that the tau targeting reagent is a 4R-preferential tau targeting reagent; or (II) at least a 70% decrease in first reporter protein messenger RNA expression and no more than a 30% decrease in second reporter protein messenger RNA expression after administering the tau targeting reagent to the non-human animal indicates that the tau targeting reagent is a 4R-preferential tau targeting reagent; or (III) a decrease of at least 70% in expression of a first reporter protein and a decrease of no more than 30% in expression of a second reporter protein after administering the tau targeting reagent to the non-human animal indicates that the tau targeting reagent is a 4R-preferential tau targeting reagent.

9. The method according to claim 7 or 8. (I) the 4R tau isoform and the 3R tau isoform are human, and optionally the 4R tau isoform and the 3R tau isoform each comprise an R5L mutation, an L237V mutation, or a G243V mutation, or optionally the 4R tau isoform and the 3R tau isoform each comprise an N279K mutation, an L284R mutation, or an S285R mutation; and / or (II) (a) the non-human animal comprises a first fusion protein comprising the 4R tau isoform fused to the first reporter protein and a second fusion protein comprising the 3R tau isoform fused to the second reporter protein; Optionally, the non-human animal comprises a first nucleic acid encoding the first fusion protein and a second nucleic acid encoding the second fusion protein, and the non-human animal expresses the first fusion protein and the second fusion protein; or (b) the non-human animal comprises a first nucleic acid comprising a coding sequence for the 4R tau isoform and a coding sequence for the first reporter protein, and a second nucleic acid comprising a coding sequence for the 3R tau isoform and a coding sequence for the second reporter protein, wherein the non-human animal expresses the 4R tau isoform, the 3R tau isoform, the first reporter protein, and the second reporter protein; Optionally, the coding sequence for the 4R tau isoform and the coding sequence for the first reporter protein are separated by a coding sequence for a first 2A peptide, and the coding sequence for the 3R tau isoform and the coding sequence for the second reporter protein are separated by a coding sequence for a second 2A peptide; Optionally, the first 2A peptide is a first P2A peptide and the second 2A peptide is a second P2A peptide.

9. The method according to claim 7 or 8. (I) the first nucleic acid and the second nucleic acid are integrated into the genome of the non-human animal; and / or (II) (a) the non-human animal comprises a viral vector comprising the first nucleic acid and the second nucleic acid; Optionally, the viral vector is a lentiviral vector or an adeno-associated viral (AAV) vector; or (b) the non-human animal comprises a first viral vector comprising the first nucleic acid and a second viral vector comprising the second nucleic acid; Optionally, the first viral vector and the second viral vector are lentiviral vectors or adeno-associated viral (AAV) vectors. The method of claim 10. (I) the non-human animal is a mammal, optionally the non-human animal is a rodent, optionally the non-human animal is a mouse or the non-human animal is a rat; and / or (II) the 4R tau isoform, the first reporter protein, the 3R tau isoform, and the second reporter protein are expressed in neurons of the central nervous system of the non-human animal; and / or (III) The non-human animal comprises fibrillar tau inclusions.

9. The method according to claim 7 or 8. (I) The first reporter protein is a first fluorescent reporter protein, the second reporter protein is a second fluorescent reporter protein, and the evaluating comprises immunofluorescence staining or flow cytometry; or (II) the assessing step includes assessing tau hyperphosphorylation or tau aggregation; (III) the tau targeting reagent is an RNAi agent or an antisense oligonucleotide; (IV) the tau targeting reagent is an intrabody; or (V) the tau targeting reagent is a nuclease agent, optionally wherein the nuclease agent comprises a Cas protein and a guide RNA designed to target a guide RNA target sequence within a tau coding sequence.

9. The method of any one of claims 1, 2, 7 and 8. (I) The first reporter protein is a first fluorescent reporter protein, and the second reporter protein is a second fluorescent reporter protein; Optionally, the first reporter protein is eYFP and the second reporter protein is mCherry; and / or (II) (a) the 4R tau isoform is a 2N4R tau isoform and the 3R tau isoform is a 2N3R tau isoform; Optionally, the 4R tau isoform comprises the sequence set forth in SEQ ID NO: 13 and the 3R tau isoform comprises the sequence set forth in SEQ ID NO: 14; or (b) the 4R tau isoform is a 1N4R tau isoform and the 3R tau isoform is a 1N3R tau isoform; Optionally, the 4R tau isoform comprises the sequence set forth in SEQ ID NO: 23, 27, 31, or 47, and the 3R tau isoform comprises the sequence set forth in SEQ ID NO: 24, 28, 32, or 49.

9. The method of any one of claims 1, 2, 7 and 8.

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