Non-human animals containing a humanized TRKB locus

Abstract

To provide suitable non-human animals providing the true human target or a close approximation of the true human target of human-TRKB-targeting reagents, thereby enabling testing of the efficacy and mode of action of such agents in live animals as well as pharmacokinetic and pharmacodynamics studies.SOLUTION: Provided is a mouse comprising a genetically modified endogenous TrkB locus, wherein the genetically modified endogenous TrkB locus encodes a tropomyosin receptor kinase B (TrkB) protein, wherein the TrkB protein comprises an endogenous TrkB signal peptide, endogenous TrkB cytoplasmic domain, endogenous TrkB transmembrane domain, and human TrkB extracellular domain, wherein the entire extracellular domain is encoded by a segment of the endogenous TrkB locus that has been detected and replaced with an orthologous human TrkB sequence.SELECTED DRAWING: Figure 4

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application No. 62 / 592,905, filed November 30, 2017, and U.S. Patent Application No. 62 / 661,373, filed April 23, 2018, each of which is incorporated by reference in its entirety for all purposes.

[0002] Reference to sequence listings submitted as text files via EFS Web The sequence listing in file 523380SEQLIST.txt is 154 kilobytes, was created on November 30, 2018, and is hereby incorporated by reference into this specification. [Background technology]

[0003] Tropomyosin receptor kinase B (TRKB) is a promising target for neuroprotection in neurodegenerative diseases such as glaucoma. TRKB is one of the most widely distributed neurotrophic receptors (NTRs) in the brain, which is highly enriched in the neocortex, hippocampus, striatum and brainstem. Binding of brain-derived neurotrophic factor (BDNF) to the TRKB receptor induces its dimerization by conformational change and autophosphorylation of tyrosine residues in the intracellular domain, leading to the activation of signaling pathways involving mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3-kinase (PI3K) and phospholipase C-γ (PLC-γ). Summary of the Invention [Means for solving the problem]

[0004] TRKB is important for neuronal survival, differentiation and function, and TRKB agonists may have therapeutic potential in many neurological, psychiatric and metabolic disorders. However, there remains a need for suitable non-human animals that provide the true human target or a close approximation of the true human target of human TRKB-targeting reagents, thereby enabling the examination of the efficacy and mechanism of action of such agents in living animals, as well as pharmacokinetic and pharmacodynamic studies.

[0005] Non-human animals comprising a humanized TRKB locus are provided, along with methods of using such non-human animals. Non-human animal genomes or cells comprising a humanized TRKB locus are also provided.

[0006] In one aspect, a non-human animal genome, a non-human animal cell, or a non-human animal comprising a humanized TRKB locus is provided, which may comprise a genetically modified endogenous TrkB locus encoding a TRKB protein, the TRKB protein comprising a cytoplasmic domain, a transmembrane domain, and an extracellular domain, the whole or a part of the extracellular domain being encoded by a segment of the endogenous TrkB locus that has been deleted and replaced with an orthologous human TRKB sequence.

[0007] In one embodiment, a non-human animal is provided that comprises a humanized TrkB locus, which can comprise a genetically modified endogenous TrkB locus that encodes a TRKB protein, the TRKB protein comprising a cytoplasmic domain, a transmembrane domain and an extracellular domain, the entire or a portion of the extracellular domain being encoded by a segment of the endogenous TrkB locus that has been deleted and replaced with an orthologous human TRKB sequence.

[0008] In another aspect, a non-human animal cell is provided that comprises in its genome a genetically modified endogenous TrkB locus encoding a TRKB protein, the TRKB protein including a cytoplasmic domain, a transmembrane domain and an extracellular domain, wherein all or a portion of the extracellular domain is encoded by a segment of the endogenous TrkB locus that has been deleted and replaced with an orthologous human TRKB sequence.

[0009] In another aspect, a non-human animal genome is provided that comprises a genetically modified endogenous TrkB locus encoding a TRKB protein, the TRKB protein comprising a cytoplasmic domain, a transmembrane domain and an extracellular domain, wherein all or a portion of the extracellular domain is encoded by a segment of the endogenous TrkB locus that has been deleted and replaced with an orthologous human TRKB sequence.

[0010] In some such non-human animal genomes, non-human animal cells or non-human animals, the TRKB protein comprises a human TRKB extracellular domain. Optionally, the extracellular domain comprises the sequence set forth in SEQ ID NO: 60. Optionally, the entire extracellular domain is encoded by a segment of the endogenous TrkB locus that has been deleted and replaced with the orthologous human TRKB sequence, and optionally the coding sequence for the extracellular domain comprises the sequence set forth in SEQ ID NO: 72.

[0011] In some such non-human animal genomes, non-human animal cells or non-human animals, the TRKB protein comprises an endogenous signal peptide. Optionally, the signal peptide comprises the sequence set forth in SEQ ID NO: 51 or 55. Optionally, the entire signal peptide is encoded by the endogenous TrkB sequence, and optionally the coding sequence for the signal peptide comprises the sequence set forth in SEQ ID NO: 63 or 67.

[0012] In some such non-human animal genomes, non-human animal cells or non-human animals, the TRKB protein comprises an endogenous TRKB transmembrane domain. Optionally, the transmembrane domain comprises the sequence set forth in SEQ ID NO: 53 or 57. Optionally, the entire transmembrane domain is encoded by an endogenous TrkB sequence, and optionally the coding sequence for the transmembrane domain comprises the sequence set forth in SEQ ID NO: 65 or 69.

[0013] In some such non-human animal genomes, non-human animal cells or non-human animals, the TRKB protein comprises an endogenous TRKB cytoplasmic domain. Optionally, the cytoplasmic domain comprises the sequence set forth in SEQ ID NO: 54 or 58. Optionally, the entire cytoplasmic domain is encoded by an endogenous TrkB sequence, and optionally the coding sequence for the cytoplasmic domain comprises the sequence set forth in SEQ ID NO: 66 or 70.

[0014] In some such non-human animal genomes, non-human animal cells or non-human animals, the TRKB protein comprises an endogenous TRKB signal peptide, an endogenous TRKB transmembrane domain and an endogenous TRKB cytoplasmic domain. Optionally, the signal peptide comprises the sequence set forth in SEQ ID NO:51, the transmembrane domain comprises the sequence set forth in SEQ ID NO:53 and the cytoplasmic domain comprises the sequence set forth in SEQ ID NO:54. Optionally, the signal peptide comprises the sequence set forth in SEQ ID NO:55, the transmembrane domain comprises the sequence set forth in SEQ ID NO:57 and the cytoplasmic domain comprises the sequence set forth in SEQ ID NO:58. Optionally, the entire signal peptide, the entire transmembrane domain and the entire cytoplasmic domain are encoded by an endogenous TrkB sequence. Optionally, the coding sequence for the signal peptide comprises the sequence set forth in SEQ ID NO:63, the coding sequence for the transmembrane domain comprises the sequence set forth in SEQ ID NO:65 and the coding sequence for the cytoplasmic domain comprises the sequence set forth in SEQ ID NO:66. Optionally, the coding sequence for the signal peptide comprises the sequence set forth in SEQ ID NO:67, the coding sequence for the transmembrane domain comprises the sequence set forth in SEQ ID NO:69, and the coding sequence for the cytoplasmic domain comprises the sequence set forth in SEQ ID NO:70.

[0015] In some such non-human animal genomes, non-human animal cells or non-human animals, the TRKB protein is a chimeric non-human animal / human TRKB protein. Optionally, the extracellular domain is a human TRKB extracellular domain, the transmembrane domain is an endogenous TRKB protein transmembrane domain, and the cytoplasmic domain is an endogenous TRKB protein cytoplasmic domain. Optionally, the TRKB protein comprises a sequence as set forth in SEQ ID NO: 4 or 5. Optionally, the coding sequence of the genetically modified TrkB locus encoding the TRKB protein comprises a sequence as set forth in SEQ ID NO: 12 or 13.

[0016] Some such non-human animal genomes, non-human animal cells or non-human animals are heterozygous for the genetically modified endogenous TrkB locus. Some such non-human animal genomes, non-human animal cells or non-human animals are homozygous for the genetically modified endogenous TrkB locus.

[0017] Some such non-human animals are mammals. Optionally, the non-human animal is a rodent. Optionally, the rodent is a rat or a mouse.

[0018] Some such non-human animals are rats. Optionally, the TRKB protein comprises the sequence set forth in SEQ ID NO: 5. Optionally, the coding sequence of the genetically modified TrkB locus encoding the TRKB protein comprises the sequence set forth in SEQ ID NO: 13.

[0019] Some such non-human animals are mice. Optionally, the TRKB protein comprises the sequence set forth in SEQ ID NO: 4. Optionally, the coding sequence of the genetically modified TrkB locus encoding the TRKB protein comprises the sequence set forth in SEQ ID NO: 12.

[0020] In another aspect, a method for evaluating the activity of a human TRKB-targeting reagent in vivo using the non-human animal described above is provided. Some such methods include (a) administering a human TRKB-targeting reagent to the non-human animal, and (b) evaluating the activity of the human TRKB-targeting reagent in the non-human animal.

[0021] In some such methods, the activity assessed is neuroprotective activity.

[0022] In some such methods, step (a) comprises injecting a human TRKB targeting reagent into the non-human animal.

[0023] In some such methods, step (b) comprises assessing changes in one or more or all of body weight, body composition, metabolism and locomotion compared to a control non-human animal. Optionally, assessing changes in body composition comprises assessing lean mass and / or fat mass compared to a control non-human animal. Optionally, assessing changes in metabolism comprises assessing changes in food consumption and / or water consumption.

[0024] In some such methods, step (b) comprises assessing TRKB phosphorylation, and / or activation of the MAPK / ERK and PI3K / Akt pathways relative to a control non-human animal.

[0025] In some such methods, step (b) comprises assessing neuroprotective activity. In some such methods, step (b) comprises assessing neuroprotective activity and the non-human animal is a rat. In some such methods, step (b) comprises assessing retinal ganglion cell viability. Optionally, assessing retinal ganglion cell viability comprises assessing retinal ganglion cell density. Optionally, retinal ganglion cell density is measured in retinas excised and stained for retinal ganglion cells. Optionally, retinal ganglion cell viability is assessed in a complete optic nerve transection model following optic nerve injury. Optionally, retinal ganglion cell viability is assessed in an optic nerve crush model.

[0026] In some such methods, the human TRKB targeting reagent is an antigen binding protein. Optionally, the antigen binding protein is a human TRKB agonist antibody. In some such methods, the human TRKB targeting reagent is a small molecule. Optionally, the small molecule is a human TRKB agonist.

[0027] In another aspect, a targeting vector is provided for generating a genetically modified endogenous TrkB locus encoding a TRKB protein, the TRKB protein being encoded by a segment of the endogenous TrkB locus comprising a cytoplasmic domain, a transmembrane domain and an extracellular domain, wherein all or a portion of the extracellular domain has been deleted and replaced with an orthologous human TRKB sequence, the targeting vector comprising an insert nucleic acid comprising the orthologous human TRKB sequence flanked by a 5' homology arm that targets a 5' target sequence in the endogenous TrkB locus and a 3' homology arm that targets a 3' target sequence in the endogenous TrkB locus.

[0028] In another aspect, a method of making any of the non-human animals described above is provided. Some such methods include: (a) introducing into a non-human animal pluripotent cell that is not a one-cell stage embryo (i) an exogenous repair template comprising an insert nucleic acid flanked by a 5' homology arm that hybridizes to a 5' target sequence in an endogenous TrkB locus and a 3' homology arm that hybridizes to a 3' target sequence in an endogenous TrkB locus, the insert nucleic acid comprising an orthologous human TRKB sequence, and (ii) a nuclease agent that targets a target sequence within the endogenous TrkB locus, such that the genome is modified to include a genetically modified endogenous TrkB locus; (b) introducing the modified non-human animal pluripotent cell into a host embryo; and (c) implanting the host embryo into a surrogate mother to produce a genetically modified F0 generation non-human animal comprising a genetically modified endogenous TrkB locus. Optionally, the pluripotent cell is an embryonic stem (ES) cell. Optionally, the nuclease agent is a Cas9 protein and a guide RNA that targets a guide RNA target sequence in endogenous TrkB locus. Optionally, step (a) further comprises introducing a second guide RNA that targets a second guide RNA target sequence in endogenous TrkB locus into the non-human animal pluripotent cell. Optionally, the exogenous repair template is a large targeting vector that is at least 10 kb long, or the exogenous repair template is a large targeting vector whose 5' homology arm and 3' homology arm are combined to be at least 10 kb long.

[0029] Some such methods include (a) introducing into a non-human animal one-cell stage embryo (i) an exogenous repair template comprising an insert nucleic acid flanked by a 5' homology arm that hybridizes to a 5' target sequence in an endogenous TrkB locus and a 3' homology arm that hybridizes to a 3' target sequence in an endogenous TrkB locus, the insert nucleic acid comprising an orthologous human TRKB sequence, and (ii) a nuclease agent that targets a target sequence within the endogenous TrkB locus, such that the genome is modified to include a genetically modified endogenous TrkB locus, and (b) implanting the modified non-human animal one-cell stage embryo into a surrogate mother to produce a genetically modified F0 generation non-human animal comprising a genetically modified endogenous TrkB locus. Optionally, the nuclease agent is a Cas9 protein and a guide RNA that targets a guide RNA target sequence within the endogenous TrkB locus. Optionally, step (a) further comprises introducing into the non-human one-cell stage embryo a second guide RNA that targets a second guide RNA target sequence within the endogenous TrkB locus. [Brief description of the drawings]

[0030] [Figure 1] Figure 1 (not to scale) shows a schematic diagram of the targeting scheme for humanization of the region of the mouse TrkB (mouse Ntrk2) locus that encodes the extracellular domain of TRKB. The top of the figure shows the endogenous mouse TrkB (mouse Ntrk2) locus, and the bottom of the figure shows the large targeting vector.

[0031] [Diagram 2] Figure 2 (not to scale) shows a schematic of the TAQMAN® assay for screening humanization of the mouse TrkB (mouse Ntrk2) locus. The gain of allele (GOA) assay includes 7138hU and 7138hD. The loss of allele (LOA) assay includes 7138U and 7138D.

[0032] [Diagram 3]FIG. 3 shows Western blots assessing total and phospho-TRKB levels in homozygous humanized TRKB mice at 1 hour, 4 hours, and 18 hours after direct hippocampal injection of the TRKB agonist antibody H4H9816P2 or an isotype control antibody.

[0033] [Figure 4] Figure 4 (not to scale) shows a schematic diagram of the targeting scheme for humanization of the region of the rat TrkB (rat Ntrk2) locus that encodes the extracellular domain of TRKB. The top of the figure shows the endogenous rat TrkB (rat Ntrk2) locus, and the bottom of the figure shows the large targeting vector.

[0034] [Diagram 5] FIG. 5 (not to scale) shows a schematic diagram of a TAQMAN® assay for screening humanization of the rat TrkB (rat Ntrk2) locus and the location of guide RNAs for targeting the rat TrkB (rat Ntrk2) locus (guide target sequences depicted in SEQ ID NOs: 41-44). Gain of allele (GOA) assays include 7138hU and 7138hD. Loss of allele (LOA) assays include rnoTU, rnoTM, and rnoTD. CRISPR assays designed to encompass regions disrupted by CRISPR / Cas9 targeting include rnoTGU and rnoTGD. Retention assays include rnoTAU2 and rnoTAD2.

[0035] [Figure 6-1] FIG. 6 shows an alignment of mouse, rat, and human TRKB (NTRK2) proteins. [Figure 6-2] FIG. 6 shows an alignment of mouse, rat, and human TRKB (NTRK2) proteins.

[0036] [Figure 7]FIG. 7 shows Western blots of phospho-TrkB, total TrkB, phospho-Akt, total AKT, phospho-ERK, and total ERK in primary cortical neurons isolated from 1-day-old homozygous humanized TRKB pups after treatment with various TrkB agonist antibodies or BDNF at 15 min or 2 h.

[0037] [Figure 8] FIG. 8 shows the pharmacokinetic profile of anti-TrkB antibody H4H9816P2 in homozygous TrkB hu / hu and wild-type mice.

[0038] [Figure 9] Figure 9 shows cell survival in differentiated human neuroblastoma SH-SY5Y cells treated with various doses of TrkB agonist antibodies or BDNF. TrkB mAb1 is H4H9816P2, and TrkB mAb2 is a control TrkB agonist antibody with affinity for human TrkB, rat TrkB, and mouse TrkB. A human isotype control antibody was used as a negative control. Data were normalized to serum-free medium without antibody.

[0039] [Figure 10] Figure 10 shows cell survival in mouse primary retinal ganglion cells treated with various doses of TrkB agonist antibody or BDNF. TrkB mAb2 is a control TrkB agonist antibody with affinity for human TrkB, rat TrkB, and mouse TrkB. Data were normalized to serum-free medium without antibody.

[0040] [Figure 11]Figures 11A and 11B show the density of retinal ganglion cells in excised retinas stained for retinal ganglion cells from wild-type rats and mice, respectively, after optic nerve section and treatment with BDNF, TrkB agonist antibody, isotype control antibody, or vehicle control. Rats were intravitreally administered BDNF (5 μg), TrkB agonist antibody (18 μg), isotype control antibody (18 μg), or vehicle control on days 3 and 10 after optic nerve section. Mice were intravitreally administered BDNF (2.5 μg), TrkB agonist antibody (10 μg), isotype control antibody (10 μg), or vehicle control on days 3 and 10 after optic nerve section. TrkB mAb2 is a control TrkB agonist antibody with affinity for human TrkB, rat TrkB, and mouse TrkB.

[0041] [Figure 12] Figures 12A and 12B show the density of retinal ganglion cells in excised retinas stained for retinal ganglion cells from wild-type mice and rats, respectively, after optic nerve section or optic nerve crush and treatment with various doses of BDNF. Figure 12A shows the dose response of BDNF in the optic nerve crush (ONC) model in WT mice. Figure 12B shows the dose response of BDNF from 0.13 μg to 30 μg in the optic nerve section model in WT rats.

[0042] [Figure 13-1] Figure 13A shows the density of retinal ganglion cells in excised retinas stained for retinal ganglion cells from homozygous TrkB, heterozygous TrkB, or wild-type TrkB rats administered intravitreally with TrkB agonist or isotype control antibody 3 and 10 days after optic nerve transection (****=p<0.0001, ***p<0.001, two-way ANOVA). Retinas were excised 14 days after transection. TrkB mAb1 is H4H9816P2.

[0043] FIG. 13B shows the density of retinal ganglion cells in uninjured eyes excised from homozygous TrkB rats, heterozygous TrkB rats, or wild-type TrkB rats.

[0044] [Figure 13-2] FIG. 13C shows the body weight of human TRKB homozygous mice administered a TrkB agonist antibody (H4H9816P2, TrkB) or an isotype control antibody (REGN1945, control).

[0045] [Figure 14] Figure 14 shows the density of retinal ganglion cells in excised retinas stained for retinal ganglion cells from human TRKB homozygous rats administered intravitreally with TrkB agonist antibody (hTrkB, H4H9816P2) or isotype control antibody (REGN1945) on days 3 and 10 after optic nerve transection. Retinas were excised 14 days after transection.

[0046] [Figure 15] Figures 15A and 15B show the density of retinal ganglion cells in excised retinas stained for retinal ganglion cells from human TRKB homozygous rats intravitreally administered various TrkB agonist antibodies (H4H9816P2-L9, H4H9814P-L9, H4H9780P-L5, or a combination of all three) or an isotype control antibody (REGN1945) on days 3 and 10 after optic nerve transection (**p<0.01, Kruskal-Wallis test compared to isotype control antibody). Retinas were excised 14 days after transection. Figure 15A includes naive controls (uninjured contralateral eyes) and Figure 15B does not.

[0047] [Figure 16] Figure 16 shows the density of retinal ganglion cells in excised retinas stained for retinal ganglion cells from wild-type rats that were intravitreally administered various TrkB agonist antibodies (H4H9780P and H4H9814P) or an isotype control antibody (REGN1945) on days 3 and 10 after optic nerve transection. Retinas were excised 14 days after transection.

[0048] [Figure 17] Figures 17A and 17B show the density of retinal ganglion cells in excised retinas stained for retinal ganglion cells from human TRKB homozygous mice intravitreally administered TrkB agonist antibody (H4H9780P) or isotype control antibody (REGN1945) on days 3 and 10 after optic nerve transection. Retinas were excised 14 days after transection. Figure 17A includes a normal control (non-injured contralateral eye) and Figure 17B does not.

[0049] FIG. 17C shows the body weight of human TRKB homozygous mice administered a TrkB agonist antibody (H4H9780P) or an isotype control antibody (REGN1945). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0050] definition The terms "protein," "polypeptide," and "peptide," used interchangeably herein, include polymeric forms of amino acids of any length, including coded and non-coded amino acids, and 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 that has a specific function or structure.

[0051] Proteins are said to have an "N-terminus" and a "C-terminus". The term "N-terminus" refers to the beginning of a protein or polypeptide, which ends 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), which ends with a free carboxyl group (-COOH).

[0052] 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 terms include single-stranded, double-stranded, and multistranded 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.

[0053] Nucleic acids are said to have a "5' end" and a "3' end" because mononucleotides react to make oligonucleotides in such a way that the 5' phosphate of one mononucleotide pentose ring is attached in one direction to its neighbor's 3' oxygen via a phosphodiester linkage. An end of an oligonucleotide is referred to as the "5' end" if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring. An end of an oligonucleotide is referred to as the "3' end" if its 3' oxygen is not linked to the 5' phosphate of another mononucleotide pentose ring. A nucleic acid sequence can also be said to have a 5' and a 3' end, even if it is internal to a larger oligonucleotide. In either a linear or circular DNA molecule, separate elements are referred to as being "upstream" or 5' of the "downstream" or 3' element.

[0054] 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 and can be inherited by its progeny. Any protocol can be used for stable incorporation of a nucleic acid into the genome of a cell.

[0055] The term "targeting vector" refers to a recombinant nucleic acid that can be introduced into a target location within the genome of a cell by homologous recombination, non-homologous end joining mediated ligation, or any other recombination means.

