Transferrin receptor transgenic model

Abstract

To provide a chimeric transferrin receptor (TfR) polynucleotide, polypeptide, chimeric TfR transgenic animal model, and uses thereof.SOLUTION: Provided are: a chimeric transferrin receptor (TfR) polypeptide comprising a natural mouse transferrin binding site and a heterologous apical domain having a specific amino acid sequence; a polynucleotide encoding the same; a chimeric TfR transgenic animal model; and a method of using the animal model to identify therapeutic agents that can cross the blood-brain barrier.SELECTED DRAWING: None

Description

Background Art

[0001] Background of the Invention The blood-brain barrier (BBB) restricts the use of macromolecular therapeutics that require brain exposure by preventing the transfer of most macromolecules from the periphery to the brain. The transferrin receptor (TfR) is highly expressed at the BBB and can be used to transport therapeutics such as those described above across the BBB via receptor-mediated transcytosis. For the purpose of evaluating the ability of potential therapeutics to cross the BBB, a mouse model in which the mouse TfR has been replaced with the full-length human TfR cDNA has been previously developed. However, these transgenic mice were unhealthy and exhibited abnormally high TfR expression, low red blood cell counts, and high serum iron concentrations. Yu et al., Science Trans. Med., 6(261):261ra154(2014) (Non-Patent Document 1). As a result, these existing mouse models are not suitable for use as tools for evaluating therapeutics that can cross the BBB for the treatment of brain diseases, and models that more closely resemble the expression and phenotype of endogenous TfR are needed.

Prior Art Documents

Non-Patent Documents

[0002]

Non-Patent Document 1

Summary of the Invention

[0003] Brief Summary of the Invention In one aspect, the present disclosure provides a polynucleotide comprising a nucleic acid sequence encoding a chimeric transferrin receptor (TfR) polypeptide comprising a non-human mammalian transferrin binding site and a heterologous apical domain having an amino acid sequence that is at least 80% identical to SEQ ID NO: 1. In some embodiments, the heterologous apical domain comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the heterologous apical domain comprises the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

[0004] In some embodiments, the non-human mammalian transferrin binding site is a native (e.g., of the same species as the transmembrane and / or intracellular domain of TfR) transferrin binding site, e.g., a native mouse transferrin binding site. In some embodiments, the chimeric TfR polypeptide has at least 80% amino acid sequence identity with SEQ ID NO: 3, or at least 85%, 90%, or 95% identity. In some embodiments, the chimeric TfR polypeptide comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the region of the nucleic acid sequence encoding the heterologous apical domain of the chimeric TfR polypeptide has at least 70% nucleotide sequence identity with SEQ ID NO: 2. In some embodiments, the region of the nucleic acid sequence encoding the heterologous apical domain of the chimeric TfR polypeptide comprises the nucleotide sequence of SEQ ID NO: 2. In some embodiments, the polynucleotide encoding the chimeric TfR comprises exons and introns of the mouse transferrin receptor gene, and the nucleic acid sequence encoding the heterologous apical domain is positioned after the fourth exon of the mouse transferrin receptor gene so as to replace the apical binding domain of the mouse transferrin receptor gene.

[0005] In another aspect, provided herein is a chimeric TfR polypeptide comprising a non-human mammalian transferrin binding site and a heterologous apical domain having an amino acid sequence that is at least 80% identical to SEQ ID NO: 1. In some embodiments, the chimeric TfR polypeptide comprises a native TfR polypeptide in which only the native apical domain has been replaced by a heterologous apical domain. In some embodiments, the chimeric TfR polypeptide comprises a native TfR binding site and an apical binding domain that is heterologous to the native TfR binding site, for example, in addition to the apical domain, at least one domain or region thereof comprises a non-native amino acid sequence. In some embodiments, the heterologous apical domain comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the heterologous apical domain comprises the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. In some embodiments, the chimeric TfR has at least 80%, 90%, 95%, or 98% amino acid sequence identity with SEQ ID NO: 3. In some embodiments, the chimeric TfR polypeptide comprises the amino acid sequence of SEQ ID NO: 3.