[0056] The term "viral vector" refers to a recombinant nucleic acid that contains at least one element of viral origin and contains elements sufficient or permissive for packaging into a viral vector particle. The vector and / or particle can be used to transfer DNA, RNA or other nucleic acid to cells, either ex vivo or in vivo. Many forms of viral vectors are known.

[0057] The term "wild-type" includes entities having a structure and / or activity that is found in a normal (as opposed to mutant, diseased, altered, etc.) state or context. Wild-type genes and polypeptides often exist in multiple alternative forms (e.g., alleles).

[0058] The term "endogenous" refers to a nucleic acid sequence that is naturally present in a cell or a non-human animal. For example, an endogenous TrkB sequence of a non-human animal refers to the native TrkB sequence that is naturally present at the TrkB locus in the non-human animal.

[0059] 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 with respect to a particular developmental stage and environmental conditions of the cell. An exogenous molecule or sequence can include, for example, a mutated version of a corresponding endogenous sequence in the cell, such as a humanized version of an endogenous sequence, or can include a sequence that corresponds to an endogenous sequence in the cell, but in a different form (i.e., not in 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 under particular environmental conditions.

[0060] The term "heterologous" when used in the context of a nucleic acid or protein indicates that the nucleic acid or protein comprises at least two moieties that are not naturally found together in the same molecule. For example, the term "heterologous" when used in reference to a portion of a nucleic acid or a portion of a protein indicates that the nucleic acid or protein comprises two or more subsequences that are not found in the same relationship to each other (e.g., linked together) in nature. As an example, a "heterologous" region of a nucleic acid vector is a segment of nucleic acid that is not found associated with the other molecule in nature, within or attached to another nucleic acid molecule. For example, a heterologous region of a nucleic acid vector can include a coding sequence that is adjacent to a sequence that is not found associated with that coding sequence in nature. Similarly, a "heterologous" region of a protein is a segment of amino acids that is not found associated with the other peptide molecule in nature, within or attached to another peptide molecule (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.

[0061] "Codon optimization" refers to 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 multiplicity of three-base pair codon combinations that specify amino acids, and generally by replacing at least one codon of the native sequence with a codon that is more frequently or most frequently used in the host cell's genes while maintaining the native amino acid sequence. For example, a nucleic acid encoding a Cas9 protein can be modified to use instead 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, compared to the naturally occurring nucleic acid sequence. Codon usage tables are readily available, for example, in the "Codon Usage Database". Such tables can be adapted in a number of ways. See Nakamura et al. (2000) Nucleic Acids Research 28:292, which is incorporated herein by reference in its entirety for all purposes. Computer algorithms for codon optimization of a particular sequence for expression in a particular host are also available (see, eg, Gene Forge).

[0062] The term "locus" refers to a gene (or significant sequence), DNA sequence, specific location of a polypeptide coding sequence, or location on a chromosome of an organism's genome. For example, "TrkB locus" can refer to the TrkB gene, TrkB DNA sequence, specific location of a TrkB coding sequence, or TrkB location on a chromosome of an organism's genome identified for where such a sequence is located. "TrkB locus" can include regulatory elements of the TrkB gene, including, for example, enhancers, promoters, 5' and / or 3' untranslated regions (UTRs), or combinations thereof.

[0063] The term "gene" refers to a DNA sequence in a chromosome that encodes a product (e.g., an RNA product and / or a polypeptide product) and includes coding regions interrupted by non-coding introns, as well as sequences located adjacent to the coding regions at both the 5' and 3' ends such that the gene corresponds to a full-length mRNA (including 5' and 3' untranslated sequences). The term "gene" also includes other non-coding sequences, including regulatory sequences (e.g., promoters, enhancers, and transcription factor binding sites), polyadenylation signals, internal ribosome entry sites, silencers, insulating sequences, and matrix attachment regions. These sequences may be located near (e.g., within 10 kb) or at distant sites, and affect the level or rate of transcription and translation of the gene.

[0064] 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 specific locus. A genotype is described as homozygous when there are two identical alleles at a particular locus, and heterozygous when the two alleles are different.

[0065] A "promoter" is a regulatory region of DNA, usually containing a TATA box that can direct RNA polymerase II to begin RNA synthesis at the appropriate transcription initiation site for a particular polynucleotide sequence. A promoter can contain other regions that affect the rate of transcription initiation as well. The promoter sequences disclosed herein modulate the transcription of operably linked polynucleotides. The promoter can be active in one or more of the cell types disclosed herein (e.g., eukaryotic cells, non-human mammalian cells, human cells, rodent cells, pluripotent cells, one-cell stage embryos, differentiated cells, or combinations thereof). The promoter can be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a temporally restricted promoter (e.g., a developmentally regulated promoter), or a spatially restricted promoter (e.g., a cell-specific or tissue-specific promoter). Examples of promoters can be found, for example, in WO2013 / 176772, which is incorporated herein by reference in its entirety for all purposes.

[0066] "Operable linkage" or "operably linked" includes the juxtaposition of two or more components (e.g., a promoter and another sequence element) that allows for the possibility that both components function normally and that at least one of the components can mediate a function exerted by at least one of the other components. For example, a promoter can be operably linked to a coding sequence if the promoter controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. Operable linkage can include such sequences that are in close proximity to each other or that act in trans (e.g., regulatory sequences can act at a distance to control transcription of the coding sequence).

[0067] The term "variant" refers to a nucleotide sequence that differs (e.g., by one nucleotide) from the most prevalent sequence in a population, or a protein sequence that differs (e.g., by one amino acid) from the most prevalent sequence in a population.

[0068] The term "fragment", when referring to a protein, refers to a protein that is shorter or has fewer amino acids than the full-length protein. The term "fragment", when referring to a nucleic acid, refers to a nucleic acid that is shorter or has fewer nucleotides than the full-length nucleic acid. A fragment can be, for example, an N-terminal fragment (i.e., a portion of the C-terminal end of a protein is removed), a C-terminal fragment (i.e., a portion of the N-terminal end of a protein is removed), or an internal fragment.

[0069] "Sequence identity" or "identity" in the context of two polynucleotide or polypeptide sequences refers to residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When the percentage of sequence identity is used with reference to proteins, residue positions that are not identical often differ by conservative amino acid substitutions, in which an amino acid residue is substituted for another amino acid residue with similar chemical properties (e.g., charge or hydrophobicity), thus not changing the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity can 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 rather than complete mismatches, thereby increasing the percentage sequence identity. Thus, for example, where identical amino acids are given a score of 1 and non-conservative substitutions are given a score of zero, conservative substitutions are given a score between zero and 1. Scoring of conservative substitutions is calculated, for example, as implemented in the program PC / GENE (Intelligenetics, Mountain View, California).

[0070] "Percentage of sequence identity" includes values ​​determined by comparing two optimally aligned sequences (maximum number of perfectly matched residues) over a comparison window, where the portion of the polynucleotide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence (no additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue exists in both sequences to obtain the number of matched positions, dividing the number of matched 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 contains a heterologous sequence linked to it), the comparison window is the full length of the shorter of the two sequences being compared.

[0071] Unless otherwise stated, 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 which produces alignments having identical nucleotide or amino acid residue matches and identical percent sequence identity for any two sequences in question when compared to corresponding alignments produced by GAP version 10.

[0072] The term "conservative amino acid substitution" refers to the replacement of an amino acid that is normally present in a sequence with a different amino acid that has a 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 polar (hydrophilic) residue, such as between arginine and lysine, between glutamine and asparagine, or between glycine and serine. Moreover, the replacement of a basic residue, such as lysine, arginine, or histidine, with another basic residue, or the replacement of an acidic residue, such as aspartic acid or glutamic acid, with another acidic residue, are additional examples of conservative substitutions. Examples of non-conservative substitutions include the substitution of a polar (hydrophilic) residue, such as cysteine, glutamine, glutamic acid or lysine, with a non-polar (hydrophobic) amino acid residue, such as isoleucine, valine, leucine, alanine or methionine, and / or the substitution of a polar residue for a non-polar residue. Exemplary amino acid categorizations are summarized below. [Table 24]

[0073] A "homologous" sequence (e.g., a nucleic acid sequence) includes a sequence that is either 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. Homologous genes, for example, typically originate 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.

[0074] The term "in vitro" includes an artificial environment and processes or reactions that occur within an artificial environment (e.g., a test tube). The term "in vivo" includes a natural environment (e.g., a cell or 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.

[0075] The term "reporter gene" refers to a nucleic acid having a sequence encoding a gene product (typically an enzyme) that is easily and quantifiably assayed when a construct 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) the factors necessary for activation of the promoter and / or enhancer element. Examples of reporter genes include, but are not limited to, the gene encoding beta-galactosidase (lacZ), the bacterial chloramphenicol acetyltransferase (cat) gene, the firefly luciferase gene, the gene encoding beta-glucuronidase (GUS), and genes encoding fluorescent proteins. "Reporter protein" refers to the protein encoded by the reporter gene.

[0076] The term "fluorescent reporter protein" as used herein means a reporter protein detectable by virtue of its fluorescence, where the fluorescence can come from either the reporter protein directly, the activity of the reporter protein in a fluorogenic substrate, or 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, tagged GFP, turboGFP, eGFP, Emerald, Azami Green, monomeric Azami, etc.). 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, mPlu m, 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 any other suitable fluorescent protein whose presence in a cell can be detected by flow cytometry methods.

[0077] The term "recombination" includes any process of genetic information exchange between two polynucleotides and can occur by any mechanism. Recombination in response to double-strand breaks (DSBs) occurs primarily through two conserved DNA repair pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). See Kasparek & Humphrey (2011) Seminars in Cell & Dev. Biol. 22:886-897, the entirety of which is incorporated herein by reference for all purposes. Similarly, repair of target nucleic acid mediated by exogenous donor nucleic acid can include any process of genetic information exchange between two polynucleotides.

[0078] NHEJ involves the repair of double-strand breaks in nucleic acid by direct ligation of the cut ends to each other or to exogenous sequences without the need for homologous template. The ligation of non-adjacent sequences by NHEJ can often result in deletion, insertion or translocation near the site of double-strand break. For example, NHEJ can also result in targeted integration of exogenous donor nucleic acid through direct ligation of the cut ends by the ends of exogenous donor nucleic acid (i.e., NHEJ-based capture). Such NHEJ-mediated targeted integration can be preferred for the insertion of exogenous donor nucleic acid when the homology-directed repair (HDR) pathway is not readily available (e.g., in non-dividing cells, primary cells, and cells that perform poor homology-based DNA repair). In addition, in contrast to homology-directed repair, knowledge of large regions of sequence identity adjacent to the cut site is not required, which can be beneficial when attempting targeted insertion into organisms with genomes with limited genome sequence knowledge. Integration can proceed via blunt-end ligation between the exogenous donor nucleic acid and the cleaved genomic sequence, or via sticky-end (i.e., with 5' or 3' overhangs) ligation using an exogenous donor nucleic acid flanked by overhangs compatible with those generated by the nuclease agent in the cleaved genomic sequence.See, for example, US2011 / 020722, WO2014 / 033644, WO2014 / 089290, and Maresca et al. (2013) Genome Res. 23(3):539-546, each of which is incorporated herein by reference in its entirety for all purposes.If blunt ends are ligated, excision of the target and / or donor may be required to generate regions of microhomology required for fragment ligation, which may result in undesired changes in the target sequence.

[0079] Recombination can also occur through homology-directed repair (HDR) or homologous recombination (HR). HDR or HR includes forms of nucleic acid repair that may require nucleotide sequence homology, and uses a "donor" molecule as a template for repair of a "target" molecule (i.e., a molecule that has undergone double-strand break), resulting in the transfer of genetic information from the donor to the target. Without wishing to be bound by any particular theory, such transfer may involve mismatch correction of the heteroduplex DNA that forms between the broken target and the donor, and / or synthesis-dependent strand annealing, in which the donor is used to resynthesize the genetic information that will become part of the target, and / or related processes. In some cases, the donor polynucleotide, a portion of the donor polynucleotide, a copy of the donor polynucleotide, or a portion of a copy of the donor polynucleotide integrates into the target DNA. See Wang et al. (2013) Cell 153:910-918; Mandalos et al. (2012) PLOS ONE 7:e45768:1-9; and Wang et al. (2013) Nat Biotechnol. 31:530-532, each of which is incorporated by reference in its entirety for all purposes.

[0080] 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).

[0081] As used herein, the phrase "anti-TRKB antibody" includes both monovalent antibodies with a single specificity, and bispecific antibodies comprising a first arm that binds to TRKB and a second arm that binds to a second (target) antigen, where the anti-TRKB arm comprises, for example, any of the HCVR / LCVR or CDR sequences depicted in Table 22 herein. The phrase "anti-TrkB antibody" also includes antibody-drug conjugates (ADCs) comprising an anti-TRKB antibody or an antigen-binding portion thereof conjugated to a drug or toxin (i.e., a cytotoxic agent). The phrase "anti-TRKB antibody" also includes antibody-radionuclide conjugates (ARCs) comprising an anti-TRKB antibody or an antigen-binding portion thereof conjugated to a radionuclide.

[0082] The term "anti-TRKB antibody" as used herein means any antigen-binding molecule or molecular complex that contains at least one complementarity determining region (CDR) that specifically binds to or interacts with TRKB or a portion of TRKB. The term "antibody" includes immunoglobulin molecules that contain four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain contains a heavy chain variable region (herein referred to as HCVR or VVR). H The heavy chain constant region is made up of three domains: H 1. C H 2 and C H Each light chain comprises a light chain variable region (herein, LCVR or V L The light chain constant region comprises one domain (C L 1) is included. H and V L The regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). H and V Lis composed of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the present invention, the FRs of the anti-TRKB antibody (or antigen-binding portion thereof) can be identical to the human germline sequence or can be naturally or artificially modified. An amino acid consensus sequence can be defined based on a side-by-side analysis of two or more CDRs.

[0083] The term "antibody" as used herein also includes antigen-binding fragments of full-length antibody molecules. An "antigen-binding portion" of an antibody, an "antigen-binding fragment" of an antibody, and other terms as used herein include any enzymatically obtainable, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be obtained from a full-length antibody molecule using any suitable standard technique, such as, for example, proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding the antibody variable and, optionally, constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, to place one or more variable and / or constant domains in a suitable configuration, or to introduce codons, to create cysteine ​​residues, to modify, add or delete amino acids, etc.

[0084] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues mimicking the hypervariable regions of an antibody (e.g., isolated complementarity determining regions (CDRs), such as CDR3 peptides), or constrained FR3-CDR3-FR4 peptides. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs) and shark variable IgNAR domains, are also encompassed within the term "antigen-binding fragments" as used herein.

[0085] An antigen-binding fragment of an antibody will typically contain at least one variable domain. The variable domain may be of any size or amino acid composition and will generally contain at least one CDR contiguous with or in frame with one or more framework sequences. L Domain associated with V H In an antigen-binding fragment having a domain, H and V L The domains can be in any suitable configuration relative to each other. For example, the variable regions can dimerize into V H -V H , V H -V L or V L -V L Alternatively, the antigen-binding fragment of the antibody may contain a monomeric V H or V L It may contain domains.

[0086] In certain embodiments, an antigen-binding fragment of an antibody can contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that can be found in an antigen-binding fragment of an antibody of the invention include: (i) V H -C H 1;(ii)V H -C H 2;(iii)V H -C H 3;(iv)V H -C H 1-C H 2;(v)V H -C H 1-C H 2-C H 3;(vi)V H -C H 2-C H 3;(vii)V H -C L ;(viii)V L -C H 1;(ix)V L -C H 2;(x)V L -C H 3;(xi)V L -C H 1-C H 2;(xii)V L -C H 1-C H 2-C H 3;(xiii)V L -C H 2-C H 3; and (xiv) V L -C LIn any configuration of the variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains can be directly linked to each other or can be linked by a complete or partial hinge or linker region. The hinge region can consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that provide a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments of the antibodies of the present invention can be linked to each other and / or to one or more monomeric V H Or V L The variable and constant domains may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above, non-covalently associated (e.g., by disulfide bond(s)) with the domain.

[0087] As with intact antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies will typically contain at least two different variable domains, each capable of specifically binding to a separate antigen or a different epitope on the same antigen. Any of the multispecific antibody formats, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of the antigen-binding fragments of antibodies of the present invention using routine techniques.

[0088] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule known as the paratope. A single antigen can have more than one epitope. Thus, different antibodies can bind to different areas of the antigen and have different biological effects. Epitopes can be either conformational or linear. Conformational epitopes are generated by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are epitopes generated by adjacent amino acid residues in a polypeptide chain. In certain circumstances, epitopes can include carbohydrate, phosphoryl, or sulfonyl moieties in an antigen.

[0089] The terms "specifically bind" or "specifically bind to" and the like mean that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding is at least about 1×10 -6 M or less (e.g., a smaller K D In one embodiment, the specific binding of a molecule to a target protein can be characterized by a specific binding affinity (where a specific binding affinity is indicative of tighter binding). Methods for determining whether two molecules specifically bind are well known and include, for example, equilibrium dialysis, surface plasmon resonance, and others. As described herein, antibodies that specifically bind to TRKB have been identified by surface plasmon resonance, e.g., BIACORE™. Furthermore, multispecific antibodies that bind to a TRKB protein and one or more additional antigens, or bispecific antibodies that bind to two different regions of TRKB, are nevertheless considered to be "specifically binding" antibodies as used herein.

[0090] The anti-TRKB antibodies disclosed herein may contain one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains. Such mutations can be easily ascertained by comparing the amino acid sequences disclosed herein with sequences available, for example, from public antibody sequence databases. Once obtained, antibodies and antigen-binding fragments containing one or more mutations can be easily tested for one or more desired properties, such as, for example, improved binding specificity, increased binding affinity, improved or enhanced antagonist or agonist biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are included.

[0091] Also included are anti-TRKB antibodies that comprise variants of any of the HCVR, LCVR and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the invention includes anti-TRKB antibodies that have HCVR, LCVR and / or CDR amino acid sequences that have 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc., conservative amino acid substitutions compared to any of the HCVR, LCVR and / or CDR amino acid sequences set out in, e.g., Table 22.

[0092] The term "substantial identity" or "substantially identical" when referring to a nucleic acid or fragment thereof in the context of an anti-TRKB antibody refers to the presence of nucleotide sequence identity at least about 95%, more preferably at least about 96%, 97%, 98% or 99% of the nucleotide bases when optimally aligned with another nucleic acid (or its complementary strand) by appropriate nucleotide insertion or deletion, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as described below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule can, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0093] The term "substantial similarity" or "substantially similar" as applied to polypeptides in the context of anti-TRKB antibodies means that two peptide sequences share at least 95% sequence identity, and even more preferably at least 98% or 99% sequence identity, when optimally aligned, such as by the programs GAP or BESTFIT, using default gap weights. Preferably, non-identical residue positions differ by conservative amino acid substitutions. A "conservative amino acid substitution" as applied to polypeptides in the context of anti-TRKB antibodies is a substitution in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity can be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, which is incorporated by reference in its entirety for all purposes. Examples of groups of amino acids having side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains, which are cysteine ​​and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic-aspartic, and asparagine-glutamine.Alternatively, a conservative replacement is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445, which is incorporated by reference in its entirety for all purposes. A "moderately conservative" replacement is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0094] A composition or method that "comprising" or "including" one or more recited elements can include other elements not specifically recited. For example, a composition that "comprises" or "includes" a protein can contain the protein alone or in combination with other ingredients. The transitional phrase "consisting essentially of" means that the claims should be interpreted to include the specified elements recited in the claims, as well as elements that do not materially affect the basic and novel feature(s) of the claimed invention. Thus, the term "consisting essentially of" is not intended to be interpreted as equivalent to "comprising" when used in the claims of the present invention.

[0095] "Optionally" or "as needed" means that the event or circumstance described thereafter may or may not occur, and that the description includes instances in which the event or circumstance occurs as well as instances in which it does not occur.

[0096] Designation of a range of values ​​includes all integers within or defining the range, and all subranges defined by integers in the range.

[0097] Unless otherwise clear from the context, the term "about" encompasses values ​​within the standard tolerance of measurement (eg, SEM) of the stated value.

[0098] 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").

[0099] The term "or" refers to any one member of a particular list, and also includes any combination of members of that list.

[0100] 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.

[0101] Statistically significant means p≦0.05.

[0102] Detailed Description I. Overview Disclosed herein are non-human animal genomes, non-human animal cells and non-human animals that contain a humanized TRKB locus, and methods of using such non-human animal cells and animals. The non-human animal cells or animals that contain a humanized TRKB locus express a human TRKB protein or a chimeric TRKB protein that contains one or more fragments of the human TRKB protein (e.g., all or a portion of the human TRKB extracellular domain).

[0103] Humanized TRKB alleles (e.g., resulting from one-for-one, total or partial replacement of non-human animal genomic DNA with orthologous human genomic DNA) would provide a true human target or a close approximation of the true human target of human TRKB-targeting reagents (e.g., agonistic antibodies or agonistic small molecules designed to target human TRKB), thereby enabling testing of efficacy and mechanism of action of such agents in living animals, as well as pharmacokinetic and pharmacodynamic studies. For example, as shown in the examples disclosed herein, intravitreal administration of a human TRKB agonistic antibody has a significant neuroprotective effect after optic nerve injury in humanized TrkB rats.

[0104] II. Non-human animals containing a humanized TRKB locus The non-human animal genomes, non-human animal cells and non-human animals disclosed herein comprise a humanized TRKB locus. The cells or non-human animals comprising the humanized TRKB locus express a human TRKB protein or a partially humanized chimeric TRKB protein in which one or more fragments of the native TRKB protein are replaced with a corresponding fragment (e.g., all or part of the extracellular domain) from human TRKB.

[0105] A.TRKB The cells and non-human animals described herein contain a humanized TRKB locus. TRKB (also known as BDNF-NT-3 growth factor receptor, GP145-TrkB, Trk-B, TrkB, neurotrophic tyrosine kinase receptor type 2, TrkB tyrosine kinase, tropomyosin-related kinase B, tropomyosin receptor kinase B, neurotrophic receptor tyrosine kinase 2 and NTRK2) is encoded by the TRKB gene (also known as NTRK2, OBHD, TRK-B and GP145-TRKB). TRKB is a receptor tyrosine kinase involved in the development and maturation of the central and peripheral nervous system through the regulation of neuronal survival, proliferation, migration, differentiation, and synaptogenesis and plasticity. TRKB is a receptor for BDNF / brain-derived neurotrophic factor and NTF4 / neurotrophin-4. Alternatively, TRKB can bind to NTF3 / neurotrophin-3, which is less efficient at activating the receptor but regulates neuronal survival through TRKB. After ligand binding, TRKB undergoes homodimerization, autophosphorylation and activation. The canonical isoforms of TRKB are expressed in the central and peripheral nervous systems. In the central nervous system (CNS), expression is observed in the cerebral cortex, hippocampus, thalamus, choroid plexus, granular layer of the cerebellum, brainstem and spinal cord. In the peripheral nervous system, it is expressed in many cranial ganglia, ophthalmic nerves, vestibular system, multiple facial structures, submandibular gland and dorsal root ganglion.

[0106] Human TRKB maps to human 9q21.33 on chromosome 9 (NCBI RefSeq gene ID 4915; assembly GRCh38.p7; location NC_000009.12 (84668368..85027070)). The gene is reported to have 23 exons. Wild-type human TRKB protein has been assigned UniProt accession number Q16620. At least seven isoforms are known (Q16620-1 to Q16620-7). The sequence of one isoform, Q16620-4 (identical to NCBI accession number NP_006171.2), is set forth in SEQ ID NO:3. The mRNA (cDNA) encoding the canonical isoform has been assigned NCBI accession number AF410899.1 and is set forth in SEQ ID NO:8. Another example of an mRNA (cDNA) encoding a human TRKB isoform is assigned RefSeq mRNA ID NM_006180.4. An exemplary coding sequence (CDS) is set forth in SEQ ID NO: 11. The full-length human TRKB protein, set forth in SEQ ID NO: 3, has 838 amino acids, including a signal peptide (amino acids 1-31), an extracellular domain (amino acids 32-430), a transmembrane domain (amino acids 431-454), and a cytoplasmic domain (amino acids 455-838). The delineation between these domains is as specified in UniProt. Reference to human TRKB includes all allelic forms and isoforms, along with the canonical (wild-type) form. Any other form of human TRKB has amino acids numbered for maximum alignment with the wild-type form, and the aligned amino acids are assigned the same number. Another exemplary isoform of human TRKB is Q16620-1 (identical to NCBI accession number NP_001018074.1), which is set forth in SEQ ID NO: 75. The mRNA (cDNA) encoding this isoform has been assigned NCBI accession number NM_001018064.2 and is set forth in SEQ ID NO: 76. An exemplary coding sequence (CDS) for this isoform (CCDS ID CCDS35050.1) is set forth in SEQ ID NO: 77.

[0107] Rat TrkB maps to rat chromosome 17 at 17p14 (NCBI RefSeq gene ID 25054; assembly Rnor_6.0; location NC_005116.4 (5934651..6245778, complement)). The gene is reported to have 23 exons. Wild-type rat TRKB protein has been assigned UniProt accession number Q63604. At least three isoforms are known (Q63604-1 to Q63604-3). The sequence of the canonical isoform, Q63604-1 (identical to NCBI accession number NP_036863.1), is set forth in SEQ ID NO:2. The mRNA (cDNA) encoding the canonical isoform has been assigned NCBI accession number NM_012731.2 and is set forth in SEQ ID NO:7. Another example of an mRNA (cDNA) encoding a rat TRKB isoform is assigned RefSeq mRNA ID M55291. An exemplary coding sequence (CDS) is set forth in SEQ ID NO: 10. The canonical full-length rat TRKB protein set forth in SEQ ID NO: 2 has 821 amino acids, including a signal peptide (amino acids 1-31), an extracellular domain (amino acids 32-429), a transmembrane domain (amino acids 430-453), and a cytoplasmic domain (amino acids 454-821). The delineation between these domains is as specified in UniProt. References to rat TRKB include all allelic forms and isoforms, along with the canonical (wild-type) form. Any other form of rat TRKB has amino acids numbered for maximum alignment with the wild-type form, and the aligned amino acids are assigned the same number.

[0108] Mouse TrkB maps to mouse chromosome 12 at 13B1;1331.2 cM (NCBI RefSeq gene ID 18212; assembly GRCm38.p4(GCF_000001635.24); location NC_000079.6(58806569..59133970)). The gene is reported to have 23 exons. The wild-type mouse TRKB protein has been assigned UniProt accession number P15209. At least four isoforms are known (P15209-1 to P15209-4). The sequence of the canonical isoform, P15209-1 (identical to NCBI accession numbers NP_001020245.1 and NP_001269890.1), is set forth in SEQ ID NO:1. An exemplary mRNA (cDNA) isoform encoding the canonical isoform has been assigned NCBI accession number NM_001025074.2 and is set forth in SEQ ID NO:6. An exemplary coding sequence (CDS) (CCDS ID CCDS26573.1) is set forth in SEQ ID NO:9. The canonical full-length mouse TRKB protein set forth in SEQ ID NO:1 has 821 amino acids including a signal peptide (amino acids 1-31), an extracellular domain (amino acids 32-429), a transmembrane domain (amino acids 430-453) and a cytoplasmic domain (amino acids 454-821). The delineation between these domains is as specified in UniProt. Reference to mouse TRKB includes all allelic forms and isoforms along with the canonical (wild type) form. Any other form of mouse TRKB has amino acids numbered for maximum alignment with the wild type form, and the aligned amino acids are assigned the same number.

[0109] B. Humanized TRKB locus The humanized TRKB locus can be a TrkB locus in which the entire TrkB gene is replaced with the corresponding orthologous human TRKB sequence, or a TrkB locus in which only a portion of the TrkB gene is replaced with the corresponding orthologous human TRKB sequence (i.e., humanized). If necessary, the corresponding orthologous human TRKB sequence is modified to be codon-optimized based on the codon usage in the non-human animal. The replaced (i.e., humanized) region can include coding regions such as exons, non-coding regions such as introns, non-translated regions or regulatory regions (e.g., promoters, enhancers, or transcriptional repressor binding elements), or any combination thereof. As an example, exons corresponding to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or all 23 exons of the human TRKB gene can be humanized. For example, exons corresponding to exons 3-10 of the human TRKB gene can be humanized, including the segment of exon 2 (coding exon 1) from the codon encoding amino acid 33, beginning immediately after the signal peptide. Alternatively, the region of TrkB that encodes the epitope recognized by an anti-human TRKB antigen binding protein, or the region targeted by a human TRKB targeting reagent (e.g., a small molecule), can be humanized. Similarly, introns corresponding to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or all 22 introns of the human TRKB gene can be humanized or can remain endogenous. For example, introns corresponding to the intron between exons 2 and 10 of the human TRKB gene (i.e., introns 2-9, between coding exon 1 and exon 10) can be humanized, including, if desired, a portion of the intron following exon 10 (i.e., intron 10). The adjacent untranslated regions containing regulatory sequences may also be humanized or remain endogenous.For example, the 5' untranslated region (UTR), the 3' UTR, or both the 5' UTR and the 3' UTR can be humanized, or the 5' UTR, the 3' UTR, or both the 5' UTR and the 3' UTR can remain endogenous. In a specific example, both the 5' UTR and the 3' UTR remain endogenous. Depending on the degree of replacement with orthologous sequences, regulatory sequences such as promoters can be endogenous or can be provided by replacing human orthologous sequences. For example, a humanized TRKB locus can include an endogenous non-human animal TrkB promoter.

[0110] One or more or all of the regions encoding the signal peptide, the cytoplasmic domain, the transmembrane domain or the extracellular domain may be humanized, or one or more of such regions may remain endogenous. Exemplary coding sequences for mouse TRKB signal peptide, extracellular domain, transmembrane domain and cytoplasmic domain are set forth in SEQ ID NOs: 63-66, respectively. Exemplary coding sequences for rat TRKB signal peptide, extracellular domain, transmembrane domain and cytoplasmic domain are set forth in SEQ ID NOs: 67-70, respectively. Exemplary coding sequences for human TRKB signal peptide, extracellular domain, transmembrane domain and cytoplasmic domain are set forth in SEQ ID NOs: 71-74, respectively.

[0111] For example, all or a portion of the region of the TrkB locus that encodes the signal peptide can be humanized, and / or all or a portion of the region of the TrkB locus that encodes the extracellular domain can be humanized, and / or all or a portion of the region of the TrkB locus that encodes the transmembrane domain can be humanized, and / or all or a portion of the region of the TrkB locus that encodes the cytoplasmic domain can be humanized. In one example, all or a portion of the region of the TrkB locus that encodes the extracellular domain is humanized. Optionally, the CDS of the human TRKB extracellular domain comprises, consists essentially of, or consists of a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 72 (or a degenerate thereof). The TRKB protein can retain the activity of native TRKB (e.g., retain the ability to become phosphorylated, retain the ability to activate downstream signaling pathways such as the PI3K / AKT and MAPK / ERK pathways, or retain the ability to regulate neuronal survival, proliferation, migration, differentiation, or synaptogenesis and plasticity, or to produce any of the phenotypes disclosed elsewhere herein). For example, the region of the TrkB locus encoding the extracellular domain can be humanized such that a chimeric TRKB protein is produced having an endogenous signal peptide, an endogenous cytoplasmic domain, an endogenous transmembrane domain, and a humanized extracellular domain.

[0112] One or more of the regions encoding the signal peptide, the cytoplasmic domain, the transmembrane domain or the extracellular domain may remain endogenous. For example, the regions encoding the signal peptide and / or the cytoplasmic domain and / or the transmembrane domain may remain endogenous. Optionally, the CDS of the endogenous TRKB signal peptide comprises, consists essentially of, or consists of a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 63 or 67 (or a degenerate thereof). Optionally, the CDS of the endogenous TRKB transmembrane domain comprises, consists essentially of, or consists of a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 65 or 69 (or a degenerate thereof). Optionally, the CDS of the endogenous TRKB cytoplasmic domain comprises, consists essentially of, or consists of a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 66 or 70 (or a degenerate thereof). In each case, the TRKB protein can retain the activity of native TRKB.

[0113] The TRKB protein encoded by the humanized TRKB locus may include one or more domains derived from a human TRKB protein and / or one or more domains derived from an endogenous (i.e., native) TRKB protein. Exemplary amino acid sequences of mouse TRKB signal peptide, extracellular domain, transmembrane domain and cytoplasmic domain are set forth in SEQ ID NOs: 51-54, respectively. Exemplary amino acid sequences of rat TRKB signal peptide, extracellular domain, transmembrane domain and cytoplasmic domain are set forth in SEQ ID NOs: 55-58, respectively. Exemplary amino acid sequences of human TRKB signal peptide, extracellular domain, transmembrane domain and cytoplasmic domain are set forth in SEQ ID NOs: 59-62, respectively.

[0114] The TRKB protein can include one or more or all of the human TRKB signal peptide, the human TRKB extracellular domain, the human TRKB transmembrane domain, and the human TRKB cytoplasmic domain. In one example, the TRKB protein can include the human TRKB extracellular domain.

[0115] The TRKB protein encoded by the humanized TRKB locus can also include one or more domains derived from an endogenous (i.e., native) non-human animal TRKB protein. As an example, the TRKB protein encoded by the humanized TRKB locus can include a signal peptide derived from an endogenous (i.e., native) non-human animal TRKB protein, and / or a cytoplasmic domain derived from an endogenous (i.e., native) non-human animal TRKB protein, and / or a transmembrane domain derived from an endogenous (i.e., native) non-human animal TRKB protein.

[0116] A domain in a chimeric TRKB protein derived from a human TRKB protein may be encoded by a fully humanized sequence (i.e., the entire sequence encoding the domain is replaced with an orthologous human TRKB sequence) or a partially humanized sequence (i.e., a portion of the sequence encoding the domain is replaced with an orthologous human TRKB sequence, and the remaining endogenous (i.e., native) sequence encoding the domain encodes the same amino acids as the orthologous human TRKB sequence, such that the encoded domain is identical to that domain in the human TRKB protein). Similarly, a domain in a chimeric protein derived from an endogenous TRKB protein may be encoded by a fully endogenous sequence (i.e., the entire sequence encoding the domain is an endogenous TrkB sequence) or a partially humanized sequence (i.e., a portion of the sequence encoding the domain is replaced with an orthologous human TRKB sequence, and the orthologous human TRKB sequence encodes the same amino acids as the replaced endogenous TrkB sequence, such that the encoded domain is identical to that domain in the endogenous TRKB protein). For example, a portion of the region of the TrkB locus that encodes a transmembrane domain (e.g., encoding the N-terminal region of the transmembrane domain) can be replaced with an orthologous human TRKB sequence, such that the amino acid sequence of the region of the transmembrane domain encoded by the orthologous human TRKB sequence is identical to the corresponding endogenous amino acid sequence.

[0117] As an example, the TRKB protein encoded by the humanized TRKB locus can include a human TRKB extracellular domain. Optionally, the human TRKB extracellular domain comprises, consists essentially of, or consists of a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 60. The TRKB protein retains the activity of native TRKB (e.g., retains the ability to become phosphorylated, retains the ability to activate downstream signaling pathways such as the PI3K / AKT and MAPK / ERK pathways, or retains the ability to regulate neuronal survival, proliferation, migration, differentiation, or synaptogenesis and plasticity, or to produce any of the phenotypes disclosed elsewhere herein). As another example, the TRKB protein encoded by the humanized TRKB locus can include an endogenous non-human animal TRKB cytoplasmic domain (e.g., mouse TRKB cytoplasmic domain or rat TRKB cytoplasmic domain). Optionally, the non-human animal TRKB cytoplasmic domain comprises, consists essentially of, or consists of a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 54 or 58. As another example, the TRKB protein encoded by the humanized TRKB locus can comprise an endogenous non-human animal TRKB transmembrane domain (e.g., mouse TRKB transmembrane domain or rat TRKB transmembrane domain). Optionally, the non-human animal TRKB transmembrane domain comprises, consists essentially of, or consists of a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 53 or 57. As another example, the TRKB protein encoded by the humanized TRKB locus can comprise an endogenous non-human animal TRKB signal peptide (e.g., mouse TRKB signal peptide or rat TRKB signal peptide). Optionally, the non-human animal TRKB signal peptide comprises, consists essentially of, or consists of a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:51 or 55.In each case, the TRKB protein can retain the activity of native TRKB. For example, the TRKB protein encoded by the humanized TRKB locus can comprise, essentially consist of, or consist of a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 4 or 5. Optionally, the TRKB CDS encoded by the humanized TRKB locus can comprise, essentially consist of, or consist of a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 12 or 13 (or a degenerate thereof). In each case, the TRKB protein can retain the activity of native TRKB.

[0118] Optionally, the humanized TRKB locus can include other elements. Examples of such elements can include a selection cassette, a reporter gene, a recombinase recognition site, or other elements. Alternatively, the humanized TRKB locus can lack other elements (e.g., can lack a selection marker or selection cassette). Examples of suitable reporter genes and reporter proteins are disclosed elsewhere herein. An example of a suitable selection marker is neomycin phosphotransferase (neomycin phosphotransferase). r ), hygromycin B phosphotransferase (hyg r ), puromycin N-acetyltransferase (puro r ), blasticidin S deaminase (bsr r), xanthine / guanine phosphoribosyltransferase (gpt) and herpes simplex virus thymidine kinase (HSV-k). Examples of recombinases include Cre, Flp and Dre recombinase. An example of a Cre recombinase gene is Crei, in which the two exons encoding the Cre recombinase are separated by an intron to prevent its expression in prokaryotic cells. Such recombinases can further include a nuclear localization signal that facilitates localization to the nucleus (e.g., NLS-Crei). Recombinase recognition sites include nucleotide sequences that are recognized by site-specific recombinases and can function as substrates for recombination events. Examples of recombinase recognition sites include FRT, FRT11, FRT71, attp, att, rox, and lox sites such as loxP, lox511, lox2272, lox66, lox71, loxM2 and lox5171.

[0119] Other elements, such as reporter genes or selection cassettes, can be self-deleting cassettes flanked by recombinase recognition sites. See, for example, US8,697,851 and US2013 / 0312129, each of which is incorporated herein by reference in its entirety for all purposes. By way of example, the self-deleting cassette can include a Crei gene (containing two exons encoding Cre recombinase, separated by an intron) operably linked to a mouse Prm1 promoter, and a neomycin resistance gene operably linked to a human ubiquitin promoter. The Prm1 promoter can be used to specifically delete the self-deleting cassette in male germ cells of F0 animals. The polynucleotide encoding the selection marker can be operably linked to a promoter that is active in the cells being targeted. Examples of promoters are described elsewhere herein. As another specific example, a self-deletion selection cassette can comprise a hygromycin resistance gene coding sequence operably linked to one or more promoters (e.g., both the human ubiquitin and EM7 promoters), followed by a polyadenylation signal, followed by a Crei coding sequence operably linked to one or more promoters (e.g., the mPrm1 promoter), followed by another polyadenylation signal, with the entire cassette flanked by loxP sites.

[0120] The humanized TRKB locus may be a conditional allele. For example, the conditional allele may be a multifunctional allele, as described in US2011 / 0104799, the entirety of which is incorporated herein by reference for all purposes. For example, the conditional allele may include: (a) a trigger sequence in sense orientation relative to the transcription of the target gene; (b) a drug selection cassette (DSC) in sense or antisense orientation; (c) a nucleotide sequence of interest (NSI) in antisense orientation; and (d) an inversion-conditional module (COIN, which utilizes an exon-splitting intron and an invertible gene trap-like module) in reverse orientation. For example, see US2011 / 0104799. The conditional allele can further comprise recombinable units that recombine after exposure to a first recombinase to form a conditional allele that (i) lacks the initiator sequence and DSC, and (ii) contains the NSI in a sense orientation and the COIN in an antisense orientation. See, e.g., US2011 / 0104799.

[0121] An exemplary humanized TRKB locus (e.g., a humanized mouse TrkB locus or a humanized rat TrkB locus) is a locus in which the region in exon 2 / coding exon 1, starting immediately after the signal peptide from the codon encoding amino acid 33 (or the codon corresponding to the codon encoding amino acid 33 in mouse TrkB, rat TrkB or human TRKB when optimally aligned with mouse TrkB, rat TrkB or human TRKB CDS, respectively), to exon 10 (or the exon corresponding to mouse TrkB, rat TrkB or human TRKB exon 10 when optimally aligned with mouse TrkB, rat TrkB or human TRKB CDS, respectively), optionally including a portion of intron 10, is replaced with the corresponding human sequence. The replaced region encodes the extracellular domain of TRKB. See Figures 1 and 4 and SEQ ID NOs: 4 and 5.

[0122] C. Non-human animal genomes, non-human animal cells and non-human animals containing a humanized TRKB locus Provided are non-human animal genomes, non-human animal cells and non-human animals comprising the humanized TRKB locus described elsewhere herein. The genome, cell or non-human animal can be male or female. The genome, cell or non-human animal can be heterozygous or homozygous with respect to the humanized TRKB locus. Diploid organisms have two alleles at each locus. Each pair of alleles represents a genotype at a specific locus. A genotype is described as homozygous when there are two identical alleles at a particular locus, and as heterozygous when the two alleles are different.

[0123] The non-human animal genome or cell provided herein can be any non-human animal genome or cell that contains, for example, a genomic locus that is homologous or orthologous to the TrkB locus or the human TRKB locus. The genome can be derived from or the cell can be a eukaryotic cell, including, for example, a fungal cell (e.g., yeast), a plant cell, an animal cell, a mammalian cell, a non-human mammalian cell, and a human cell. The term "animal" includes any member of the animal kingdom, including, for example, mammals, fish, reptiles, amphibians, birds, and worms. The mammalian cell can be, for example, a non-human mammalian cell, a rodent cell, a rat cell, a mouse cell, or a hamster cell. Other non-human mammals include, for example, non-human primates, monkeys, apes, orangutans, cats, dogs, rabbits, horses, bulls, deer, bison, livestock (e.g., bovine species such as cows, steers, etc.; ovine species such as sheep, goats, etc.; and porcine species such as pigs and wild boars). Birds include, for example, chickens, turkeys, ostriches, geese, ducks, etc. Domesticated animals and farm animals are also included. The term "non-human" excludes humans.

[0124] The cells may be in any kind of undifferentiated or differentiated state. For example, the cells may be totipotent, pluripotent (e.g., human pluripotent cells, or non-human pluripotent cells, such as mouse embryonic stem (ES) cells or rat ES cells), or non-pluripotent. Totipotent cells include undifferentiated cells that can give rise to any cell type, and pluripotent cells include undifferentiated cells that retain the ability to develop into two or more differentiated cell types. Such pluripotent and / or totipotent cells may 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).

[0125] The cell provided herein may be an embryonic cell (e.g., a sperm or an oocyte). The cell may be a mitotically competent or mitotically inactive cell, a meiotically competent or meiotically inactive cell. Similarly, the cell may be a primary somatic cell or a cell that is not a primary somatic cell. A somatic cell includes any cell that is not a gamete, an embryonic cell, a gametocyte, or an undifferentiated stem cell. For example, the cell may be a neuron, such as a hippocampal neuron or a cortical neuron.

[0126] Suitable cells provided herein also include primary cells. Primary cells include cells or cell cultures directly isolated from organisms, organs or tissues. Primary cells include cells that are not transformed and are not immortal. This includes any cell obtained from organisms, organs or tissues that have not been previously subjected to tissue culture, or that have been previously subjected to tissue culture but cannot be subjected to tissue culture indefinitely. Such cells can be isolated by conventional techniques and include, for example, hippocampal neurons or cortical neurons.

[0127] Other suitable cells provided herein include immortalized cells. Immortalized cells include cells derived from multicellular organisms that do not normally proliferate indefinitely, but that can escape normal cellular senescence and instead continue to divide due to mutations or alterations. Such mutations or alterations can occur naturally or can be induced intentionally. Specific examples of immortalized cell lines are neuroblastoma cell lines such as N18TG2 or T48, or cell lines such as NIH-3T3 cell lines. Many types of immortalized cells are known. Immortalized or primary cells include cells that are typically used for culture or for expressing recombinant genes or proteins.

[0128] The cells provided herein also include one-cell stage embryos (i.e., fertilized oocytes or zygotes). Such one-cell stage embryos can be from any genetic background (e.g., for mice, BALB / c, C57BL / 6, 129, or combinations thereof), can be fresh or frozen, and can be derived from natural mating or in vitro fertilization.