[0006] In a further aspect, provided herein are host cells that express a chimeric transferrin receptor as described above. In some embodiments, the host cell comprises a polynucleotide encoding a chimeric transferrin receptor polypeptide. In some embodiments, the host cell is a mouse cell. In some embodiments, the chimeric TfR polypeptide expressed by the host cell comprises (a) a heterologous apical domain replacing the endogenous apical domain of the TfR polypeptide, and (b) an endogenous transferrin binding site. In some embodiments, the heterologous apical domain has an amino acid sequence that is at least 80% identical to SEQ ID NO: 1. In some embodiments, the host cell expresses a chimeric TfR in which only the apical domain of the endogenous TfR is replaced by the heterologous apical domain. In some aspects, the host cell expresses a chimeric TfR comprising an endogenous TfR binding site and a heterologous apical domain that, for example, in addition to the apical domain, comprises at least one domain or region thereof that contains a non-natural amino acid sequence. In some embodiments, the heterologous apical domain comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid sequence encoding the heterologous apical domain intracellularly comprises the nucleotide sequence of SEQ ID NO: 2. In some embodiments, the host cell is a mouse cell. In some embodiments, the nucleic acid sequence encoding the apical domain intracellularly is located after the fourth exon of the mouse transferrin receptor gene. In some embodiments, the host cell is ex vivo. In some embodiments, the host cell is an embryonic stem cell. In some embodiments, the genome of the host cell comprises a deletion of the apical domain of the native TfR.

[0007] In a further aspect, the present disclosure provides a non-human transgenic animal that expresses a chimeric TfR polypeptide, the chimeric TfR polypeptide comprising a heterologous apical domain that replaces the apical domain of a TfR polypeptide that is endogenous to the non-human transgenic animal. In some embodiments, the genome of the non-human transgenic animal comprises a transferrin receptor gene encoding a heterologous apical domain that replaces the apical domain of the endogenous TfR of the non-human transgenic animal. In some embodiments, the non-human transgenic animal expresses a chimeric TfR comprising a heterologous apical domain that replaces the native domain of the TfR of the non-human transgenic animal and a native transferrin binding site. In some embodiments, the non-human transgenic animal expresses a chimeric transferrin receptor in which only the apical domain of the endogenous transferrin receptor is replaced by a heterologous apical domain. In some embodiments, the non-human transgenic animal expresses a chimeric TfR polypeptide comprising an endogenous TfR binding site and an apical binding domain that is heterologous to the endogenous TfR binding site, wherein, for example, at least one domain or region in addition to the apical domain comprises a non-natural amino acid sequence. In some embodiments, the non-human transgenic animal comprises host cells as described above. In some embodiments, the transgenic animal is a rodent. In some embodiments, the transgenic animal is a mouse or a rat. In some embodiments, the transgenic animal is homozygous for the chimeric TfR. In some embodiments, the transgenic animal is heterozygous for the chimeric TfR.

[0008] In another aspect, provided herein is a method for screening an apical domain-binding polypeptide (ADBP) that binds to a chimeric TfR, the method comprising contacting a candidate ADBP with the chimeric TfR polypeptide described above and determining the amount of candidate ADBP that binds to the chimeric TfR polypeptide. In some embodiments, the step of contacting the candidate ADBP with the chimeric TfR polypeptide comprises contacting the ADBP with a host cell that expresses the chimeric TfR polypeptide. In some embodiments, the step of contacting the candidate ADBP with the chimeric TfR polypeptide comprises contacting the ADBP with an endothelium that expresses the chimeric TfR polypeptide. In some embodiments, the endothelium is a blood-brain barrier endothelium. In some embodiments, the amount of candidate ADBP that binds to the chimeric TfR polypeptide is determined by an immunoassay. In some embodiments, the amount of candidate ADBP that binds to the chimeric TfR polypeptide is determined by surface plasmon resonance. In some embodiments, the contacting step is performed in vivo. In some embodiments, the candidate ADBP is bound to an effector molecule. In some embodiments, the effector molecule is a small molecule, RNA, DNA, or polypeptide. In some embodiments, the effector molecule is a polypeptide. In some embodiments, the polypeptide is an antibody or an antigen-binding fragment thereof.