[0129] The cells provided herein can be normal, healthy cells, or can be diseased or mutant cells.

[0130] Non-human animals comprising the humanized TRKB locus described herein can be produced by methods described elsewhere herein. 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, monkeys, apes, orangutans, cats, dogs, horses, bulls, deer, bison, sheep, rabbits, rodents (e.g., mice, rats, hamsters, and guinea pigs), and livestock (e.g., bovine species such as cows and steers; ovine species such as sheep and goats; and porcine species such as pigs and wild boars). Birds include, for example, chickens, turkeys, ostriches, geese, and ducks. Domesticated animals and agricultural animals are also included. The term "non-human animal" excludes humans. Preferred non-human animals include rodents, such as, for example, mice and rats.

[0131] Non-human animals can be from any genetic background. For example, suitable mice can be from 129 strain, C57BL / 6 strain, 129 and C57BL / 6 mix, BALB / c strain, or 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) Mammalian Genome 10:836, which is incorporated herein by reference in its entirety for all purposes. Examples of C57BL strains include C57BL / A, C57BL / An, C57BL / GrFa, C57BL / Kal_wN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr and C57BL / Ola. Suitable mice can also be derived from a mix of the 129 strains mentioned above and the C57BL / 6 strains mentioned above (e.g., 50% 129 and 50% C57BL / 6). Similarly, suitable mice can be derived from a mix of the 129 strains mentioned above or a mix of the BL / 6 strains mentioned above (e.g., 129S6 (129 / SvEvTac) strain).

[0132] Similarly, rats can be from any rat strain, including, for example, the ACI rat strain, the Dark Agouti (DA) rat strain, the Wistar rat strain, the LEA rat strain, the Sprague Dawley (SD) rat strain, or the Fischer rat strain, such as Fisher F344 or Fischer F6. Rats can also be obtained from strains derived from a mix of two or more of the strains listed above. For example, suitable rats can be from the DA strain or the ACI strain. The ACI rat strain has a white abdomen and paws and an RT1 av1 The dark agouti (DA) rat strain is characterized as having the black agouti containing haplotype. Such strains are available from a variety of sources, including Harlan Laboratories. The dark agouti (DA) rat strain is characterized as having the agouti coat and the RT1 av1 The rat is characterized as having a haplotype. Such rats can be obtained from various sources, including Charles River and Harlan Laboratories. Some suitable rats can be derived from purebred rat strains. For example, see US2014 / 0235933, the entirety of which is incorporated herein by reference for all purposes.

[0133] III. Methods of Using Non-Human Animals Containing a Humanized TRKB Locus to Evaluate the Efficacy of Human TRKB Targeting Reagents In Vivo or Ex Vivo Various methods are provided for using non-human animals comprising a humanized TRKB locus as described elsewhere herein to evaluate or optimize the delivery or efficacy of a human TRKB targeting reagent (e.g., a therapeutic agonist molecule) in vivo or ex vivo. Because the non-human animals comprise a humanized TRKB locus, the non-human animals will more accurately reflect the efficacy of the human TRKB targeting reagent.

[0134] A. Methods for testing the efficacy of human TRKB targeting reagents in vivo or ex vivo Various methods are provided for evaluating the delivery or efficacy of a human TRKB targeting reagent in vivo using a non-human animal comprising a humanized TRKB locus as described elsewhere herein. Such methods can include (a) introducing a human TRKB targeting reagent into the non-human animal, and (b) evaluating the activity of the human TRKB targeting reagent.

[0135] The human TRKB targeting reagent can be a human TRKB targeting antibody or antigen binding protein, or any other large or small molecule that targets human TRKB. Alternatively, the human TRKB targeting reagent can be any biological or chemical agent that targets the human TRKB locus (human TRKB gene), human TRKB mRNA, or human TRKB protein. Examples of human TRKB targeting reagents are disclosed elsewhere herein.

[0136] Such human TRKB targeting reagents can be administered by any delivery method (e.g., injection, AAV, LNP or HDD) and any administration route disclosed in more detail elsewhere herein. Means and administration routes for delivering therapeutic molecules are disclosed in more detail elsewhere herein. In certain methods, the reagents are delivered by injection (e.g., direct hippocampal injection, subcutaneous injection or intravitreal injection).

[0137] Methods for evaluating the activity of human TRKB targeting reagents are well known and are provided elsewhere herein. In some methods, evaluating the activity (e.g., agonistic or inhibitory activity) of human TRKB targeting reagents comprises evaluating TRKB activity (e.g., TRKB phosphorylation, TRKB-mediated activation of downstream signaling pathways, or TRKB-induced phenotype) as disclosed elsewhere herein. Activity evaluation can be performed in any cell type, any tissue type, or any organ type as disclosed elsewhere herein. In some methods, activity evaluation is in brain tissue (e.g., hippocampus or striatum) or neurons (e.g., retinal ganglion cells, hippocampal neurons, or cortical neurons).

[0138] When the TRKB targeting reagent is a genome editing reagent (e.g., a nuclease agent), the method can include evaluating the modification of the humanized TRKB locus. For example, the evaluating can include sequencing (e.g., next generation sequencing) the humanized TRKB locus in one or more cells isolated from the non-human animal. The evaluating can include isolating a target organ (e.g., brain) or tissue from the non-human animal and evaluating the modification of the humanized TRKB locus in the target organ or tissue. The evaluating can also include evaluating the modification of the humanized TRKB locus in two or more different cell types within the target organ or tissue. Similarly, the evaluating can 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 the humanized TRKB locus in the non-target organ or tissue.

[0139] Such methods may also include measuring the expression level of mRNA produced by the humanized TRKB locus, or measuring the expression level of a protein encoded by the humanized TRKB locus. For example, protein levels can be measured in a particular cell, tissue or organ type (e.g., brain), or secretion levels can be measured in serum. Methods for assessing expression of TRKB mRNA or protein expressed from the humanized TRKB locus are provided elsewhere herein and are well known.

[0140] Using the various methods provided above for assessing activity in vivo, the activity of human TRKB targeting reagents can also be assessed ex vivo, as described elsewhere herein.

[0141] B. Methods for optimizing the delivery or efficacy of human TRKB targeting reagents in vivo or ex vivo Various methods are provided for optimizing the delivery of human TRKB targeting reagents to cells or non-human animals, or for optimizing the activity or efficacy of human TRKB targeting reagents in vivo. Such methods may, for example, include: (a) performing the above-described method of testing the efficacy of human TRKB targeting reagents in a first non-human animal or a first cell; (b) varying a variable and performing the method with the varied variable in a second non-human animal (i.e., of the same species) or a second cell; and (c) comparing the activity of the human TRKB targeting reagent in step (a) with the activity of the human TRKB targeting reagent in step (b) and selecting the method that results in higher efficacy or activity.

[0142] Methods for measuring the delivery, efficacy or activity of human TRKB targeting reagents are disclosed elsewhere herein. Higher efficacy can mean different things depending on the desired effect in the non-human animal or cell. For example, higher efficacy can mean higher activity and / or higher specificity. Higher activity can be, for example, activity in activating TRKB or activity in inhibiting TRKB. This can refer to a higher percentage of cells being targeted within a particular target cell type (e.g., neurons such as retinal ganglion cells) or within a particular target tissue or organ (e.g., brain). Higher specificity can refer to higher specificity for TRKB compared to off-target effects, higher specificity for the targeted cell type, or higher specificity for the targeted tissue or organ type.

[0143] The variable that is changed can be any parameter. As an example, the variable that is changed can be the packaging or delivery method by which the human TRKB targeting reagent(s) is introduced into the cell or non-human animal. Examples of delivery methods are disclosed elsewhere herein. As another example, the variable that is changed can be the administration route for the introduction of the human TRKB targeting reagent(s) into the cell or non-human animal. Examples of administration routes are disclosed elsewhere herein.

[0144] As another example, the variable that is changed can be the concentration or amount of human TRKB targeting reagent(s) that is introduced. As another example, the variable that is changed can be the timing of introducing the human TRKB targeting reagent(s) relative to the timing of evaluating the activity or effectiveness of the reagent. As another example, the variable that is changed can be the number or frequency at which the human TRKB targeting reagent(s) is introduced. As another example, the variable that is changed can be the human TRKB targeting reagent(s) that is introduced (e.g., comparing one reagent to a different reagent).

[0145] C. Human TRKB targeting reagents The human TRKB targeting reagent can be any reagent that targets human TRKB protein, human TRKB gene or human TRKB mRNA. The human TRKB targeting reagent can be, for example, an agonist (i.e., a molecule that indirectly or directly activates human TRKB) or an antagonist (i.e., an inhibitor or inhibitory reagent that blocks human TRKB activity). In a specific example, the human TRKB targeting reagent is a TRKB agonist. The human TRKB targeting reagent in the method disclosed herein can be a known human TRKB targeting reagent, a putative human TRKB targeting reagent (e.g., a candidate reagent designed to target human TRKB), or a reagent that is being screened for human TRKB targeting activity.

[0146] For example, the human TRKB targeting reagent can be an antigen binding protein (e.g., an agonist antibody) that targets an epitope of the human TRKB protein. An example of such a reagent is the TRKB agonist antibody H4H9816P2. Other anti-TRKB antibodies are disclosed elsewhere herein. In some cases, the anti-TRKB antibody has a K of less than about 200 nM as measured by surface plasmon resonance at 25° C. or 37° C. D In other cases, the anti-TRKB antibody binds to human TRKB with a K of less than about 600 pM, less than about 300 pM, less than about 200 pM, less than about 150 pM, less than about 100 pM, less than about 80 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM, less than about 10 pM, less than about 5 pM, less than about 3 pM, or less than about 1 pM. DIn some cases, the anti-TRKB antibody binds to human TRKB with a dissociation half-life (t1 / 2) of greater than about 10 minutes as measured by surface plasmon resonance at 25° C. or 37° C. In other cases, the anti-TRKB antibody binds to human TRKB with a t1 / 2 of greater than about 20 minutes, greater than about 50 minutes, greater than about 100 minutes, greater than about 120 minutes, greater than about 150 minutes, greater than about 300 minutes, greater than about 350 minutes, greater than about 400 minutes, greater than about 450 minutes, greater than about 500 minutes, greater than about 550 minutes, greater than about 600 minutes, greater than about 700 minutes, greater than about 800 minutes, greater than about 900 minutes, greater than about 1000 minutes, greater than about 1100 minutes, or greater than about 1200 minutes. As a specific example, an anti-TRKB antibody may comprise a set of six CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) selected from the group set forth in Table 22, or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0147] Other human TRKB targeting reagents include small molecules (e.g., agonists) that target human TRKB protein. Examples of small molecule TRKB agonists include 7,8-dihydroxyflavone (7,8-DHF), deoxygedunin, LM22A-4 (N,N',N''-tris(2-hydroxyethyl)-1,3,5-benzenetricarboxamide) and LM22B-10 (2-[[4-[[4-[bis-(2-hydroxyethyl)-amino]-phenyl]-(4-chloro-phenyl)-methyl]-phenyl]-(2-hydroxy-ethyl)-amino]-ethanol). For example, see Liu et al. (2015) Translational Neurodegeneration 5:2; Massa et al. (2010) J. Clin. Invest. 120(5):1774-1785; and Yang et al. (2016) Neuropharmacology 110:343-361, each of which is incorporated herein by reference in its entirety for all purposes. An example of a TRKB targeting reagent that is an inhibitor is K252a. For example, see Yang et al. (2016) Neuropharmacology 110:343-361, each of which is incorporated herein by reference in its entirety for all purposes.

[0148] Other human TRKB targeting reagents include peptides or peptidomimetics (e.g., agonists) that target human TRKB protein. Examples of peptidomimetics that function as human TRKB agonists are disclosed, for example, in O'Leary et al. (2003) J. Biol. Chem. 278(28):25738-25744, the entire contents of which are incorporated herein by reference for all purposes.

[0149] Other human TRKB targeting reagents can 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 human TRKB gene. Similarly, a human TRKB targeting reagent can be an exogenous donor nucleic acid (e.g., a targeting vector or single-stranded oligodeoxynucleotide (ssODN)) designed to recombine with the human TRKB gene.

[0150] Other human TRKB targeting reagents can include antisense oligonucleotides (e.g., siRNA or shRNA) that target human TRKB mRNA. Antisense oligonucleotides (ASOs) or antisense RNAs are short synthetic strings of nucleotides designed to selectively bind to the RNA encoding the targeted protein, thereby preventing expression of the targeted protein by preventing translation. These compounds bind to RNA with high affinity and selectivity through well-characterized Watson-Crick base pairing (hybridization). RNA interference (RNAi) is an endogenous cellular mechanism for controlling gene expression in which small interfering RNA (siRNA) bound to the RNA-induced silencing complex (RISC) mediates cleavage of target messenger RNA (mRNA).

[0151] The non-human animals disclosed herein can also be used to evaluate the activity of any other known or putative human TRKB targeting reagents. Similarly, the non-human animals disclosed herein can be used to screen any other molecules for human TRKB targeting activity.

[0152] D. Administering a Human TRKB Targeting Reagent to a Non-Human Animal or Cell The methods disclosed herein can include introducing various molecules (e.g., human TRKB targeting reagents such as antibodies or small molecules) into a non-human animal or cell, including nucleic acids, proteins, nucleic acid-protein complexes, peptidomimetics, antigen-binding proteins, or small molecules. "Introducing" includes providing a molecule (e.g., a nucleic acid or a protein or a small molecule) to a cell or non-human animal in such a manner that it reaches the interior of the cell or the interior of a cell in the non-human animal. The introduction can be achieved by any means. When multiple components are introduced, the multiple components can be introduced simultaneously or sequentially in any combination. In addition, two or more of the components can be introduced into a cell or non-human animal by the same delivery method or different delivery methods. Similarly, two or more of the components can be introduced into a non-human animal by the same administration route or different administration routes.

[0153] Molecules introduced into non-human animals or cells can be provided in compositions that include carriers that increase the stability of the introduced molecule (e.g., extend the period under given storage conditions (e.g., -20°C, 4°C, or ambient temperature) that degradation products remain below a threshold, such as less than 0.5% by weight of the starting nucleic acid or protein; or increase stability in vivo). Non-limiting examples of such carriers include poly(lactic acid) (PLA) microspheres, poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres, liposomes, micelles, reverse micelles, lipid cochleates, and lipid microtubules.

[0154] Various methods and compositions are provided herein to allow the introduction of human TRKB targeting reagent into cells or non-human animals.The methods for introducing nucleic acid into various cell types are known, including, for example, stable transfection method, transient transfection method and virus-mediated method.

[0155] Transfection protocols and protocols for introducing nucleic acid sequences into cells can vary. Non-limiting transfection methods include liposomes; nanoparticles; calcium phosphate (Graham et al. (1973) Virology 52 (2): 456-67, Bacchetti et al. (1977) Proc. Natl. Acad. Sci. USA 74 (4): 1590-4 and Kriegler, M (1991). Transfer and Expression: A Laboratory Manual. New York: WH Freeman and Company. pp. 96-97); dendrimers; or chemical-based transfection methods using cationic polymers such as DEAE-dextran or polyethyleneimine. Non-chemical methods include electroporation, sono-poration, and optical transfection. Particle-based transfection includes the use of a gene gun or magnet-assisted transfection (Bertram (2006) Current Pharmaceutical Biotechnology 7, 277-28). Viral methods can also be used for transfection.

[0156] The introduction of human TRKB targeting reagent 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 through the nuclear membrane into the nucleus. 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 by regular electroporation). In one example, nucleofection is performed using the LONZA® NUCLEOFECTOR™ system.

[0157] Introduction of human TRKB targeting reagents into cells (e.g., zygotes) can also be achieved by microinjection. In zygotes (i.e., one-cell stage embryos), microinjection can be into the maternal and / or paternal pronuclei, or into the cytoplasm. If microinjection is only into one pronucleus, the paternal pronucleus is preferred due to its larger size. Microinjection of mRNA is preferably into the cytoplasm (e.g., to deliver mRNA directly to the translation machinery), while microinjection of proteins or protein- or RNA-encoding polynucleotides is preferably into the nucleus / pronucleus. Alternatively, microinjection can be performed by injection into both the nucleus / pronucleus and the cytoplasm: the needle can first be introduced into the nucleus / pronucleus and a first amount can be injected, and while removing the needle from the one-cell stage embryo, a second amount can be injected into the cytoplasm. If the protein is injected into the cytoplasm and needs to be targeted to the nucleus, it can contain a nuclear localization signal to ensure delivery to the nucleus / pronucleus. The method for carrying out microinjection is well known.See, for example, Nagy et al. (Nagy A, Gertsenstein M, Vintersten K, Behringer R., 2003, Manipulating the Mouse Embryo. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press);See also Meyer et al. (2010) Proc. Natl. Acad. Sci. USA 107:15022-15026 and Meyer et al. (2012) Proc. Natl. Acad. Sci. USA 109:9354-9359.

[0158] Other methods for introducing human TRKB targeting reagents into cells or non-human animals can 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 acid or protein can be introduced into cells or non-human animals in carriers such as poly(lactic acid) (PLA) microspheres, poly(D,L-lactic-co-glycolic 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) and lipid nanoparticle-mediated delivery.

[0159] The introduction of human TRKB targeting reagents into cells or non-human animals can be achieved by hydrodynamic delivery (HDD). Hydrodynamic delivery has emerged as a method for intracellular DNA delivery in vivo. For gene delivery to parenchymal cells, only the essential DNA sequence needs to be injected via selected blood vessels, eliminating the safety concerns associated with current viral and synthetic vectors. When injected into the bloodstream, DNA can reach cells in different tissues that are accessible to the blood. Hydrodynamic delivery uses the force generated by the rapid injection of a large volume of solution into the incompressible blood in the circulation to overcome the physical barriers of endothelium and cell membranes that prevent large, membrane-impermeable compounds from entering parenchymal cells. In addition to the delivery of DNA, this method is useful for the efficient intracellular delivery of RNA, proteins and other small compounds in vivo. See, for example, Bonamassa et al. (2011) Pharm. Res. 28(4):694-701, which is incorporated herein by reference in its entirety for all purposes.

[0160] Introduction of human TRKB targeting reagents can also be achieved by virus-mediated delivery, such as AAV-mediated delivery or lentivirus-mediated delivery. Other exemplary viruses / viral vectors include retroviruses, adenoviruses, vaccinia viruses, poxviruses, and herpes simplex viruses. The viruses can infect dividing cells, non-dividing cells, or both dividing and non-dividing cells. The viruses can integrate into the host genome or not. Such viruses can also be engineered to have reduced immunity. The viruses can be replication-competent or replication-deficient (e.g., lacking one or more genes required for additional rounds of virion replication and / or packaging). The viruses can cause transient expression, sustained expression (e.g., for at least 1 week, 2 weeks, 1 month, 2 months, or 3 months), or permanent expression. Exemplary viral titers (e.g., AAV titers) are 10 12 , 10 13 , 10 14 , 10 15 and 10 16 Contains vector genomes / mL.

[0161] The ssDNA AAV genome consists of two open reading frames, Rep and Cap, flanked by two inverted terminal sequences that allow the 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. Such genes (E4, E2a and VA) mediated AAV replication. For example, the transfer plasmid, Rep / Cap and the helper plasmid can be transfected into HEK293 cells containing adenovirus genes E1+ to produce infectious AAV particles. Alternatively, Rep, Cap and adenovirus helper genes can be combined into a single plasmid. Similar packaging cells and methods can be used for other viruses, such as retroviruses.

[0162] Multiple serotypes of AAV have been identified. These serotypes differ in the type of cells they infect (i.e., their tropism), allowing preferential transduction of specific cell types. Serotypes for CNS tissues include AAV1, AAV2, AAV4, AAV5, AAV8, and AAV9. Serotypes for heart tissues include AAV1, AAV8, and AAV9. Serotypes for kidney tissues include AAV2. Serotypes for lung tissues include AAV4, AAV5, AAV6, and AAV9. Serotypes for pancreatic tissues include AAV8. Serotypes for photoreceptor cells include AAV2, AAV5, and AAV8. Serotypes for retinal pigment epithelium tissues include AAV1, AAV2, AAV4, AAV5, and AAV8. Serotypes for skeletal muscle tissues include AAV1, AAV6, AAV7, AAV8, and AAV9. Serotypes for liver tissue include AAV7, AAV8 and AAV9, in particular AAV8.

[0163] 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 capsid from serotype 5. The use of pseudotyped viruses can improve transduction efficiency and alter tropism. Hybrid capsids from different serotypes can also be used to alter viral tropism. For example, AAV-DJ contains hybrid capsids from eight serotypes and exhibits high infectivity across a wide range of cell types in vivo. AAV-DJ8 is another example that exhibits the properties of AAV-DJ but with enhanced brain uptake. AAV serotypes can also be modified by mutation. Examples of mutational modifications of AAV2 include Y444F, Y500F, Y730F, and S662V. Examples of mutational modifications of AAV3 include Y705F, Y731F, and T492V. Examples of mutational modifications of AAV6 include S663V and T492V. Other pseudotyped / modified AAV variants include AAV2 / 1, AAV2 / 6, AAV2 / 7, AAV2 / 8, AAV2 / 9, AAV2.5, AAV8.2 and AAV / SASTG.

[0164] To accelerate transgene expression, self-complementary AAV (scAAV) variants can be used. Because AAV depends on the cell's DNA replication machinery to synthesize the complementary strand of AAV's single-stranded DNA genome, transgene expression can be delayed. To address this delay, scAAV can be used to contain complementary sequences that can spontaneously anneal upon infection, eliminating the requirement for host cell DNA synthesis.

[0165] To increase packaging capacity, a longer transgene can be split between two AAV transfer plasmids, the first with a 3' splice donor and the second with a 5' splice acceptor. After co-infection of cells, the viruses can form a concatemer and be spliced ​​together to express the full-length transgene. This allows for expression of a longer transgene, but at a lower expression efficiency. A similar method to increase capacity utilizes homologous recombination. For example, a transgene can be split between two transfer plasmids, both of which have substantial sequence overlap, such that co-expression induces homologous recombination and expression of the full-length transgene.