[0009] In yet another aspect, provided herein is a method for measuring the amount of ADBP that binds to a chimeric TfR polypeptide, the method comprising contacting the ADBP with the chimeric TfR polypeptide disclosed above and determining the amount of ADBP bound to the chimeric TfR polypeptide by immunoassay or surface plasmon resonance.

[0010] In yet another aspect, provided herein is a method of screening for an ADBP that crosses the blood-brain barrier, the method comprising: (a) administering to a non-human transgenic animal disclosed herein an ADBP that binds to an apical domain having at least 80% amino acid sequence identity with SEQ ID NO: 1; and (b) measuring the presence or activity of the ADBP in the brain of the non-human transgenic animal. In some embodiments, the ADBP is bound to an effector molecule. In some embodiments, the effector molecule is a small molecule, RNA, DNA, or polypeptide. In some embodiments, the polypeptide is an antibody or an antigen-binding fragment thereof. In some embodiments, the determining step comprises performing a quantitative immunoassay. In some embodiments, the measuring step comprises contacting the brain or brain tissue of the animal with an agent that binds to the effector molecule to determine the level of the effector molecule in the brain. In some embodiments, the measuring step comprises measuring the pharmacodynamic (PD) effect of the effector molecule. In some embodiments, the effector molecule is an anti-BACE1 antibody or an antigen-binding fragment thereof, and the measuring step comprises measuring the level of soluble ABeta40 in the brain. In some embodiments, the effector molecule is an antibody or an antigen-binding fragment thereof that binds to a target in the brain.

[0011] In another aspect, provided herein is a method of monitoring an ADBP that crosses the blood-brain barrier, the method comprising: (a) administering to a non-human transgenic animal disclosed herein an ADBP that binds to an apical domain having at least 80% amino acid sequence identity with SEQ ID NO: 1; and (b) measuring the presence or activity of the ADBP in the brain of the non-human transgenic animal. In some embodiments, the ADBP is bound to an effector molecule. In some embodiments, the effector molecule is a small molecule, RNA, DNA, or polypeptide. In some embodiments, the polypeptide is an antibody or an antigen-binding fragment thereof. In some embodiments, the determining step comprises performing a quantitative immunoassay. In some embodiments, the determining step comprises contacting the effector molecule with an agent that binds to the effector molecule and determining the level of the effector molecule present in the brain. In some embodiments, the effector molecule is an antibody or an antigen-binding fragment thereof that binds to a target in the brain. In some embodiments, the measuring step comprises measuring the PD effect of the effector molecule that binds to the target. In some embodiments, the effector molecule is an anti-BACE1 antibody or an antigen-binding fragment thereof, and the measuring step comprises measuring the level of soluble ABeta40 in the brain.

[0012] In yet another aspect, provided herein is a method of generating a transgenic non-human single-cell embryo that expresses a chimeric transferrin receptor (TfR) polypeptide, the method comprising replacing the apical domain of the endogenous TfR of the non-human single-cell embryo with a heterologous apical domain having at least 80% identity with SEQ ID NO:1. In some embodiments, replacing the apical domain is effected by homologous recombination. In some embodiments, the method comprises contacting a donor DNA comprising a Cas9 protein, at least one single guide RNA (sgRNA), and a nucleic acid sequence encoding the heterologous apical domain, wherein the heterologous apical domain coding sequence replaces the apical domain of the endogenous TfR within the genome of the non-human single-cell embryo, and the heterologous apical domain is flanked by a left homologous arm and a right homologous arm. In some embodiments, the heterologous apical domain is codon-optimized for expression in the non-human single-cell embryo. In some embodiments, the non-human single-cell embryo is a mouse embryo. In some embodiments, the donor DNA is positioned downstream of the fourth exon of the mouse transferrin receptor gene.