[0166] The introduction of human TRKB targeting reagents can also be achieved by lipid nanoparticle (LNP)-mediated delivery. Lipid formulations can protect biological molecules from degradation while improving their cellular uptake. Lipid nanoparticles are particles that contain multiple lipid molecules physically associated with each other by intermolecular forces. This includes microspheres (including unilamellar and multilamellar vesicles, e.g., liposomes), the dispersed phase in emulsions, micelles, or the internal phase in suspensions. Such lipid nanoparticles can be used to encapsulate one or more nucleic acids or proteins for delivery. Formulations containing cationic lipids are useful for the delivery of 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 that nanoparticles can exist in vivo. Examples of suitable cationic lipids, neutral lipids, anionic lipids, helper lipids and stealth lipids can be found in WO2016 / 010840A1, the entirety of which is incorporated herein by reference for all purposes. 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.

[0167] Delivery mechanism can be selected to reduce immunogenicity. For example, when multiple components are delivered, they can be delivered by different mechanisms (e.g., bimodal delivery). These different mechanisms can impart different pharmacodynamic or pharmacokinetic properties to the delivered molecule of interest. For example, different mechanisms can result in different tissue distribution, different half-life, or different temporal distribution. Some delivery mechanisms (e.g., delivery of nucleic acid vectors that persist in cells by autonomous replication or genomic integration) result in more persistent expression and presence of molecules, while other delivery mechanisms are transient and less persistent (e.g., delivery of RNA or protein).

[0168] In vivo administration can be by any suitable route, including, for example, parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal or intramuscular. Systemic administration mechanisms include, for example, oral and parenteral routes. Examples of parenteral routes include intravenous, intraarterial, intraosseous, intramuscular, intradermal, subcutaneous, intranasal and intraperitoneal routes. An example is intravenous injection. Nasal drops and intravitreal injection are other examples. Local administration mechanisms include, for example, intrathecal, intracerebroventricular, intraparenchymal (e.g., localized intraparenchymal delivery to the striatum (e.g., into the caudate nucleus or putamen), cerebral cortex, precentral gyrus, hippocampus (e.g., into the dentate gyrus or CA3 region), temporal cortex, amygdala, frontal cortex, thalamus, cerebellum, medulla, hypothalamus, optic tectum, tegmentum, or substantia nigra), intraocular, intraorbital, subconjuctival, intravitreal, subretinal, and transscleral routes. Significantly smaller amounts of components (compared to systemic approaches) can be effective when administered locally (e.g., intraparenchymal or intravitreal) compared to when administered systemically (e.g., intravenously). Local administration mechanisms can also reduce or eliminate the incidence of potentially toxic side effects that may occur when therapeutically effective amounts of components are administered systemically. In specific examples, the human TRKB targeting reagent is administered by direct hippocampal injection, subcutaneous injection, or intravitreal injection.

[0169] The composition comprising human TRKB targeting reagent can be formulated using one or more physiologically and pharmaceutically 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 other components of the formulation and is not substantially harmful to its recipient.

[0170] The frequency of administration and the number of dosages may depend on the half-life and route of administration of the human TRKB targeting reagent, among other factors. The introduction of the human TRKB targeting reagent into cells or non-human animals can be performed one or more times over a period of time. For example, the introduction can be performed at least twice over a period of time, at least three times over a period of time, at least four times over a period of time, at least five times over a period of time, at least six times over a period of time, at least seven times over a period of time, at least eight times over a period of time, at least nine times over a period of time, at least ten times over a period of time, at least eleven times over a period of time, at least twelve times over a period of time, at least thirteen times over a period of time, at least fourteen times over a period of time, at least fifteen times over a period of time, at least sixteen times over a period of time, at least seventeen times over a period of time, at least eighteen times over a period of time, at least nineteen times over a period of time, or at least twenty times over a period of time.

[0171] E. In vivo or ex vivo measurement of delivery, activity or efficacy of human TRKB targeting reagents The methods disclosed herein may further comprise detecting or measuring the activity of the human TRKB targeting reagent. Measuring the activity (e.g., agonist or inhibitor activity) of such a reagent may comprise measuring TRKB activity. TRKB activity may be measured by any known means. For example, TRKB phosphorylation may be assessed (e.g., in the brain or neurons), activation of downstream pathways such as PI3K / AKT and MAPK / ERK by TRKB may be assessed (e.g., in the brain or neurons, such as primary cortical neurons), or cell survival may be assessed (e.g., neuronal cell survival, such as retinal ganglion cell survival). For example, phosphorylation or activation of downstream signaling pathways may be assessed 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, or 18 hours after dosing. An increase in TRKB phosphorylation, activation of downstream signaling pathways, or cell survival may be indicative of TRKB activation, while a decrease may be indicative of TRKB inhibition.

[0172] In non-human animals, the evaluating step can include evaluating one or more or all of body weight, body composition, metabolism, and locomotion (e.g., at 12, 24, 48, 72, 96, or 120 hours after dosing) compared to a control non-human animal. See, for example, Lin et al. (2008) PLoS ONE 3(4):e1900; Rios et al. (2013) Trends in Neurosciences 36(2):83-90; and Zorner et al. (2003) Biol. Psychiatry 54:972-982, each of which is incorporated herein by reference in its entirety for all purposes. Evaluating changes in body composition can include, for example, evaluating lean mass and / or fat mass. Evaluating changes in metabolism can include, for example, evaluating changes in food consumption and / or water consumption. A decrease in body weight, fat mass, lean mass, food intake and water intake can be indicative of TRKB activation, whereas an increase can be indicative of TRKB inhibition. An increase in locomotion can be indicative of TRKB activation, whereas a decrease can be indicative of TRKB inhibition.

[0173] The evaluating step can include evaluating neuroprotective activity. As an example, cell survival can be evaluated in non-human animals. For example, rodent retinal ganglion cells (RGCs) are often used to study neurodegenerative processes associated with axonal pathology and to assay neuroprotective therapies. See, for example, Nadal-Nicolas et al. (2009) Invest. Ophthalmol. Vis. Sic. 50(8):3860-3868, which is incorporated herein by reference in its entirety for all purposes. Retinal ganglion cell survival / viability can be evaluated after treatment with human TRKB targeting reagents compared to control non-human animals (e.g., in a complete optic nerve section model after optic nerve injury). For example, retinal ganglion cell survival / viability can be evaluated in a complete optic nerve section model after optic nerve injury. See, for example, Nadal-Nicolas et al. (2009) Invest. Ophthalmol. Vis. Sic. 50(8):3860-3868, which is incorporated herein by reference in its entirety for all purposes. As another example, retinal ganglion cell survival / viability can be assessed in an optic nerve crush model. In this model, crush injury to the optic nerve leads to progressive retinal ganglion cell apoptosis. See, for example, Tang et al. (2011) J. Vis. Exp. 50:2685, which is incorporated herein by reference in its entirety for all purposes. Retinal ganglion cell survival / viability can be assessed, for example, by measuring retinal ganglion cell density (e.g., in retinas excised and stained for retinal ganglion cells). Increased survival / viability can be indicative of TRKB activation, while decreased survival / viability can be indicative of TRKB inhibition.

[0174] When the human TRKB targeting reagent is a genome editing reagent, the measuring step can include evaluating the humanized TRKB locus for modification. Various methods can be used to identify cells with targeted genetic modifications. The screening can include a quantitative assay to evaluate the modification of parental chromosome alleles (MOA). For example, the quantitative assay can be performed by 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 a non-targeted 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 probe(s), INVADER® probe, TAQMAN® molecular beacon probe or ECLIPSE™ probe technology (see, for example, US2005 / 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 is sometimes referred to as "NGS" or "massively parallel sequencing" or "high-throughput sequencing". NGS can be used as a screening tool in addition to MOA assays to reveal the exact nature of the targeted genetic modification and whether it is consistent across cell types or tissue types or organ types.

[0175] The step of evaluating in a non-human animal can be performed in any cell type from any tissue or organ. For example, evaluation can be performed in multiple cell types from the same tissue or organ (e.g., brain), or in cells from multiple locations within a tissue or organ (e.g., hippocampus and striatum). 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 human TRKB targeting reagent. As another example, evaluation can be performed in multiple types of tissue 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.

[0176] If the reagent is designed to inactivate the humanized TRKB locus, affect expression of the humanized TRKB locus, or prevent translation of humanized TRKB mRNA, the measuring step can include evaluating humanized TRKB mRNA or protein expression, which can be done in the brain or in specific cell types (e.g., neurons, such as retinal ganglion cells).

[0177] IV. METHODS FOR GENERATING NON-HUMAN ANIMALS COMPRISING A HUMANIZED TRKB LOCUS Various methods are provided for producing non-human animal genomes, non-human animal cells or non-human animals comprising humanized TRKB locus as disclosed elsewhere herein. Any convenient method or protocol for producing genetically modified organisms is suitable for producing such genetically modified non-human animals. See, for example, Cho et al. (2009) Current Protocols in Cell Biology 42:19.11:19.11.1-19.11.22 and Gama Sosa et al. (2010) Brain Struct. Funct. 214(2-3):91-109, each of which is incorporated herein by reference in its entirety for all purposes. Such genetically modified non-human animals can be generated, for example, by targeted gene knock-in at the TrkB locus.

[0178] For example, a method for producing a non-human animal comprising a humanized TRKB locus can include the steps of (1) modifying the genome of a pluripotent cell to comprise a humanized TRKB locus, (2) identifying or selecting a genetically modified pluripotent cell comprising a humanized TRKB locus, (3) introducing the genetically modified pluripotent cell into a non-human animal host embryo, and (4) implanting and gestation the host embryo in a surrogate mother. For example, a method for producing a non-human animal comprising a humanized TRKB locus can include the steps of (1) modifying the genome of a pluripotent cell to comprise a humanized TRKB locus, (2) identifying or selecting a genetically modified pluripotent cell comprising a humanized TRKB locus, (3) introducing the genetically modified pluripotent cell into a non-human animal host embryo, and (4) gestation the host embryo in a surrogate mother. If desired, the host embryo containing the modified pluripotent cells (e.g., non-human ES cells) can be incubated to the blastocyst stage prior to implantation and gestation in a surrogate mother to produce an F0 non-human animal. The surrogate mother can then produce an F0 generation non-human animal that contains a humanized TRKB locus.

[0179] The method can further include identifying a cell or animal that has a modified target genomic locus. A variety of methods can be used to identify cells and animals that have a targeted genetic modification.

[0180] The step of modifying the genome can utilize, for example, an exogenous repair template (e.g., a targeting vector) to modify the TrkB locus to include the humanized TRKB locus disclosed herein. As an example, the targeting vector can be for generating a humanized TRKB gene in an endogenous TrkB locus (e.g., an endogenous non-human animal TrkB locus), and the targeting vector includes a 5' homology arm that targets a 5' target sequence in the endogenous TrkB locus and a 3' homology arm that targets a 3' target sequence in the endogenous TrkB locus. The exogenous repair template can also include a nucleic acid insert that includes a segment of DNA to be integrated into the TrkB locus. The integration of the nucleic acid insert into the TrkB locus can result in the addition of a nucleic acid sequence of interest in the TrkB locus, the deletion of a nucleic acid sequence of interest in the TrkB locus, or the replacement (i.e., deletion and insertion) of a nucleic acid sequence of interest in the TrkB locus. The homology arms can flank an insert nucleic acid containing a human TRKB sequence to generate a humanized TRKB locus (e.g., to delete a segment of the endogenous TrkB locus and replace it with an orthologous human TRKB sequence).

[0181] Exogenous repair template can be for non-homologous end joining mediated insertion or homologous recombination.Exogenous repair template can include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), which can be single-stranded or double-stranded, and can be in linear or circular form.For example, repair template can be single-stranded oligodeoxynucleotide (ssODN).

[0182] The exogenous repair template can also contain heterologous sequences that are not present in the untargeted endogenous TrkB locus. For example, the exogenous repair template can contain a selection cassette, such as a selection cassette flanked by recombinase recognition sites.

[0183] Some exogenous repair templates include homology arms. When the exogenous repair template nucleic acid also includes a nucleic acid insert, the homology arms can flank the nucleic acid insert. For ease of reference, the homology arms are referred to herein as 5' and 3' (i.e., upstream and downstream) homology arms. This terminology refers to the relative position of the homology arms to the nucleic acid insert in the exogenous repair template. The 5' and 3' homology arms correspond to regions in the TrkB locus, referred to herein as "5' target sequence" and "3' target sequence", respectively.

[0184] Homologous arms and target sequences "correspond" or "are corresponding" to each other if the two regions share a sufficient level of sequence identity with each other to act as substrates for homologous recombination reaction. The term "homology" includes DNA sequences that are identical or share sequence identity with corresponding sequences. The sequence identity between a given target sequence and the corresponding homologous arms found in an exogenous repair template can be any degree of sequence identity that allows homologous recombination to occur. For example, the amount of sequence identity shared by the homology arms (or fragments thereof) of the exogenous repair template and the target sequence (or fragments thereof) can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, so that the sequences undergo homologous recombination.Furthermore, the corresponding regions of homology between the homology arms and the corresponding target sequence can be of any length sufficient to promote homologous recombination.In some targeting vectors, the intended mutation in the endogenous TrkB locus is contained in the insert nucleic acid flanked by the homology arms.

[0185] In cells other than one-cell stage embryos, the exogenous repair template can be a "large targeting vector" or "LTVEC", which includes a targeting vector that includes homology arms that correspond to and are derived from larger nucleic acid sequences than those typically used by other approaches intended to perform homologous recombination in cells. LTVEC also includes targeting vectors that include nucleic acid inserts with larger nucleic acid sequences than those typically used by other approaches intended to perform homologous recombination in cells. For example, LTVEC allows modification of large loci that cannot be accommodated by traditional plasmid-based targeting vectors due to their size limitations. For example, the targeted locus can be a cellular locus that cannot be targeted using conventional methods or that can be targeted inaccurately or with significantly lower efficiency in the absence of a nick or double-strand break induced by a nuclease agent (e.g., Cas protein) (i.e., the 5' and 3' homology arms can correspond to it). LTVEC can be of any length, typically at least 10 kb in length. The sum of the 5' and 3' homology arms in a LTVEC is typically at least 10 kb.

[0186] The screening step can include, for example, a quantitative assay to evaluate the modification of alleles (MOA) of parental chromosomes. For example, the quantitative assay can be performed by 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-targeted reference locus. The primer set can include a fluorescent probe that recognizes the amplified sequence.

[0187] Other examples of suitable quantitative assays include fluorescence-mediated in situ hybridization (FISH), comparative genomic hybridization, isothermal DNA amplification, quantitative hybridization to immobilized probe(s), INVADER® probe, TAQMAN® molecular beacon probe or ECLIPSE™ probe technology (see, e.g., US2005 / 0144655, which is incorporated by reference in its entirety for all purposes).

[0188] An example of a suitable pluripotent cell is an embryonic stem (ES) cell (e.g., a mouse ES cell or a rat ES cell). The modified pluripotent cell can be generated by recombination, for example, by (a) introducing into a cell one or more exogenous donor nucleic acids (e.g., a targeting vector) including, for example, an insert nucleic acid flanked by 5' and 3' homology arms corresponding to 5' and 3' targeting sites, the insert nucleic acid including a human TRKB sequence to generate a humanized TRKB locus, and (b) identifying at least one cell that includes in its genome the insert nucleic acid integrated into the endogenous TrkB locus (i.e., identifying at least one cell that includes a humanized TRKB locus). The modified pluripotent cells can be generated by recombination, for example, by: (a) introducing into a cell one or more targeting vectors comprising an insert nucleic acid flanked by 5' and 3' homology arms corresponding to 5' and 3' target sites, where the insert nucleic acid comprises a humanized TRKB locus; and (b) identifying at least one cell that comprises within its genome the insert nucleic acid integrated into the target genomic locus.

[0189] Alternatively, modified pluripotent cells can be generated by: (a) introducing into a cell one or more exogenous donor nucleic acids (e.g., a targeting vector) that optionally include: (i) a nuclease agent that induces a nick or double-stranded break at a target site within the endogenous TrkB locus; and (ii) an insert nucleic acid flanked by 5' and 3' homology arms that correspond to the 5' and 3' target sites that are positioned sufficiently close to the nuclease target site, e.g., the insert nucleic acid includes a human TRKB sequence to generate a humanized TRKB locus; and (c) identifying at least one cell that includes within its genome the insert nucleic acid integrated into the endogenous TrkB locus (i.e., identifying at least one cell that includes a humanized TRKB locus). Alternatively, modified pluripotent cells can be generated by (a) introducing into a cell one or more targeting vectors comprising (i) a nuclease agent that induces a nick or double-stranded break at a recognition site within a target genomic locus; and (ii) an insert nucleic acid flanked by 5' and 3' homology arms corresponding to 5' and 3' target sites positioned sufficiently close to the recognition site, where the insert nucleic acid comprises a humanized TRKB locus, and (c) identifying at least one cell that comprises a modification (e.g., integration of the insert nucleic acid) at the target genomic locus. Any nuclease agent that induces a nick or double-stranded break at the desired recognition site may 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, for example, US2013 / 0309670 and US2015 / 0159175, each of which is incorporated herein by reference in its entirety for all purposes.

[0190] Donor cells can be introduced into any stage of host embryo, such as blastocyst stage or pre-morula stage (i.e., 4-cell stage or 8-cell stage). Progeny that can transmit genetic modification through germline are generated. See, for example, U.S. Patent No. 7,294,754, the entirety of which is incorporated herein by reference for all purposes.

[0191] Alternatively, the method of producing a non-human animal described elsewhere herein can include (1) modifying the genome of a one-cell stage embryo to include a humanized TRKB locus using the methods described above for modifying pluripotent cells, (2) selecting a genetically modified embryo, and (3) implanting and gestating the genetically modified embryo in a surrogate mother. Alternatively, the method of producing a non-human animal described elsewhere herein can include (1) modifying the genome of a one-cell stage embryo to include a humanized TRKB locus using the methods described above for modifying pluripotent cells, (2) selecting a genetically modified embryo, and (3) gestating the genetically modified embryo in a surrogate mother. Progeny capable of transmitting the genetic modification through the germline are generated.

[0192] Nuclear transfer techniques can also be used to generate non-human mammals. Briefly, methods for nuclear transfer can include the steps of (1) enucleating an oocyte or providing an enucleated oocyte, (2) isolating or providing a donor cell or nucleus to be combined with the enucleated oocyte, (3) inserting a cell or nucleus into the enucleated oocyte to form a reconstituted cell, (4) implanting the reconstituted cell into the uterus of an animal to form an embryo, and (5) allowing the embryo to develop. In such methods, oocytes are generally retrieved from dead animals, but can also be isolated from either the oviducts and / or ovaries of living animals. Oocytes can be matured in a variety of well-known media prior to enucleation. Enucleation of oocytes can be performed in a number of well-known manners. Insertion of a donor cell or nucleus into an enucleated oocyte to form a reconstituted cell can be done by microinjection of the donor cell under the zona pellucida prior to fusion. Fusion can be induced by application of DC electric pulses across the contact / fusion plane (electrofusion), by exposure of 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, chemical-induced shocks, permeabilization with sperm, increasing levels of divalent cations in the oocyte, and decreasing phosphorylation of cellular proteins in the oocyte (as by kinase inhibitors). The activated reconstituted cells or embryos can be cultured in well-known media and then transferred to the uterus of an animal. See, for example, US2008 / 0092249, WO1999 / 005266, US2004 / 0177390, WO2008 / 017234, and U.S. Patent No. 7,612,250, each of which is incorporated herein by reference in its entirety and for all purposes.

[0193] Various methods provided herein allow for the generation of genetically modified non-human F0 animals, where the cells of the genetically modified F0 animals contain a humanized TRKB locus. It is recognized that the number of cells in the F0 animal that have a humanized TRKB locus will vary depending on the method used to generate the F0 animal. For example, the introduction of donor ES cells into pre-morula stage embryos (e.g., 8-cell stage mouse embryos) from corresponding organisms by the VELOCIMOUSE® method allows a greater percentage of the cell population of the F0 animal to contain cells with a nucleotide sequence of interest that contains a targeted genetic modification. For example, at least 50%, 60%, 65%, 70%, 75%, 85%, 86%, 87%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cell contribution of the non-human F0 animal can contain a cell population with a targeted modification.

[0194] The cells of the genetically modified F0 animal can be heterozygous for the humanized TRKB locus or can be homozygous for the humanized TRKB locus.

[0195] All patent applications, websites, other publications, accession numbers, etc. cited above or below are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual item was specifically and individually indicated to be incorporated herein by reference. Where different versions of a sequence are associated with an accession number at different times, the version associated with that accession number at the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or, if applicable, the filing date of the priority application referencing that accession number. Similarly, where different versions of a publication, website, etc. are published at different times, the version most recently published at the effective filing date of this application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the present invention can be used in combination with any other, unless otherwise indicated. Although the present invention has been described in some detail by way of illustration and example, for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

[0196] 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 codes for amino acids. Nucleotide sequences follow the standard convention of beginning at the 5'-end of the sequence and proceeding forward to the 3'-end (i.e., left to right on each line). Only one strand of each nucleotide sequence is shown, but the complementary strand is understood to be included by reference to either of the displayed strands. Where a nucleotide sequence encoding an amino acid sequence is provided, it is understood that codon-degenerate variants thereof that encode the same amino acid sequence are also provided. Amino acid sequences follow the standard convention of beginning at the amino terminus of the sequence and proceeding forward to the carboxy terminus (i.e., left to right on each line).

[0197] [Table 1-1] [Table 1-2] [Table 1-3] EXAMPLES

[0198] Example 1: Generation of mice containing a humanized TRKB locus A large targeting vector (LTVEC) was made that contains a 5' homology arm that contains 41.6 kb of the mouse TrkB locus and a 3' homology arm that contains 62.4 kb of the mouse TrkB locus, and the 65.7 kb region of the mouse TrkB gene that encodes the mouse TRKB extracellular domain was replaced with the corresponding 74.4 kb of human TRKB sequence. Information on mouse and human TRKB is shown in Table 2. A description of the construction of the large targeting vector is shown in Table 3. The construction and use of the large targeting vector (LTVEC) derived from bacterial artificial chromosome (BAC) DNA by bacterial homologous recombination (BHR) reaction using VELOCIGENE® genetic engineering technology is described, for example, in U.S. Patent No. 6,586,251 and Valenzuela et al. (2003) Nat. Biotechnol. 21(6):652-659, each of which is incorporated herein by reference in its entirety for all purposes. Generation of LTVECs by in vitro assembly methods is described, for example, in U.S. Patent Application Publication No. 2015 / 0376628 and WO2015 / 200334, each of which is incorporated by reference in its entirety for all purposes.