[0013] In yet another aspect, provided herein is a method of generating a non-human transgenic animal, the method comprising: (a) transplanting the transgenic non-human single-cell embryo disclosed above into a pseudopregnant female of the same animal species as the non-human single-cell embryo; and (b) selecting a non-human transgenic animal from the offspring born to the female, wherein the non-human transgenic animal comprises a chimeric transferrin receptor (TfR) polypeptide in which the apical domain of the endogenous TfR is replaced with a heterologous apical domain having an amino acid sequence with at least 80% identity with SEQ ID NO:1.

[0014] In yet another aspect, provided herein is a method for generating a non-human transgenic animal that expresses a chimeric transferrin receptor (TfR) polypeptide, the method comprising: (a) introducing into the germ cells of an animal a polynucleotide encoding an apical domain having at least 80% identity with SEQ ID NO: 1, wherein the polynucleotide targets a region of the endogenous TfR gene encoding the endogenous TfR apical domain and the polynucleotide encoding an apical domain having at least 80% identity with SEQ ID NO: 1 replaces the region of the endogenous TfR gene encoding the endogenous apical domain; and (b) developing the cell or its progeny into a non-human transgenic animal.

[0015] [Inventive Concept 1001] A polynucleotide comprising a nucleic acid sequence encoding a chimeric transferrin receptor (TfR) polypeptide comprising a non-human mammalian transferrin binding site and a heterologous apical domain having an amino acid sequence that is at least 80% identical to SEQ ID NO: 1. [Inventive Concept 1002] The polynucleotide of Inventive Concept 1001, wherein the heterologous apical domain comprises the amino acid sequence of SEQ ID NO: 1. [Inventive Concept 1003] The polynucleotide of Inventive Concept 1001, wherein the heterologous apical domain comprises the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. [Inventive Concept 1004] The polynucleotide of any one of Inventive Concepts 1001 to 1003, wherein the non-human mammalian transferrin binding site is a native mouse transferrin binding site. [Inventive Concept 1005] The polynucleotide of any one of Inventive Concepts 1001 to 1004, wherein the chimeric TfR polypeptide has at least 80% amino acid sequence identity with SEQ ID NO: 3. [Inventive Concept 1006] The polynucleotide of Inventive Concept 1005, wherein the chimeric TfR polypeptide comprises the amino acid sequence of SEQ ID NO: 3. [Inventive Concept 1007] The polynucleotide of the present invention 1001, wherein the region of the nucleic acid sequence encoding the heterologous apical domain of the chimeric TfR polypeptide has at least 70% nucleotide sequence identity with SEQ ID NO: 2. [The present invention 1008] The polynucleotide of the present invention 1007, wherein the region of the nucleic acid sequence encoding the heterologous apical domain of the chimeric TfR polypeptide contains the nucleotide sequence of SEQ ID NO: 2. [The present invention 1009] The polynucleotide according to any one of the present inventions 1001 to 1008, wherein the polynucleotide encoding the chimeric TfR contains exons and introns of the mouse transferrin receptor gene, and the nucleic acid sequence encoding the heterologous apical domain is arranged after the fourth exon of the mouse transferrin receptor gene so as to replace the apical binding domain of the mouse transferrin receptor gene. [The present invention 1010] A chimeric transferrin receptor (TfR) polypeptide encoded by the polynucleotide according to any one of the present inventions 1001 to 1009. [The present invention 1011] A host cell comprising the polynucleotide according to any one of the present inventions 1001 to 1009. [The present invention 1012] The host cell of the present invention 1011, which is a mouse cell. [The present invention 1013] A host cell expressing a chimeric transferrin receptor (TfR) polypeptide, wherein the chimeric TfR polypeptide comprises (a) a heterologous apical domain replacing the endogenous apical domain of the TfR polypeptide and (b) an endogenous transferrin binding site. [The present invention 1014] The host cell of the present invention 1013, wherein the heterologous apical domain has an amino acid sequence that is at least 80% identical to SEQ ID NO: 1. [The present invention 1015] The host cell of the present invention 1013, wherein the heterologous apical domain contains the amino acid sequence of SEQ ID NO: 1. [The present invention 1016] The host cell according to any one of the present inventions 1013, 1014, or 1015, wherein the nucleic acid sequence encoding the heterologous apical domain in the cell comprises the nucleotide sequence of SEQ ID NO: 2. [The present invention 1017] The host cell according to any one of the present inventions 1013 to 1016, which is a mouse cell. [The present invention 1018] The host cell according to the present invention 1017, wherein the nucleic acid sequence encoding the apical domain in the cell is located after the fourth exon of the mouse transferrin receptor gene. [The present invention 1019] The host cell according to any one of the present inventions 1011 to 1018, which is in ex vivo. [The present invention 1020] The host cell according to the present invention 1019, which is an embryonic stem cell. [The present invention 1021] The host cell according to any one of the present inventions 1011 to 1020, wherein the genome of the host cell comprises a deletion of the apical domain of endogenous TfR. [The present invention 1022] A non-human transgenic animal expressing a chimeric TfR polypeptide, wherein the chimeric TfR polypeptide comprises a heterologous apical domain replacing the apical domain of a TfR polypeptide endogenous to the non-human transgenic animal. [The present invention 1023] The non-human transgenic animal according to the present invention 1022, wherein the genome of the non-human transgenic animal comprises a heterologous apical domain replacing the apical domain of the natural TfR of the non-human transgenic animal. [The present invention 1024] A non-human transgenic animal comprising the host cell according to any one of the present inventions 1011 to 1021. [The present invention 1025] The non-human transgenic animal according to any one of the present inventions 1022, 1023, or 1024, which is a rodent. [The present invention 1026] The non-human transgenic animal according to the present invention 1025, which is a mouse. [The present invention 1027] The non-human transgenic animal of the present invention 1025, which is a rat. [The present invention 1028] The non-human transgenic animal according to any one of the present inventions 1022 to 1027, which is homozygous for the chimeric TfR. [The present invention 1029] The non-human transgenic animal according to any one of the present inventions 1022 to 1027, which is heterozygous for the chimeric TfR. [The present invention 1030] A method for screening an apical domain-binding polypeptide (ADBP) that binds to a chimeric TfR, comprising: contacting a candidate ADBP with the chimeric TfR polypeptide of the present invention 1010; and determining the amount of the candidate ADBP that binds to the chimeric TfR polypeptide. A method comprising the above steps. [The present invention 1031] The method of the present invention 1030, wherein the step of contacting the candidate ADBP with the chimeric TfR polypeptide comprises contacting the ADBP with a host cell expressing the chimeric TfR polypeptide. [The present invention 1032] The method of the present invention 1030, wherein the step of contacting the candidate ADBP with the chimeric TfR polypeptide comprises contacting the ADBP with an endothelium expressing the chimeric TfR polypeptide. [The present invention 1033] The method of the present invention 1032, wherein the endothelium is a blood-brain barrier endothelium. [The present invention 1034] The method according to any one of the present inventions 1030 to 1033, wherein the amount of the candidate ADBP that binds to the chimeric TfR polypeptide is determined by an immunoassay. [The present invention 1035] The method of the present invention 1030, wherein the amount of the candidate ADBP that binds to the chimeric TfR polypeptide is determined by surface plasmon resonance. [The present invention 1036] The method according to any one of 1030 to 1034 of the present invention, wherein the step of contacting is carried out in vivo. [The present invention 1037] The method according to any one of 1030 to 1036 of the present invention, wherein the candidate ADBP is bound to an effector molecule. [The present invention 1038] The method according to 1037 of the present invention, wherein the effector molecule is a small molecule, RNA, DNA, or polypeptide. [The present invention 1039] The method according to 1038 of the present invention, wherein the effector molecule is a polypeptide. [The present invention 1040] The method according to 1039 of the present invention, wherein the polypeptide is an antibody or an antigen-binding fragment thereof. [The present invention 1041] A method for measuring the amount of ADBP that binds to chimeric TfR, comprising: contacting ADBP with the chimeric TfR polypeptide of the present invention 1010; and determining the amount of ADBP bound to the chimeric TfR polypeptide by immunoassay or surface plasmon resonance. The method comprising the above steps. [The present invention 1042] A method for screening ADBP that passes through the blood-brain barrier, comprising: (a) administering an ADBP that binds to an apical domain having at least 80% amino acid sequence identity with SEQ ID NO: 1 to a non-human transgenic animal according to any one of 1022 to 1029 of the present invention; and (b) measuring the presence or activity of the ADBP in the brain of the non-human transgenic animal. The method comprising the above steps. [The present invention 1043] The method according to 1042 of the present invention, wherein the ADBP is bound to an effector molecule. [The present invention 1044] The method according to 1043 of the present invention, wherein the effector molecule is a small molecule, RNA, DNA, or polypeptide. [The present invention 1045] The method of the present invention 1044, wherein the polypeptide is an antibody or an antigen-binding fragment thereof. [The present invention 1046] The method according to any one of the present inventions 1042 to 1045, wherein the step of determining comprises performing a quantitative immunoassay. [The present invention 1047] The method of the present invention 1043, wherein the step of measuring comprises contacting the brain or brain tissue of the animal with an agent that binds to the effector molecule and determining the level of the effector molecule present in the brain. [The present invention 1048] The method of the present invention 1042, wherein the step of measuring comprises measuring the pharmacodynamic (PD) effect of the effector molecule. [The