[0199] [Table 2]

[0200] [Table 3]

[0201] Specifically, the region starting from exon 2 (coding exon 1, from amino acid 32, preserving the signal peptide) through exon 10, including the first 137 base pairs of intron 10 and all introns between exons 2 and 10 (i.e., between coding exon 1 and exon 10), was deleted from the mouse TrkB locus (preserving the mouse transmembrane domain encoded by exons 10 and 11). The region including exon 2 / coding exon 1 (from amino acid 32, starting after the signal peptide) through exon 10, including the first 177 base pairs of intron 10 and all introns between exons 2 and 10 (i.e., between coding exon 1 and exon 10), was inserted in place of the deleted rat region (preserving the rat transmembrane domain encoded by exons 10 and 11).

[0202] The sequences of the mouse TRKB signal peptide, extracellular domain, transmembrane domain, and cytoplasmic domain are set forth in SEQ ID NOs: 51-54, respectively, with the corresponding coding sequences set forth in SEQ ID NOs: 63-66, respectively. The sequences of the human TRKB signal peptide, extracellular domain, transmembrane domain, and cytoplasmic domain are set forth in SEQ ID NOs: 59-62, respectively, with the corresponding coding sequences set forth in SEQ ID NOs: 71-74, respectively. The predicted encoded chimeric TRKB protein has mouse TRKB transmembrane and intracellular domains, mouse TRKB signal peptide, and human TRKB extracellular domain. See FIG. 1. An alignment of mouse and human TRKB proteins is shown in FIG. 6. The mouse and human TrkB / TRKB coding sequences are set forth in SEQ ID NOs: 9 and 11, respectively. The mouse and human TRKB protein sequences are set forth in SEQ ID NOs: 1 and 3, respectively. The predicted chimeric mouse / human TRKB coding sequence and the predicted chimeric mouse / human TRKB protein sequences are set forth in SEQ ID NOs: 12 and 4, respectively.

[0203] To generate mutant alleles, the large targeting vector was introduced into F1H4 mouse embryonic stem cells. Following antibiotic selection, colonies were picked, expanded, and screened by TAQMAN®. See Figure 2. Allele loss assays were performed to detect loss of endogenous rat alleles, and allele gain assays were performed to detect gain of humanized alleles, using the primers and probes shown in Table 4.

[0204] [Table 4]

[0205] Allele modification (MOA) assays, including loss of allele (LOA) and gain of allele (GOA) assays, are described, for example, in US Patent Application Publication No. 2014 / 0178879, US Patent Application Publication No. 2016 / 0145646, WO2016 / 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. Loss of allele (LOA) assays reverse the logic of traditional screening and quantify the copy number in genomic DNA samples of the native locus where the mutation is introduced. In correctly targeted heterozygous cell clones, one of the two native alleles (for genes that are not on the X or Y chromosome) is detected by LOA assays, and the other allele is interrupted by the targeted modification. The same principle can be applied in reverse as a gain of allele (GOA) assay to quantitate the copy number of a targeting vector inserted in a genomic DNA sample.

[0206] F0 mice were generated using VELOCIMOUSE® method. See, for example, U.S. Patent Nos. 7,576,259, 7,659,442, 7,294,754, U.S. Patent Application Publication No. 2008 / 0078000, and Poueymirou et al. (2007) Nat. Biotechnol. 25(1):91-99, each of which is incorporated herein by reference in its entirety for all purposes. In the VELOCIMOUSE® method, targeted mouse embryonic stem (ES) cells are injected into pre-morula stage embryos, such as 8-cell stage embryos, by laser-assisted injection. This effectively obtains F0 generation mice entirely derived from ES cells. All experiments carried out on humanized TRKB mice described below were carried out on mice in which the self-deletion selection cassette was self-deleted.

[0207] Example 2 TrkB hu / hu In vivo comparison of the effects of H4H9816 and isotype control REGN1945 antibodies on body weight and metabolism in mice (MAID7139) Experimental procedure To determine the effect of the TRKB agonist antibody H4H9816P2 on body weight and composition, mice homozygous for expression of the human TRKB receptor (TrkB) that replaces the murine TRKB receptor were cultured in vitro following a single subcutaneous injection of the antibody. hu / hu Metabolic studies of TrkB in mice (TrkB agonists and TrkB knockout mice) were performed. Some of these studies were based on previous studies of TrkB agonists and TrkB knockout mice. See, e.g., Lin et al. (2008) PLoS ONE 3(4):e1900; Rios et al. (2013) Trends in Neurosciences 36(2):83-90; and Zorner et al. (2003) Biol. Psychiatry 54:972-982, each of which is incorporated by reference in its entirety for all purposes. TrkB hu / huMice (male, 20 weeks old) were initially transferred from group cages to single-cage housing for 2 weeks of acclimation. After this period, mice were transferred to metabolic cages (CLAMS, Columbus Instruments) to assess changes in food and water consumption, locomotion, energy expenditure, and respiration following antibody administration. Regular powdered chow was stored on a spring-loaded balance (Mettler Toledo, PL602E) in the floor chamber, and food consumption was measured from the change in total weight of the chow. Water was available through a drinking spout on the cage, and intake was measured by tracking the change in pump line volume (Oxymax® / CLAMS Liquid Unit). Each of these parameters was measured continuously at 16-18 min intervals by CLAMS metabolic cages throughout the duration of the study. Metabolic data were analyzed by single measures and summarized for 24-h intervals encompassing one complete light-dark cycle using OXYMAX® / CLAMS software (Columbus Instruments, v5.35). After 2 weeks of cage acclimation, mice were treated with a single subcutaneous dose of 50 mg / kg of either the TRKB agonist antibody H4H9816P2, or an IgG4 isotype control antibody, in PBS, pH 7.2. hu / hu Mice were administered naive control TrkB hu / hu Groups of mice were not injected. Mice were weighed immediately prior to dosing, and at 24, 48, 72, 96, and 120 hours after dosing. Nuclear magnetic resonance relaxometry (also referred to as quantitative magnetic resonance) was performed using an EchoMRI™-500Analyzer (EchoMRI LLC) to measure the body composition of each mouse. Prior to dosing, mice were placed in a clear plastic holder and inserted into the NMR-MRI device to measure the lean mass, fat mass, and hydration status of each subject. Measurements were taken over the course of 0.5-3.2 minutes per mouse and again approximately 120 hours after dosing.

[0208] Results and Conclusions TrkB hu / huDaily body weight monitoring was performed to determine whether a single subcutaneous injection of H4H9816P2 induces weight loss in mice. Prior to dosing, there were no significant differences in the mean body weights of the three treatment groups, as the mean pre-dose body weights ranged from 28.39 to 29.85 g (Table 5). However, 48 hours after dosing, TrkB mice treated with H4H9816P2 showed no significant differences in body weight. hu / hu Mice lost an average of 1.70 g, or 5.96% of their pre-dose body weight. At the same time point, naive and isotype control antibody-treated TrkB hu / hu The body weight of mice increased by 1.79-2.37% from their pre-dosing weight. hu / hu Mice continued to lose weight throughout the time course of the study, and by 72 and 96 hours post-dosing, the mice had lost an average of 8.42% and 11.80% of their pre-dose weight, respectively. At 120 hours post-dosing, TrkB hu / hu Mice lost an average of 12.67% of their pre-dose body weight. Conversely, naïve and isotype control-treated TrkB hu / hu Mice did not show any loss of pre-dose body weight throughout the study. hu / hu The TRKB agonist antibody H4H9816P2 inhibited TrkB, as mouse body weight was significantly reduced compared to both naive and isotype controls at 48, 72, 96, and 120 hours after dosing. hu / hu It was determined to induce significant weight loss in mice.

[0209] [Table 5]

[0210] The effect of injection of TRKB agonist antibody H4H9816P2 on body composition was also measured by performing NMR-MRI on each subject before and after dosing. hu / huThere were no significant differences in fat mass or lean mass among the three treatment groups of mice, as each group had an average of 4.19–4.75 g of fat mass and 21.32–21.70 g of lean mass (Table 6). However, after administration of the antibody, TrkB hu / hu Mice lost an average of 48.90% of their total body fat mass over the course of the study (Table 6). hu / hu Mice lost an average of 8.49% and 9.48% of their pre-drug fat mass, respectively, which was significantly lower than subjects treated with H4H9816P2 (Table 6). hu / hu Mice lost an average of 7.84% of their lean mass throughout the study, which was significantly greater than the average of 2.41% and 1.75% of pre-dose lean mass lost by the naive and isotype control antibody-treated groups, respectively (Table 6). Thus, the weight loss described is due to a TrkB hu / hu This could be explained by the significant loss of fat mass and to a lesser extent lean mass following injection of the TRKB agonist antibody H4H9816P2 in mice.

[0211] [Table 6]

[0212] TrkB hu / hu In addition to assessing the effect of injection of TRKB agonist antibody H4H9816P2 on body weight and body composition in mice, feeding, drinking, and locomotor activity were continuously measured in metabolic cages. hu / hu Mice consumed an average of 3.49–3.73 g of chow per day. However, H4H9816P2 treatment reduced TrkB hu / hu Food intake in mice was significantly reduced to 2.20 g of chow per day within 24 hours after dosing. hu / huThe average level of food intake in mice never exceeded 2.49 g of chow per day throughout the remainder of the study. hu / hu Mice continuously consumed an average of 3.62–4.07 g of chow per day (Table 7).

[0213] Similarly, there were no significant differences in daily water consumption between treatment groups prior to dosing. hu / hu Mice consumed an average of 4.67-5.55 mL of water per day in each treatment group (Table 8). hu / hu Daily water intake in mice was reduced to 2.05–3.24 mL compared with naïve and isotype control antibody-treated TrkB mice, which continuously consumed 4.50–5.77 mL of water per day throughout the study. hu / hu The TrkB agonist antibody H4H9816P2 injection reduced TrkB expression in both naive and isotype control mice. hu / hu It appears to result in a significant reduction in both food and water intake in mice.

[0214] [Table 7]

[0215] [Table 8]

[0216] To determine the effect of antibody treatment on activity, locomotion was analyzed by OXYMAX® / CLAMS software (Columbus Instruments, v5.35), which continuously measures the total number of x-plane steps taken by each mouse. One mouse showed hyperactivity before dosing and was excluded from the post-dose statistical analysis. Naïve and isotype antibody-treated subjects were continuously recorded taking an average of 11,000-15,000 steps per day throughout the study. Meanwhile, TrkB mice treated with H4H9816P2 showed significantly higher activity than controls treated with H4H9816P2. hu / hu Mice recorded 28,260 steps between 24 and 48 hours post-dosing, 21,193 and 27,028 steps between 48 and 72 hours and 72 and 96 hours post-dosing, respectively (Table 9). hu / hu Mice recorded more total steps at each time point after antibody administration, suggesting that hyperactivity was an additive effect of injection of H4H9816P2. Combined, these effects suggest that a single subcutaneous injection of the TRKB agonist antibody H4H9816P2 inhibits TrkB hu / hu These results suggest that it induced significant changes in body weight, body composition, metabolism, and locomotion in mice.

[0217] [Table 9]

[0218] Example 3 TrkB hu / hu In vivo comparison of the effects of TRKB agonist antibody H4H9816P2 and IgG4 isotype control REGN1945 on TRKB phosphorylation in the brain following stereotactic injection in mice (MAID7139) Experimental procedure Tyrosine receptor kinase B (TRKB) is activated by binding of its ligand, brain-derived neurotrophic factor (BDNF), at the extracellular receptor domain, which induces dimerization and autophosphorylation of tyrosine residues in the intracellular receptor domain, and subsequently activates cytoplasmic signaling pathways. See, e.g., Haniu et al. (1997) J. Biol. Chem. 272(40):25296-25303 and Rogalski et al. (2000) J. Biol. Chem. 275(33):25082-25088, each of which is incorporated by reference in its entirety for all purposes. To determine the effect of the TRKB agonist antibody H4H9816P2 on TRKB activation kinetics, mice (MAID7139) homozygous for the chimeric mouse / human TRKB receptor with humanized extracellular domain (TrkB hu / hu We performed a time course study of TRKB phosphorylation following direct injection into the hippocampus in mice (hereafter referred to as TrkB hu / hu Mice (N=48) received bilateral stereotaxic injections of 2 μL of vehicle (PBS), REGN1945, herein referred to as an IgG4 isotype control antibody (final concentration 27.5 mg / mL), or TRKB agonist antibody H4H9816P2 (final concentration 27.5 mg / mL) into the hippocampus at -2 mm posterior and +1.5 mm lateral to the bregma. Both injections and needle removal were performed slowly over a 5 min interval to minimize tissue damage. TrkB agonist antibodies were then anesthetized by CO2 euthanasia approximately 30 min, 1 h, 4 h, or 18 h after injection. hu / hu Mice were sacrificed. A terminal bleed was performed by cardiac puncture to collect blood, and the mice were then perfused transcardially with chilled heparinized saline. The brains were carefully removed from the skull and 2 mm of tissue surrounding the injection site was removed. 3Brain sections were excised, collected in Eppendorf tubes and stored on ice. Brain sections were then lysed in 300 μL of RIPA lysis buffer (ThermoFisher Scientific, Cat#89901) containing 2× protease inhibitors and phosphatase inhibitors (ThermoFisher Scientific, Cat#78444) and stored on ice. Lysed tissue was then homogenized, aliquoted and stored at −80° C. for further processing.

[0219] Immunoprecipitation and Western blot were performed to evaluate TRKB phosphorylation in brain tissue. Anti-human TRKB antibody H4H10108N, which does not compete for binding with H4H9816P2, was coupled to NHS-activated Sepharose beads (prepared using the manufacturer's protocol, GE Healthcare, Cat#17-0906) and washed three times with DPBS to remove any residual storage solution. Homogenized brain lysates were thawed on ice and diluted to a concentration of 1 mg / mL (brain weight vs. buffer volume) in a buffer consisting of 1% NP-40, 0.1% Tween-20, protease inhibitors, and phosphatase inhibitors in TBST. Protein concentration of homogenized brain lysates was quantified by performing a standard BCA assay according to the manufacturer's instructions (Thermo Scientific Pierce, Cat#23225). 15 μL of anti-human TRKB antibody (H4H10108N) NHS-activated Sepharose beads per 100 μg protein were added to the brain lysate solution and the mixture was incubated overnight at 4 °C with gentle shaking at 20 rpm (Thermo rotator). The next day, samples were centrifuged at 1000 × g for 1 min and then the supernatant was carefully removed. The beads were then washed twice with 400 μL of Tris-buffered saline (Bio-Rad, Cat # 1706435) (TBST) containing 1% Tween-20 (Sigma Aldrich, Cat # P9416). After careful aspirating of the wash buffer, 60 μL of 0.1% trifluoroacetic acid (TFA, Sigma Aldrich, T62200), pH 3.0 in water was added to each sample. The solution was mixed and left to stand for 2 min before being collected and transferred to another tube. This process was repeated with an additional 60 μL of 0.1% TFA, pH 3.0. The two 0.1% TFA solutions for each sample were then combined and 2 μL of 1 M Tris-HCl, pH 8.5 (ThermoFisher Scientific, Cat# 15567-027) was added.

[0220] Solutions were dried using a fast vacuum, resuspended, and reduced in 20 μL of a mixture of 1× Laemmli buffer (Bio-Rad, Cat#1610737) and 355 nM 2-mercaptoethanol (BME; Gibco, Cat#21985-023). Samples were boiled at 95 °C for 10 min and loaded onto 10-well Mini-Protean 4–15% Tris-Glycine Gels (Bio-Rad, Cat#4561086). After electrophoresis, protein samples were transferred from the Tris-Glycine gels to PVDF membranes (Bio-Rad, Cat#170-4156) via a Trans-Blot Turbo Transfer System (Bio-Rad, Cat#1704156) at a constant speed of 1.3 A and 25 V for 30 min. After transfer, the membrane was blocked with 2.5% milk in TBST (Bio-Rad, Cat#170-6406) for 1 hour at room temperature and then probed with anti-phospho-TRKB antibody (Novus, Cat#NB100-92656) diluted 1:1000 in 2.5% BSA solution or anti-TRKB primary antibody (Cell Signaling, Cat#4603) diluted 1:1000 in 2.5% milk TBST overnight at 4° C. The next day, the blot was washed with TBST and incubated with horseradish peroxidase-conjugated anti-rabbit IgG antibody (Jackson, Cat#111-035-144) diluted 1:1000 in 1% milk in TBST for 1 hour at room temperature. The blots were then washed again and developed with ECL solution (PerkinElmer, Inc. Cat#RPN2106) followed by image exposures taken every 30 seconds.

[0221] Results and Conclusions As shown in Figure 3, TrkB hu / huImmunoprecipitation of proteins from mouse brain lysates and subsequent Western blots showed that hippocampal TRKB phosphorylation was detectable in mice injected with the TRKB agonist antibody H4H9816P2, but not in mice treated with vehicle or isotype control antibody. Among the time points evaluated, TRKB phosphorylation peaked at 4 hours after stereotaxic injection in mice dosed with H4H9816P2. TRKB phosphorylation was detected by Western blot in some, but not all, mice as early as 18 hours after dosing. Conversely, injection of vehicle and IgG4 isotype control antibody did not induce TRKB phosphorylation at any time point. Total TRKB receptor levels were significantly higher in some, but not all, TrkB receptors dosed with H4H9816P2 compared to vehicle and isotype control treated mice by Western blot. hu / hu It was also shown that TRKB levels were downregulated in mice treated with H4H9816P2. Total TRKB levels appeared to be slightly downregulated 18 hours after dosing in subjects treated with H4H9816P2. Thus, these results suggest that direct injection of the TRKB agonist antibody H4H9816P2 inhibits TrkB hu / hu It has been shown that phosphorylation of hippocampal TRJB receptors is induced in mice.

[0222] Example 4 TrkB on day 1 of life hu / hu Activation of downstream signaling pathways by TrkB agonist antibodies in primary cortical neurons from mice Experimental procedure All procedures were performed in accordance with the ARVO Statement for Use of Animals in Ophthalmic and Vision Research and the Regeneron Pharmaceuticals, Inc. IACUC. Mouse primary cortical neurons were isolated and cultured from humanized TrkB mice (MAID7139). See, for example, Beaudoin et al. (2012) Nat. Protoc. 7(9):1741-1754, the entire contents of which are incorporated by reference herein for all purposes. Western blots were performed to determine the effect of TrkB agonist antibodies on downstream pathways of Akt and Erk (p-Akt, p-Erk1 / 2). Primary cortical neurons from postnatal day 1 (P1) humanized TrkB juvenile mice were cultured for 4 days (DIV-4) in NeuralQ Basal Medium (Global Stem, cat#GSM-9420) supplemented with GS21 Neural Supplement (Global Stem, cat#GSM-3100), Glutamax (Invitrogen, cat#35050-061) and penicillin / streptomycin. Cells were treated with TrkB agonist antibody H4H9816P-L1 (10 μg / mL), TrkB agonist antibody H4H9780P-L1 (10 μg / mL), TrkB agonist antibody H4H9814P-L1 (10 μg / mL), IgG4 isotype control REGN1945 (10 μg / mL), control antibody H1M8037C-L1 (10 μg / mL), or BDNF (1 μg / mL) for 15 min or 2 h. Western blots were performed to determine whether the agonists had differences in the maintenance and strength of downstream signaling. Treated cells were rinsed and scraped in cold PBS containing 1% protease and phosphatase inhibitors (Sigma). Protein concentrations were determined by Bradford protein assay (Pierce). Samples (50 μg) were separated by SDS-PAGE in 3–8% Tris-acetate reducing gels (Novex) and transferred to nitrocellulose membranes (Bio-Rad).

[0223] Membranes were incubated for 1 hour in blocking solution containing 5% milk and 0.1% Tween-20 at pH 7.6, followed by overnight incubation at 4° C. in blocking buffer containing 5% BSA, 0.1% Tween-20, and rabbit anti-phospho-Trk (Cell Signaling, cat#9141, 1:500), rabbit anti-phospho-Akt (Cell Signaling, cat#9271, 1:1000), or rabbit anti-phospho-ERK1 / 2 antibodies (Sigma, cat#E7028, 1:5000). Labeled proteins were then visualized by incubation with horseradish peroxidase (HRP)-conjugated anti-goat, anti-mouse, or anti-rabbit IgG and development with a chemiluminescent substrate for HRP (Pierce). To determine the amount of total TrkB, MAPK, or Akt present in each lane, the antibodies were stripped from the nitrocellulose membranes for 20 min in stripping buffer (Pierce), incubated with rabbit anti-TrkB (Cell Signaling, cat#4603, 1:1000), rabbit anti-Erk1 / 2 (Cell Signaling, cat#06-182, 1:1000), or rabbit anti-Akt antibodies (Cell Signaling, cat#9272, 1:1000), and then visualized as described above. Beta-actin (Sigma, cat#A5316, 1:20000) and GAPDH (Sigma, cat#G9295) were probed as sample loading controls.

[0224] material [Table 10]

[0225] [Table 11]

[0226] [Table 12]

[0227] [Table 13]

[0228] [Table 14]

[0229] Results and Conclusions As shown in Figure 7, all TrkB agonist antibodies showed activation of MAPK / ERK and PI3K / Akt pathways after 15 min of incubation, but only BDNF and H4H9814P showed TrkB phosphorylation. After 2 h of incubation, all TrkB agonist antibodies showed activation of TrkB.

[0230] Example 5 Pharmacokinetic Evaluation of Anti-TrkB Antibodies in Humanized TrkB Mice and Wild-Type Mice Experimental procedure The pharmacokinetics of the anti-TrkB antibody H4H9816P2 (lot H4H9816P2-L7) was evaluated using humanized TrkB (chimeric mice / mice homozygous for human TrkB expression, TrkB hu / hu ) (MAID7139) and wild-type (WT) mice. Cohorts included 5 mice per mouse lineage. All mice received a single subcutaneous (SC) dose of 10 mg / kg. Blood samples were collected 6 hours and 1, 2, 3, 6, 9, 16, 21, and 30 days after dosing. Blood was processed to serum and frozen at -80°C until analysis.

[0231] Circulating antibody concentrations were determined by total human IgG4 / hIgG1 antibody analysis using GyroLab xPlore™ (Gyros, Uppsala, Sweden). Briefly, biotinylated mouse anti-human IgG4 / IgG1 specific monoclonal antibody (REGN2567, lot RSCH15088) diluted to 100 μg / mL in antibody dilution buffer (0.05% Tween-20+PBS) was captured on a Gyrolab Bioaffy 200CD containing an affinity column preloaded with streptavidin coated beads (Dynospheres™). The standard used for calibration in this assay was H4H9816P at concentrations ranging from 0.488 to 2000 ng / mL in dilution buffer (0.5% BSA+PBS) containing 0.1% normal mouse serum (NMS). Serum samples were diluted 1:100 in antibody dilution buffer. Human IgG captured on anti-REGN2567 coated affinity column on CD run at room temperature was detected by addition of 0.5 μg / mL Alexa-647 conjugated mouse anti-human kappa monoclonal antibody (REGN654, lot RSCH13067) diluted in detection buffer (Rexxip F buffer) and the resulting fluorescent signal was recorded in response units (RU) by a GyroLab xPlore instrument. Sample concentrations were determined by interpolation of a standard curve fitted using a 5-parameter logistic curve fit using Gyrolab Evaluator software. The average concentration of two replicates was used for subsequent PK analysis.

[0232] PK parameters were determined by noncompartmental analysis (NCA) and extravascular dosing model using Phoenix® WinNonlin® software version 6.3 (Certara, LP, Princeton, NJ). The respective mean concentration values ​​for each antibody were used to calculate the maximum observed serum concentration (C max ), the estimated observed half-life (t 1 / 2 ), and the area under the concentration curve versus time to the final measurable concentration (AUC lastAll PK parameters, including PK parameters (K, K, K) were determined using the linear trapezoidal rule with linear interpolation and uniform weighting.

[0233] Results and Conclusions After subcutaneous administration of 10 mg / kg of anti-TrkB antibody H4H9816P2, hu / hu Similar maximum antibody concentrations (C max ) were observed (135 and 131 μg / mL, respectively). By day 9, H4H9816P2 inhibited TrkB hu / hu TrkB mice showed more rapid drug elimination than WT mice, indicating a target-mediated effect. hu / hu Antibody concentrations in WT mice were approximately 35-fold lower on day 30. Antibody exposure to H4H9816P2 in WT mice (AUC last ) is TrkB hu / hu These results were approximately 1.7-fold higher than those seen in WT mice (1730 and 1020 d*μg / mL, respectively). hu / hu Approximately three times longer half-life (T 1 / 2 ) (8.4 and 2.9 days, respectively).

[0234] A summary of the data for total anti-TrkB antibody concentrations is summarized in Table 15, mean PK parameters are listed in Table 16, and mean total antibody concentrations versus time are shown in Figure 8. In Figure 8, mice were administered a single 10 mg / kg subcutaneous dose on day 0. Serum total H4H9816P2 concentrations were measured by Gyros immunoassay. Data points at 6 hours, 1, 2, 3, 6, 9, 16, 21, and 30 days post-dose represent mean antibody concentrations. H4H9816P2 total antibody concentrations were significantly higher than TrkB hu / hu Mice are represented by filled circles and wild-type mice by filled squares. Data are plotted as mean ± SD.

[0235] [Table 15]

[0236] [Table 16]

[0237] Example 6: Generation of rats containing a humanized TRKB locus A large targeting vector was made that contains 7 kb of the 5' homology arm of the rat TrkB locus and 47 kb of the 3' homology arm of the rat TrkB locus, and the 68.5 kb region of the rat TrkB gene that encodes the rat TRKB extracellular domain was replaced with the corresponding 74.4 kb of human TRKB sequence.The creation and use of large targeting vectors (LTVEC) derived from bacterial artificial chromosome (BAC) DNA by bacterial homologous recombination (BHR) reaction using VELOCIGENE® genetic engineering technology is described, for example, in U.S. Patent No. 6,586,251 and Valenzuela et al. (2003) Nat. Biotechnol. 21(6):652-659, each of which is incorporated herein by reference in its entirety for all purposes. The generation of LTVECs by in vitro assembly methods is described, for example, in US Patent Application Publication No. 2015 / 0376628 and WO2015 / 200334, each of which is incorporated herein by reference in its entirety for all purposes. Information regarding rat and human TRKB is provided in Table 17. A description of the generation of large targeting vectors is provided in Table 18.

[0238] [Table 17]

[0239] [Table 18]

[0240] Specifically, the region starting from exon 2 (coding exon 1, from amino acid 32, preserving the signal peptide) through exon 10, including the first 50 base pairs of intron 10 and all introns between exons 2 and 10 (i.e., between coding exon 1 and exon 10), was deleted from the rat TrkB locus (preserving the rat transmembrane domain encoded by exons 10 and 11). The region including exon 2 / coding exon 1 (from amino acid 32, starting after the signal peptide) through exon 10, including the first 66 base pairs of intron 10 and all introns between exons 2 and 10 (i.e., between coding exon 1 and exon 10), was inserted in place of the deleted rat region (preserving the rat transmembrane domain encoded by exons 10 and 11).

[0241] The sequences of the rat TRKB signal peptide, extracellular domain, transmembrane domain, and cytoplasmic domain are set forth in SEQ ID NOs: 55-58, respectively, with the corresponding coding sequences set forth in SEQ ID NOs: 67-70, respectively. The sequences of the human TRKB signal peptide, extracellular domain, transmembrane domain, and cytoplasmic domain are set forth in SEQ ID NOs: 59-62, respectively, with the corresponding coding sequences set forth in SEQ ID NOs: 71-74, respectively. The predicted encoded chimeric TRKB protein has rat TRKB transmembrane and intracellular domains, rat TRKB signal peptide, and human TRKB extracellular domain. See FIG. 4. An alignment of rat and human TRKB proteins is shown in FIG. 6. The rat and human TrkB / TRKB coding sequences are set forth in SEQ ID NOs: 10 and 11, respectively. The rat and human TRKB protein sequences are set forth in SEQ ID NOs: 2 and 3, respectively. The predicted chimeric rat / human TRKB coding sequence and the predicted chimeric rat / human TRKB protein sequences are set forth in SEQ ID NOs: 13 and 5, respectively.

[0242] To generate mutant alleles, CRISPR / Cas9 components containing four guide RNAs (guide RNA target sequences shown in SEQ ID NOs: 41-44) were introduced into rat embryonic stem cells along with a large targeting vector. 6 Rat ES cells (dark agouti strain DA2B) were electroporated with the following: 2 mg TrkB LTVEC, 5 mg Cas9 expression plasmid, and 5 mg each of gRNAs, namely gU, gU2, gD, and gD2. Electroporation conditions were 400V voltage, 100 mF capacitance, and 0 W resistance. Antibiotic selection was performed with G418 at a concentration of 75 mg / mL. Colonies were picked, grown, and screened by TAQMAN®. See FIG. 5. Allele loss assays were performed to detect loss of endogenous rat alleles, allele gain assays were performed to detect gain of humanized alleles, and CRISPR and retention assays were performed using the primers and probes shown in Table 19.

[0243] [Table 19]

[0244] Allele modification (MOA) assays, including loss of allele (LOA) and gain of allele (GOA) assays, are described, for example, in US Patent Application Publication No. 2014 / 0178879, US Patent Application Publication No. 2016 / 0145646, WO2016 / 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. Loss of allele (LOA) assays reverse the logic of traditional screening and quantify the copy number in genomic DNA samples of the native locus where the mutation is introduced. In correctly targeted heterozygous cell clones, one of the two native alleles (for genes that are not on the X or Y chromosome) is detected by LOA assays, and the other allele is interrupted by the targeted modification. The same principle can be applied in reverse as a gain of allele (GOA) assay to quantitate the copy number of a targeting vector inserted in a genomic DNA sample.

[0245] The retention assay is described in US Patent Application Publication No. 2016 / 0145646 and WO2016 / 081923, each of which is incorporated herein by reference in its entirety for all purposes. The retention assay distinguishes between correctly targeted insertion of a nucleic acid insert into a target genome locus and random transgenic insertion of a nucleic acid insert into a genomic location outside the target genome locus by evaluating the copy number of 5' and 3' target sequence DNA templates corresponding to the 5' and 3' homology arms of the targeting vector, respectively. Specifically, the retention assay determines the copy number in a genomic DNA sample of the 5' target sequence DNA template intended to be retained in the modified target genome locus and / or the 3' target sequence DNA template intended to be retained in the modified target genome locus. In diploid cells, a correctly targeted clone will retain a copy number of 2. A copy number greater than 2 generally indicates random transgenic integration of the targeting vector outside the target genome locus rather than at the target genome locus. A copy number less than 1 generally indicates a large deletion that extends beyond the region targeted for deletion.

[0246] The CRISPR assay is a TAQMAN® assay designed to encompass the region interrupted by the CRISPR gRNA. When an indel (insertion or deletion) is cut and created by the CRISPR gRNA, it cannot be amplified by the TAQMAN® assay, thus reporting CRISPR cleavage.

[0247] The positive clone CB1 was thawed, expanded, and reconfirmed by TAQMAN®. CB1 was also confirmed by successful PCR from the 5' end of the human replacement sequence, past the end of the 5' homology arm, to the adjacent rat genomic sequence. The PCR amplicon was confirmed to be correct by terminal sequencing.

[0248] F0 and F1 rats were generated using the methods described in US Patent Application Publication Nos. 2014 / 0235933, 2014 / 0310828, WO2014 / 130706, and WO2014 / 172489, each of which is incorporated by reference in its entirety for all purposes. In these methods, a confirmed targeted rat ES cell clone (e.g., a dark agouti ES cell clone) is microinjected into a blastocyst (e.g., a Sprague Dawley (SD) blastocyst), which is then transferred to a pseudopregnant recipient female (e.g., a SD recipient female) for gestation by standard techniques. Chimeras are identified (e.g., by coat color), and male F0 chimeras are mated with wild-type female rats of the same pedigree (e.g., SD females). Germline (e.g., agouti) F1 young rats are then genotyped for the presence of the targeted allele. All experiments carried out in humanized TRKB rats described below were performed in rats in which the self-deleted selection cassette was self-deleted.

[0249] Example 7 TrkB hu / hu In vivo comparison of the effects of TRKB agonist antibody H4H9816P2 and IgG4 isotype control REGN1945 on retinal ganglion cell (RGC) survival in rats Experimental procedure All procedures were performed in accordance with the ARVO Statement for Use of Animals in Ophthalmic and Vision Research and the Regeneron Pharmaceuticals Inc. IACUC. Adult female TrkB humanized rats (MAID100010) aged 8–10 weeks and weighing 200–250 g each were used. All surgical procedures on rats were performed under general anesthesia using intraperitoneal injections of ketamine (63 mg / kg) and xylazine (6.0 mg / kg). Eye ointment containing erythromycin (0.5%, Bausch & Lomb) was applied to protect the cornea.

[0250] Intraorbital optic nerve axotomy and intravitreal injection. The left optic nerve (ON) was exposed intraorbitally and its dura was opened. The ON was transected approximately 1.5 mm behind the globe. Care was taken not to damage the blood supply to the retina. Intravitreal injections were performed just behind the pars plana using a pulled glass pipette connected to a 50 μL Hamilton syringe. Care was taken not to damage the lens. Any rats with significant postoperative complications (e.g. retinal ischemia, cataract) were excluded from further analysis. Animals were assigned to different experimental groups. After ON axotomy, the control group was intravitreally injected with 3 μL of isotype control REGN1945 (46.6 μg / μL) and the other groups were injected with 3 μL of anti-human TRKB antibody H4H9816P2 (45.7 μg / μL) on days 3 and 10.

[0251] Immunohistochemical staining and counting of surviving retinal ganglion cells (RGCs). BRN3A (brain-specific homeobox / POU domain protein 3A) was used as a marker for surviving retinal ganglion cells (RGCs) because it has been shown to be an efficient and reliable method for selective labeling of surviving RGCs in retinal whole mounts after ON injury. See, for example, Nadal-Nicolas et al. (2009) Invest. Ophthalmol. Vis. Sic. 50(8):3860-3868, which is incorporated by reference in its entirety for all purposes. To immunostain for BRN3A, retinas were blocked in 10% normal donkey serum and 0.5% Triton-X100 for 1 hour, and then incubated in the same medium containing BRN3A antibody (1:400, Cat#sc-31984, Santa Cruz) for 2 hours at room temperature. After further washing, retinas were incubated with Alexa594-conjugated donkey anti-goat secondary antibody (1:400, Cat#A-11058, Invitrogen) overnight at 4°C.

[0252] Results and Conclusions To evaluate the effect of TRKB agonist antibodies on RGC survival in vivo, a complete optic nerve transection model was used. TRKB agonist antibody (H4H9816P2) or isotype control antibody was applied on days 3 and 10 postoperatively. Animals were euthanized on day 14 post-axotomy. RGC density in the uninjured contralateral eye was similar for the three TRKB genotypes (homozygous humanized, heterozygous humanized, and wild type), averaging approximately 1600 / mm3 as shown in Table 20. 2 BRN3A staining was used to assess the density of viable RGCs in retinal whole mounts. hu / hu In humanized rats, TRKB agonist antibody (H4H9816P2) was found to significantly (p<0.01, Mann-Whitney test) increase RGC survival compared to controls (685±106 vs. 255±66 RGC / mm 2 ). Heterozygous TrkB hu / + There was also a significant (p<0.05, Mann-Whitney test) survival benefit of TrkB agonist Ab in humanized rats (444±90 vs. 208±50 RGC / mm 2 In wild-type rats, there was a small and non-significant increase in the number of RGCs in rats treated with TRKB agonist antibody compared to the isotype control (Table 21). In conclusion, TRKB agonist antibody (H4H9816P2) inhibits TrkB hu / hu It significantly increased the survival of RGCs in humanized rats.

[0253] [Table 20]

[0254] [Table 21]

[0255] (Example 8 Amino acid sequences of heavy and light chain variable regions of anti-TRKB antibodies used in the examples) Several fully human anti-TRKB antibodies (i.e., antibodies with human variable domains and human constant domains) were tested in the examples, including antibodies designated as H4H9780P, H4H9814P, and H4H9816P2. Table 22 shows the amino acid sequence identifiers of the heavy and light chain variable regions and CDRs of selected anti-TRKB antibodies used in the examples. Table 23 shows the nucleic acid sequence identifiers of the heavy and light chain variable regions and CDRs of selected anti-TRKB antibodies used in the examples. These antibodies are described in more detail in U.S. Patent Application Publication No. 16 / 202,881, filed November 28, 2018, which is incorporated herein by reference in its entirety for all purposes.

[0256] [Table 22]

[0257] [Table 23]

[0258] Antibodies are typically referred to herein according to the following nomenclature: an Fc prefix (e.g., "H4H") followed by a numeric identifier (e.g., "9780", "9816", etc., as shown in Table 22), followed by a "P" or "P2" suffix. The H4H prefix in the antibody designation indicates the particular Fc region isotype of the antibody. Thus, according to this nomenclature, an antibody may be referred to herein as, for example, "H4H9780P", which indicates a human IgG4 Fc region. If indicated by an initial "H" in the antibody designation, the variable region is a fully human variable region. As will be appreciated by those skilled in the art, an antibody having a particular Fc isotype can be converted to an antibody having a different Fc isotype (e.g., an antibody having a murine IgG1 Fc can be converted to an antibody having a human IgG4 Fc, etc.), but in any case, the variable domain (including the CDRs) (which is indicated by the numeric identifiers shown in Table 22) remains the same, and the antigen binding characteristics are expected to be the same or substantially similar regardless of the nature of the Fc domain.

[0259] Example 9 Neuroprotective effect of anti-human TrkB agonist antibody in humanized TrkB rats The following experiments were performed to evaluate the neuroprotective effects of the endogenous TRKB agonist, brain-derived neurotrophic factor (BDNF), and a TRKB agonist monoclonal antibody (mAb) in wild-type (WT) mice and rats, and humanized TrkB mice and rats.

[0260] The in vitro effects of BDNF and TRKB Ab were quantified by cell survival assays using the differentiated human neuroblastoma cell line SH-SY5Y. In vitro, BDNF or TRKB Ab significantly increased cell survival of retinoic acid-differentiated SH-SY5Y cells. The effect showed a bell-shaped dose response, with optimal doses of 1 μg / mL for BDNF and 10 μg / mL for TRKB Ab. Neuroblastoma cell line SH-SY5Y was cultured for 4 days in differentiation medium containing 10 μM all-trans retinoic acid. Cultures were replaced with serum-free differentiation medium containing various doses of antibody (0.01-100 μg / mL). After 2 days, CCK8 reagent was added, plates were incubated for 3-4 hours, and OD450 was measured to determine the percentage of viable cells. Data were normalized to serum-free medium without antibody. As shown in Figure 9, TRKB mAbs (TrkB mAb1 is H4H9816P2 and TrkB mAb2 is a control TrkB agonist antibody) increased survival of SH-SY5Y cells in a dose-dependent manner. The human isotype control had no effect on survival of SH-SY5Y cells. Serum-free medium without antibody resulted in 100% survival.

[0261] The retinas of P2 C57BL / 6J mice were then excised and dissociated. Retinal ganglion cells were purified by immuno-panning and cultured in 96-well plates with or without treatment. After 24 hours, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added to each well to calculate cell survival for each group. As shown in Figure 10, BDNF had a bell-shaped response curve, with an optimal dose of 1 μg / mL. TrkB mAb2 (a control TrkB agonist antibody with affinity for human TrkB, mouse TrkB, and rat TrkB) also appears to have a bell-shaped curve at higher doses, but shows neuroprotective effects.

[0262] To test the neuroprotective effects in vivo, WT and humanized TrkB mice and rats were used. Animals were injected intravitreally (IVT) with BDNF or TRKB mAb 3 and 10 days after optic nerve transection (ONT). Retinal ganglion cell (RGC) numbers were quantified by Brn3a IHC on retinal flat mounts using HALO software (Indica Labs) at 1 week in mice and 2 weeks in rats after optic nerve transection.

[0263] One or two weeks after optic nerve transection, TrkB hu / hu The death of RGCs in TrkB mice was similar to that in WT mice. hu / hu The neuroprotective effects in mice were small or not significant. In contrast, IVT TRKB TrkB in Ab hu / hu There was significant RGC neuroprotection in rats. TrkB hu / hu Weight loss was observed in mice but not in rats following IVT TRKB Ab treatment.BDNF had no effect on body weight in either mice or rats.

[0264] Figure 11A and Figure 11B show the results of experiments evaluating neuroprotection in WT mouse and rat optic nerve transection models. In Figure 11A, 8-9 week old dark agouti rats were intravitreally administered BDNF (5 μg), TrkB mAb2 (18 μg), isotype control antibody (18 μg), or vehicle control on days 3 and 10 after transection. TrkB mAb2 is a control TrkB agonist antibody with affinity for human TrkB, mouse TrkB, and rat TrkB. Retinas were excised and stained for retinal ganglion cells 14 days after transection. BDNF and TRKB mAb showed significant neuroprotection as measured by retinal ganglion cell (RGC) density. In FIG. 11B, 8-week-old C57BL / 6J WT mice were intravitreally administered BDNF (2.5 μg), TrkB mAb2 (10 μg), isotype control antibody (10 μg), or vehicle control on days 3 and 10 after transection. TrkB mAb2 is a control TrkB agonist antibody with affinity for human TrkB, mouse TrkB, and rat TrkB. There was no significant neuroprotection. Thus, BDNF and TRKB mAb treatment led to a significant increase in RGC density in excised retinas of wild-type rats after optic nerve transection, whereas no significant effect on RGC density was observed in the same model in wild-type mice.

[0265] Figures 12A and 12B show the dose response of BDNF in WT mice and rats. In Figure 12A, the dose response of BDNF in the optic nerve crush (ONC) model of WT mice shows a small window of neuroprotection. Figure 12B shows the dose response of BDNF from 0.13 μg to 30 μg in the optic nerve transection model of WT rats. There is a bell-shaped response similar to the in vitro data, with the optimal dose being 0.8 μg. Retinas were excised and retinal ganglion cells were stained 14 days after transection. That is, BDNF treatment resulted in a more pronounced dose response curve, as measured by RGC density in excised retinas of wild-type rats after optic nerve transection, compared to a less pronounced dose response curve of BDNF, as measured by RGC density in excised retinas of wild-type mice after optic nerve crush.

[0266] Next, we tested the neuroprotective effect of TRKB Ab in humanized TrkB rats. The results in Figure 13A and Figure 13B show that intravitreal injection of TRKB mAb in humanized TrkB rats with optic nerve section shows remarkable neuroprotection of retinal ganglion cells. Human TRKB homozygous rats, human TRKB heterozygous rats, or wild-type TrkB rats were intravitreally administered TrkB mAb1 or isotype control antibody (3 μL) on days 3 and 10 after optic nerve section. Retinas were excised and RGCs were stained 14 days after section. Rats were 17-19 week old females. As shown in Figure 13A, rats treated with TrkB mAb1 (H4H9816P2) showed neuroprotection in all three genotypes compared to corresponding rats treated with isotype control antibody. Rats homozygous and heterozygous for human TRKB treated with isotype control have higher RGC density than wild-type rats treated with isotype control. Figure 13B shows that there is no difference in RGC numbers between genotypes in naive eyes, and Figure 13C shows the body weight of human TRKB homozygous mice treated with TrkB agonist antibody (H4H9816P2) or isotype control antibody (REGN1945) 14 days after amputation.

[0267] Whole-mount RGC isodensity maps of rat retinas showing Brn3a-labeled cells in intact and injured and treated eyes in the three genotypes were then generated (data not shown). Whole-mount reconstructions were prepared utilizing a movable stage on a fluorescent microscope (Nikon Eclipse Ti). RGCs were counted using image analysis software (HALO®, Indica Labs, Corrales, NM, USA). Isodensity maps were generated by Matlab. Higher RGC density was observed in humanized TrkB rats treated with TrkB mAb1 (H4H9816P2) compared to rats treated with isotype control (data not shown).