present invention 1049] The method of the present invention 1048, wherein the effector molecule is an anti-BACE1 antibody or an antigen-binding fragment thereof, and the step of measuring comprises measuring the level of soluble ABeta40 in the brain. [The present invention 1050] The method of the present invention 1043, wherein the effector molecule is an antibody or an antigen-binding fragment thereof that binds to a target in the brain. [The present invention 1051] A method for monitoring ADBP passing through the blood-brain barrier, comprising: (a) administering an ADBP that binds to the apical domain having at least 80% amino acid sequence identity with SEQ ID NO: 1 to any one of the non-human transgenic animals of the present inventions 1022 to 1029; (b) measuring the presence or activity of the ADBP in the brain of the non-human transgenic animal. A method comprising the above steps. [The present invention 1052] The method of the present invention 1051, wherein the ADBP is bound to an effector molecule. [The present invention 1053] The method of the present invention 1052, wherein the effector molecule is a small molecule, RNA, DNA, or polypeptide. [The present invention 1054] The method of the present invention 1053, wherein the polypeptide is an antibody or an antigen-binding fragment thereof. [The present invention 1055] The method according to any one of the present inventions 1051 to 1054, wherein the step of determining comprises performing a quantitative immunoassay. [The present invention 1056] The method of the present invention 1055, wherein the step of determining comprises contacting the effector molecule with an agent that binds to the effector molecule and determining the level of the effector molecule present in the brain. [The present invention 1057] The method of the present invention 1052, wherein the effector molecule is an antibody or an antigen-binding fragment thereof that binds to a target in the brain. [The present invention 1058] The method of the present invention 1057, wherein the step of measuring comprises measuring the PD effect of the effector molecule that binds to the target. [The present invention 1059] The method of the present invention 1057, wherein the effector molecule is an anti-BACE1 antibody or an antigen-binding fragment thereof, and the step of measuring comprises measuring the level of soluble ABeta40 in the brain. [The present invention 1060] A method for generating a transgenic non-human single-cell embryo that expresses a chimeric transferrin receptor (TfR) polypeptide, the method comprising replacing the apical domain of the endogenous TfR of the non-human single-cell embryo with a heterologous apical domain having at least 80% identity with SEQ ID NO: 1. [The present invention 1061] The method of the present invention 1060, wherein the replacement of the apical domain is carried out by homologous recombination. [The present invention 1062] The method comprises contacting a Cas9 protein, at least one sgRNA, and a donor DNA comprising a nucleic acid sequence encoding the heterologous apical domain. The heterologous apical domain coding sequence replaces the apical domain of the endogenous TfR in the genome of the non-human single-cell embryo, and the heterologous apical domain is adjacent to a left homologous arm and a right homologous arm. The method of the present invention 1060 or 1061. [The present invention 1063] The method according to any one of the present inventions 1060 to 1062, wherein the heterologous apical domain is codon-optimized for expression in the non-human single-cell embryo. [The present invention 1064] The method according to any one of the present inventions 1060 to 1063, wherein the non-human single-cell embryo is a mouse embryo. [The present invention 1065] The method of the present invention 1064, wherein the donor DNA is located after the fourth exon of the mouse transferrin receptor gene. [The present invention 1066] A method for generating a non-human transgenic animal, (a) transplanting a transgenic non-human single-cell embryo according to any one of the present inventions 1060 to 1065 into a pseudopregnant female of the same animal species as the non-human single-cell embryo; (b) selecting a non-human transgenic animal from the offspring born from the female, wherein the non-human transgenic animal comprises a chimeric transferrin receptor (TfR) polypeptide in which the apical domain of the endogenous TfR is replaced with a heterologous apical domain having an amino acid sequence with at least 80% identity to SEQ ID NO: 1; A method comprising. [The present invention 1067] A method for generating a non-human transgenic animal that expresses a chimeric transferrin receptor (TfR) polypeptide, (a) A step of introducing a polynucleotide encoding an apical domain having at least 80% identity with SEQ ID NO: 1 into embryonic cells of an animal, wherein the polynucleotide targets a region of the endogenous TfR gene encoding the endogenous TfR apical domain and the polynucleotide encoding the apical domain having at least 80% identity with SEQ ID NO: 1 replaces the region of the endogenous TfR gene encoding the endogenous apical domain. (b) A step of developing the cells or their progeny into a non-human transgenic animal A method comprising. The foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the claimed invention. Other objects, advantages, and novel features will be readily apparent to those skilled in the art from the following detailed description of the invention.

Brief Description of the Drawings

[0016]

Figure 1

Figure 2

Figure 3

Figure 4A

Figure 4B

Figure 4C

Figure 4D

Figure 5A

Figure 5B

Figure 5C

Figure 5D

[0017] **Detailed Description of the Invention** A chimeric form of the transferrin receptor was developed that includes a non-human (e.g., mouse) mammalian transferrin binding site and an apical domain that is heterologous to the domain containing the transferrin binding site. These chimeric receptors can be expressed in transgenic animals, particularly where the transferrin binding site is derived from a transgenic animal species and the apical domain is derived from a primate (e.g., human or monkey). Accordingly, the present invention provides a polynucleotide encoding a chimeric transferrin receptor that includes a non-human mammalian transferrin binding site and an apical domain having an amino acid sequence that is at least 80% identical to SEQ ID NO:1. The present invention also provides the use of a non-human transgenic animal, e.g., a non-primate transgenic animal that expresses such a chimeric TfR, for screening polypeptides that can cross the BBB by binding to the human transferrin receptor (huTfR) in vivo. In some embodiments, the non-human transgenic animal includes a native transferrin receptor (such as a mouse transferrin receptor (mTfR)), the apical domain of which is replaced with an orthologous apical domain having an amino acid sequence that is at least 80% identical to SEQ ID NO:1, thereby leaving the native transferrin binding site and a majority, e.g., at least 70% or at least 75%, of the sequence encoding the transferrin receptor intact. Accordingly, this non-human transgenic animal retains to a maximum extent the transferrin binding function of the endogenous transferrin receptor of the non-human animal, including the ability not only to bind and transport transferrin but also to maintain appropriate iron homeostasis. As a result, the transgenic animal is healthy and suitable for use in the discovery and development of therapeutic agents for treating brain diseases.

[0018] The term As used herein, unless the context clearly dictates otherwise, the singular forms "a," "an," and "the" include plural referents. Thus, for example, reference to "an antibody" includes a combination of two or more such molecules, etc.