[0268] Taken together, the data shown in Figures 11A, 11B, 12A, 12B, and 13A-13C indicate that intravitreal administration of TRKB agonist mAb following optic nerve injury in humanized TrkB rats has a significant neuroprotective effect, in contrast to the small or unnoticeable neuroprotective effect observed following optic nerve injury in humanized TrkB mice.

[0269] To further evaluate the effect of TRKB agonist antibodies on RGC survival in the rat optic nerve transection (ONT) model, a dose-response study was performed. Human TRKB homozygous rats (MAID100010, 75% SD, 25% DA) aged 1-9 months were used. Six rats were used in each group. Human TRKB homozygous rats were intravitreally administered (3 μL) various doses of TrkB mAb1 or isotype control antibody (REGN1945) on days 3 and 10 after optic nerve transection. Retinas were excised and RGCs were stained 14 days after transection. As shown in Figure 14, TrkB mAb1 increased RGC survival in a dose-dependent manner in TrkB humanized rats.

[0270] We next compared the neuroprotective effects of various TrkB agonist antibodies in an optic nerve transection (ONT) model in human TRKB homozygous rats. Humanized TrkB rats (MAID100010, 75% SD, 25% DA) aged 8–10 weeks were used. Five to six rats were used in each group. Human TRKB homozygous rats were intravitreally administered (3 μL) H4H9816P2-L9 (10 μg), H4H9814P-L9 (10 μg), H4H9780P-L5 (10 μg), a combination of all three (3.3 μg each), or an isotype control antibody (REGN1945, 10 μg) on ​​days 3 and 10 after optic nerve transection. Retinas were excised 14 days after transection, and RGCs were stained. The results are shown in Figure 15A and Figure 15B. Each TrkB agonist antibody had a neuroprotective effect compared to an isotype control antibody. Body weights of each group were similar (data not shown).

[0271] In contrast, the TrkB agonist antibodies H4H9780P and H4H9814P did not have any neuroprotective effect in wild-type rats. Neuroprotective effects in wild-type rats were evaluated using the optic nerve transection (ONT) model. Female wild-type rats aged 8 to 10 weeks were used. Five to six rats were used in each group. Wild-type rats were intravitreally administered (3 μL) H4H9780P (120 μg), H4H9814P (120 μg), or an isotype control antibody (REGN1945, 120 μg) on ​​days 3 and 10 after optic nerve transection. Retinas were excised and RGCs were stained 14 days after transection. As shown in Figure 16, none of the TrkB agonist antibodies had a significant neuroprotective effect in wild-type rats.

[0272] Furthermore, TrkB agonist antibody (H4H9780P) had no neuroprotective effect in human TRKB homozygous mice. Five-month-old male human TRKB homozygous mice (MAID7139, 75% C57BL / 6, 25% 129) were used. Five to six mice were used in each group. Human TRKB homozygous mice were intravitreally administered (1 μL) H4H9780P (40 μg per eye) or an isotype control antibody (REGN1945, 40 μg per eye) on days 3 and 10 after optic nerve transection. Retinas were excised and RGCs were stained 14 days after transection. As shown in Figure 17A and Figure 17B, TrkB agonist antibody had no neuroprotective effect in human TRKB homozygous mice, in contrast to the neuroprotective effect seen in human TRKB homozygous rats. FIG. 17C shows the body weights of human TRKB homozygous mice given H4H9780P or an isotype control antibody 14 days after amputation. The present invention provides, for example, the following items. (Item 1) A non-human animal comprising a genetically modified endogenous TrkB locus encoding a TRKB protein, said TRKB protein comprising a cytoplasmic domain, a transmembrane domain and an extracellular domain, wherein all or part of said extracellular domain is encoded by a segment of said endogenous TrkB locus that has been deleted and replaced with an orthologous human TRKB sequence. (Item 2) The non-human animal described in item 1, wherein the TRKB protein comprises a human TRKB extracellular domain. (Item 3) The non-human animal of item 2, wherein the extracellular domain comprises the sequence set forth in SEQ ID NO: 60. (Item 4) 4. The non-human animal of item 2 or 3, wherein the entire extracellular domain is encoded by the segment of the endogenous TrkB locus that has been deleted and replaced with the orthologous human TRKB sequence, and optionally wherein the coding sequence for the extracellular domain comprises the sequence set forth in SEQ ID NO: 72. (Item 5) The non-human animal of any preceding item, wherein the TRKB protein comprises an endogenous signal peptide. (Item 6) 6. The non-human animal of item 5, wherein the signal peptide comprises a sequence as set forth in SEQ ID NO: 51 or 55. (Item 7) 7. The non-human animal according to item 5 or 6, wherein the entire signal peptide is encoded by an endogenous TrkB sequence, and optionally the coding sequence for the signal peptide comprises the sequence set forth in SEQ ID NO: 63 or 67. (Item 8) The non-human animal of any preceding item, wherein the TRKB protein comprises an endogenous TRKB transmembrane domain. (Item 9) The non-human animal of item 8, wherein the transmembrane domain comprises a sequence as set forth in SEQ ID NO: 53 or 57. (Item 10) 10. The non-human animal of item 8 or 9, wherein the entire transmembrane domain is encoded by an endogenous TrkB sequence, and optionally the coding sequence for the transmembrane domain comprises the sequence set forth in SEQ ID NO: 65 or 69. (Item 11) The non-human animal of any preceding item, wherein the TRKB protein comprises an endogenous TRKB cytoplasmic domain. (Item 12) 12. The non-human animal of item 11, wherein the cytoplasmic domain comprises a sequence as set forth in SEQ ID NO: 54 or 58. (Item 13) 13. The non-human animal of item 11 or 12, wherein the entire cytoplasmic domain is encoded by an endogenous TrkB sequence, and optionally the coding sequence for the cytoplasmic domain comprises the sequence set forth in SEQ ID NO: 66 or 70. (Item 14) The non-human animal of any preceding item, wherein the TRKB protein comprises an endogenous TRKB signal peptide, an endogenous TRKB transmembrane domain, and an endogenous TRKB cytoplasmic domain. (Item 15) the signal peptide comprises the sequence set forth in SEQ ID NO:51, the transmembrane domain comprises the sequence set forth in SEQ ID NO:53, and the cytoplasmic domain comprises the sequence set forth in SEQ ID NO:54; or the signal peptide comprises the sequence set forth in SEQ ID NO:55, the transmembrane domain comprises the sequence set forth in SEQ ID NO:57, and the cytoplasmic domain comprises the sequence set forth in SEQ ID NO:58. 15. The non-human animal according to item 14. (Item 16) the entire signal peptide, the entire transmembrane domain, and the entire cytoplasmic domain are encoded by an endogenous TrkB sequence; Optionally, the coding sequence for the signal peptide comprises the sequence set forth in SEQ ID NO:63, the coding sequence for the transmembrane domain comprises the sequence set forth in SEQ ID NO:65, and the coding sequence for the cytoplasmic domain comprises the sequence set forth in SEQ ID NO:66; or Optionally, the coding sequence for the signal peptide comprises the sequence set forth in SEQ ID NO:67, the coding sequence for the transmembrane domain comprises the sequence set forth in SEQ ID NO:69, and the coding sequence for the cytoplasmic domain comprises the sequence set forth in SEQ ID NO:70. 16. The non-human animal according to item 14 or 15. (Item 17) The non-human animal of any preceding item, wherein the TRKB protein is a chimeric non-human animal / human TRKB protein. (Item 18) 18. The non-human animal of item 17, wherein the extracellular domain is a human TRKB extracellular domain, the transmembrane domain is an endogenous TRKB protein transmembrane domain, and the cytoplasmic domain is an endogenous TRKB protein cytoplasmic domain. (Item 19) 19. The non-human animal of item 17 or 18, wherein the TRKB protein comprises the sequence set forth in SEQ ID NO: 4 or 5. (Item 20) 20. The non-human animal of item 19, wherein the coding sequence of the genetically modified TrkB locus encoding the TRKB protein comprises the sequence set forth in SEQ ID NO: 12 or 13. (Item 21) The non-human animal of any preceding item, which is heterozygous for said genetically modified endogenous TrkB locus. (Item 22) 21. The non-human animal of any one of items 1 to 20, which is homozygous for the genetically modified endogenous TrkB locus. (Item 23) 2. A non-human animal according to any preceding item which is a mammal. (Item 24) 24. The non-human animal according to item 23, which is a rodent. (Item 25) 25. The non-human animal according to item 24, which is a rat or a mouse. (Item 26) 26. The non-human animal according to item 25, which is a rat. (Item 27) 27. The non-human animal of item 26, wherein the TRKB protein comprises the sequence set forth in SEQ ID NO:5. (Item 28) 28. The non-human animal of item 27, wherein the coding sequence of the genetically modified TrkB locus encoding the TRKB protein comprises the sequence set forth in SEQ ID NO:13. (Item 29) 26. The non-human animal according to item 25, which is a mouse. (Item 30) 30. The non-human animal of item 29, wherein the TRKB protein comprises the sequence set forth in SEQ ID NO:4. (Item 31) 31. The non-human animal of item 30, wherein the coding sequence of the genetically modified TrkB locus encoding the TRKB protein comprises the sequence set forth in SEQ ID NO:12. (Item 32) 1. A method for assessing the activity of a human TRKB targeting reagent in vivo, comprising: (a) administering the human TRKB targeting reagent to the non-human animal according to any one of items 1 to 31; (b) evaluating the activity of said human TRKB targeting reagent in said non-human animal. (Item 33) 33. The method of claim 32, wherein step (a) comprises injecting the human TRKB targeting reagent into the non-human animal. (Item 34) 34. The method according to item 32 or 33, wherein step (b) comprises assessing changes in one or more or all of body weight, body composition, metabolism and locomotion compared to a control non-human animal. (Item 35) 35. The method of claim 34, wherein said step of assessing changes in body composition comprises assessing lean mass and / or fat mass relative to a control non-human animal. (Item 36) 36. The method according to item 34 or 35, wherein said step of assessing metabolic changes comprises assessing changes in food consumption and / or water consumption. (Item 37) 37. The method according to any one of items 32 to 36, wherein step (b) comprises assessing TRKB phosphorylation, and / or activation of the MAPK / ERK and PI3K / Akt pathways compared to a control non-human animal. (Item 38) The method according to any one of items 32 to 27, wherein step (b) comprises evaluating neuroprotective activity. (Item 39) 39. The method of claim 38, wherein step (b) comprises evaluating neuroprotective activity, and the non-human animal is a rat. (Item 40) 40. The method of any one of items 32 to 39, wherein step (b) comprises assessing retinal ganglion cell viability. (Item 41) 41. The method of claim 40, wherein retinal ganglion cell viability is assessed in a complete optic nerve transection model after optic nerve injury. (Item 42) 41. The method of claim 40, wherein retinal ganglion cell viability is assessed in an optic nerve crush model. (Item 43) 43. The method according to any one of items 32 to 42, wherein the human TRKB targeting reagent is an antigen-binding protein. (Item 44) The method of claim 43, wherein the antigen-binding protein is a human TRKB agonist antibody. (Item 45) 43. The method of any one of items 32 to 42, wherein the human TRKB targeting reagent is a small molecule. (Item 46) 46. ​​The method of claim 45, wherein the small molecule is a human TRKB agonist. (Item 47) A method for producing the non-human animal according to any one of items 1 to 31, comprising: (a) A non-human animal pluripotent cell that is not a one-cell stage embryo, (i) an exogenous repair template comprising an insert nucleic acid flanked by a 5' homology arm that hybridizes to a 5' target sequence at an endogenous TrkB locus and a 3' homology arm that hybridizes to a 3' target sequence at the endogenous TrkB locus, wherein the insert nucleic acid comprises an orthologous human TRKB sequence; and (ii) a nuclease agent that targets a target sequence within the endogenous TrkB locus. wherein the genome is modified to contain said genetically modified endogenous TrkB locus; (b) introducing the modified non-human animal pluripotent cells into a host embryo; (c) implanting the host embryo into a surrogate mother to produce a genetically modified F0 generation non-human animal comprising the genetically modified endogenous TrkB locus. A method comprising: (Item 48) 48. The method of claim 47, wherein the pluripotent cells are embryonic stem (ES) cells. (Item 49) 49. The method of claim 47 or 48, wherein the nuclease agent is a Cas9 protein and a guide RNA that targets a guide RNA target sequence within the endogenous TrkB locus. (Item 50) 50. The method of claim 49, wherein step (a) further comprises introducing into the non-human animal pluripotent cell a second guide RNA that targets a second guide RNA target sequence within the endogenous TrkB locus. (Item 51) 51. The method of any one of items 47 to 50, wherein the exogenous repair template is a large targeted vector having a length of at least 10 kb, or the exogenous repair template is a large targeted vector, the sum of the 5' homology arm and the 3' homology arm of which is at least 10 kb in length. (Item 52) A method for producing the non-human animal according to any one of items 1 to 31, comprising: (a) A non-human one-cell embryo is (i) an exogenous repair template comprising an insert nucleic acid flanked by a 5' homology arm that hybridizes to a 5' target sequence at an endogenous TrkB locus and a 3' homology arm that hybridizes to a 3' target sequence at the endogenous TrkB locus, wherein the insert nucleic acid comprises an orthologous human TRKB sequence; and (ii) a nuclease agent that targets a target sequence within the endogenous TrkB locus. wherein the genome is modified to contain said genetically modified endogenous TrkB locus; (b) implanting the modified non-human animal one-cell stage embryo into a surrogate mother to produce a genetically modified F0 generation non-human animal comprising the genetically modified endogenous TrkB locus. A method comprising: (Item 53) 53. The method of claim 52, wherein the nuclease agent is a Cas9 protein and a guide RNA that targets a guide RNA target sequence within the endogenous TrkB locus. (Item 54) 54. The method of claim 53, wherein step (a) further comprises introducing into the non-human one-cell stage embryo a second guide RNA that targets a second guide RNA target sequence within the endogenous TrkB locus. (Item 55) A non-human animal cell comprising a genetically modified endogenous TrkB locus encoding a TRKB protein, said TRKB protein comprising a cytoplasmic domain, a transmembrane domain and an extracellular domain, wherein all or part of said extracellular domain is encoded by a segment of said endogenous TrkB locus that has been deleted and replaced with an orthologous human TRKB sequence. (Item 56) A non-human animal genome comprising a genetically modified endogenous TrkB locus encoding a TRKB protein, said TRKB protein comprising a cytoplasmic domain, a transmembrane domain and an extracellular domain, wherein all or part of the extracellular domain is encoded by a segment of said endogenous TrkB locus that has been deleted and replaced with an orthologous human TRKB sequence. (Item 57) 1. A targeting vector for generating a genetically modified endogenous TrkB locus encoding a TRKB protein, comprising: the TRKB protein comprises a cytoplasmic domain, a transmembrane domain and an extracellular domain, and wherein all or part of the extracellular domain is encoded by a segment of the endogenous TrkB locus that has been deleted and replaced with an orthologous human TRKB sequence; the targeting vector comprises an insert nucleic acid comprising the orthologous human TRKB sequence flanked by a 5' homology arm that targets a 5' target sequence at the endogenous TrkB locus and a 3' homology arm that targets a 3' target sequence at the endogenous TrkB locus; Targeting vectors.

Claims

1. 1. A mouse comprising a genetically modified endogenous TrkB locus, wherein the genetically modified endogenous TrkB locus encodes a tropomyosin receptor kinase B (TRKB) protein; A mouse, wherein the TRKB protein comprises an endogenous TRKB signal peptide, an endogenous TRKB cytoplasmic domain, an endogenous TRKB transmembrane domain and a human TRKB extracellular domain, wherein the entire extracellular domain is encoded by a segment of the endogenous TrkB locus that has been deleted and replaced with an orthologous human TRKB sequence.

2. The mouse of claim 1 , wherein the human TRKB extracellular domain comprises the sequence set forth in SEQ ID NO:

60.

3. The mouse of claim 1 or 2, wherein the coding sequence for the human TRKB extracellular domain comprises the sequence set forth in SEQ ID NO:

72.

4. The mouse of any one of claims 1 to 3, wherein the signal peptide comprises the sequence set forth in SEQ ID NO:

51.

5. The mouse of any one of claims 1 to 4, wherein the entire signal peptide is encoded by an endogenous TrkB sequence.

6. The mouse of claim 5 , wherein the coding sequence for the signal peptide comprises the sequence set forth in SEQ ID NO:

63.

7. The mouse of any one of claims 1 to 6, wherein the transmembrane domain comprises the sequence set forth in SEQ ID NO:

53.

8. The mouse of any one of claims 1 to 7, wherein the entire transmembrane domain is encoded by an endogenous TrkB sequence.

9. The mouse of claim 8, wherein the coding sequence for the transmembrane domain comprises the sequence set forth in SEQ ID NO:

65.

10. The mouse of any one of claims 1 to 9, wherein the cytoplasmic domain comprises the sequence set forth in SEQ ID NO:

54.

11. The mouse of any one of claims 1 to 10, wherein the entire cytoplasmic domain is encoded by an endogenous TrkB sequence.

12. The mouse of claim 11, wherein the coding sequence for the cytoplasmic domain comprises the sequence set forth in SEQ ID NO:

66.

13. the signal peptide comprises the sequence set forth in SEQ ID NO:51, the transmembrane domain comprises the sequence set forth in SEQ ID NO:53, and the cytoplasmic domain comprises the sequence set forth in SEQ ID NO:

54. A mouse according to any one of claims 1 to 12.

14. the entire signal peptide, the entire transmembrane domain, and the entire cytoplasmic domain are encoded by an endogenous TrkB sequence; A mouse according to any one of claims 1 to 13.

15. the coding sequence for the signal peptide comprises the sequence set forth in SEQ ID NO:63, the coding sequence for the transmembrane domain comprises the sequence set forth in SEQ ID NO:65, and the coding sequence for the cytoplasmic domain comprises the sequence set forth in SEQ ID NO:

66. The mouse of claim 14.

16. The mouse of any one of claims 1 to 15, wherein the TRKB protein comprises the sequence set forth in SEQ ID NO:

4.

17. The mouse of claim 16, wherein the coding sequence of the genetically modified TrkB locus that encodes the TRKB protein comprises the sequence set forth in SEQ ID NO:

12.

18. The mouse of any one of claims 1 to 17, which is heterozygous for the genetically modified endogenous TrkB locus.

19. The mouse of any one of claims 1 to 17, which is homozygous for the genetically modified endogenous TrkB locus.

20. A method for evaluating the activity of a human TRKB targeting reagent in vivo, comprising: (a) administering the human TRKB targeting reagent to a mouse according to any one of claims 1 to 19; (b) evaluating the activity of the human TRKB targeting reagent in the mouse.

21. 21. The method of claim 20, wherein step (a) comprises injecting the mouse with the human TRKB targeting reagent.

22. 22. The method of claim 20 or 21, wherein step (b) comprises assessing changes in one or more or all of the following compared to control mice: body weight, body composition, metabolism and locomotion.

23. 23. The method of claim 22, wherein said step of assessing changes in body composition comprises assessing lean mass and / or fat mass compared to control mice.

24. 24. The method of claim 22 or 23, wherein said step of assessing metabolic changes comprises assessing changes in food consumption and / or water consumption.

25. The method of any one of claims 20 to 24, wherein step (b) comprises assessing TRKB phosphorylation, and / or activation of the MAPK / ERK and PI3K / Akt pathways compared to control mice.

26. The method of any one of claims 20 to 25, wherein step (b) comprises assessing neuroprotective activity.

27. The method of any one of claims 20 to 26, wherein step (b) comprises assessing retinal ganglion cell viability.

28. 28. The method of claim 27, wherein retinal ganglion cell viability is assessed in a complete optic nerve transection model following optic nerve injury.

29. 28. The method of claim 27, wherein retinal ganglion cell viability is assessed in an optic nerve crush model.

30. The method of any one of claims 20 to 29, wherein the human TRKB targeting reagent is an antigen binding protein.

31. 31. The method of claim 30, wherein the antigen binding protein is a human TRKB agonist antibody.

32. The method of any one of claims 20 to 31, wherein the human TRKB targeting reagent is a small molecule.

33. The method of claim 32, wherein the small molecule is a human TRKB agonist.

34. A method for producing a mouse according to any one of claims 1 to 19, comprising the steps of: (I) (a) modifying the genome of a pluripotent mouse cell to contain the genetically modified endogenous TrkB locus; (b) identifying or selecting said genetically modified pluripotent mouse cells that contain said genetically modified endogenous TrkB locus; (c) introducing the genetically modified pluripotent mouse cells into a mouse host embryo; and (d) implanting the mouse host embryo into a surrogate mouse mother and allowing it to gestate; or (II) (a) modifying the genome of a mouse one-cell stage embryo to contain the genetically modified endogenous TrkB locus; (b) selecting said genetically modified mouse one-cell stage embryos that contain said genetically modified endogenous TrkB locus; and (c) implanting the genetically modified mouse one-cell stage embryo into a surrogate mouse mother and allowing it to gestate. A method comprising:

35. A mouse cell comprising a genetically modified endogenous TrkB locus, said genetically modified endogenous TrkB locus encoding a tropomyosin receptor kinase B (TRKB) protein; A mouse cell, wherein the TRKB protein comprises an endogenous TRKB signal peptide, an endogenous TRKB cytoplasmic domain, an endogenous TRKB transmembrane domain and a human TRKB extracellular domain, the entire extracellular domain being encoded by a segment of the endogenous TrkB locus that has been deleted and replaced with an orthologous human TRKB sequence.

36. 1. A composition comprising a targeting vector for generating a genetically modified endogenous TrkB locus, said genetically modified endogenous TrkB locus comprising replacement of an endogenous mouse TrkB genomic sequence encoding a mouse tropomyosin receptor kinase B (TRKB) protein extracellular domain with a corresponding human TRKB genomic sequence encoding a human TRKB protein extracellular domain; the genetically modified endogenous TrkB locus encodes a TRKB protein, the TRKB protein comprising an endogenous TRKB signal peptide, an endogenous TRKB cytoplasmic domain, an endogenous TRKB transmembrane domain, and a human TRKB extracellular domain; the targeting vector comprises an insert nucleic acid comprising the human TRKB genomic sequence flanked by a 5' homology arm that targets a 5' target sequence in the endogenous TrkB locus and a 3' homology arm that targets a 3' target sequence in the endogenous TrkB locus; composition.

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