[0019] As used herein, the terms "about" and "approximately" when used to modify a numerical value or an amount specified by a range indicate a reasonable deviation from the numerical value and values known to those skilled in the art, e.g., ±20%, ±10%, or ±5% are within the intended range of the recited value.

[0020] As used herein, "transferrin receptor" refers to transferrin receptor protein 1. The human transferrin receptor 1 polypeptide sequence is set forth in SEQ ID NO: 6. Transferrin receptor protein 1 sequences from other species are also known (e.g., chimpanzee, accession number XP_003310238.1; rhesus monkey, NP_001244232.1; dog, NP_001003111.1; cow, NP_001193506.1; mouse, NP_035768.1; rat, NP_073203.1; and chicken, NP_990587.1). The term "transferrin receptor" also encompasses allelic variants of the exemplary reference sequences, e.g., human sequences encoded by the gene at the transferrin receptor protein 1 chromosomal locus. The full-length transferrin receptor protein includes a short N-terminal intracellular region, a transmembrane region, and a large extracellular domain. The extracellular domain is characterized by three domains: a protease-like domain, a helical domain, and an apical domain.

[0021] As used herein, the term "chimeric TfR" refers to a transferrin receptor protein in which all or a sub-region of the apical domain has been replaced with the corresponding apical domain region from a heterologous transferrin receptor.

[0022] As used herein, "transferrin binding site" refers to the region within the helical and protease-like domains of the TfR protein that mediates the binding of transferrin, e.g., iron-bound transferrin, to the receptor. The transferrin binding site is distal to the apical domain.

[0023] As used herein, the term "non-human mammalian transferrin binding site" refers to a sequence from the transferrin binding site of the native transferrin receptor of a non-human mammal, or a functional derivative thereof that is capable of binding to native non-human mammalian transferrin. In some embodiments, the transferrin binding site of a non-human mammal comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to the transferrin binding site of the native transferrin receptor of the non-human mammal. Examples of non-human mammals include mice, rats, rabbits, cows, sheep, dogs, cats, horses, pigs, non-human primates, and the like.

[0024] As used herein, "huTfR 頂端+ / + mouse" refers to a transgenic mouse in which the apical domain of the mouse transferrin receptor has been replaced with the apical domain of the human transferrin receptor, and the transgenic mouse is homozygous for the transgene.

[0025] As used herein, "huTfR 頂端+ / - mouse" refers to a transgenic mouse in which the apical domain of the mouse transferrin receptor has been replaced with the apical domain of the human transferrin receptor, and the transgenic mouse is heterozygous for the transgene.

[0026] As used herein, the terms "wild-type," "native," and "naturally-occurring" with respect to a transferrin receptor or a domain thereof refer to a transferrin receptor or a domain thereof having a naturally-occurring sequence.

[0027] As used herein, an "endogenous" transferrin receptor or a domain thereof refers to a transferrin receptor that occurs naturally in a cell or non-human animal, i.e., a transferrin receptor in which there has been no genetic modification to the cell or animal.

[0028] As used herein, the term "heterologous" with respect to a domain of the transferrin receptor, such as the apical domain, means that a domain of the transferrin receptor is expressed outside of its natural context, e.g., is separated from a transferrin receptor sequence that is normally adjacent in nature or is adjacent (or contiguous) to a transferrin receptor sequence that is not normally adjacent.

[0029] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids.

[0030] Natural amino acids are those that are encoded by the genetic code and those that are modified later, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Naturally occurring α-amino acids include, but are not limited to, alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Stereoisomers of naturally occurring α-amino acids include, but are not limited to, D-alanine (D-Ala), D-cysteine (D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof.

[0031] In this specification, amino acids may be referred to by either their commonly known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Commission on Biochemical Nomenclature.

[0032] The terms "polypeptide", "peptide", and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. This term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. The amino acid polymers can include full-length L-amino acids, full-length D-amino acids, or mixtures of L and D amino acids.

[0033] A "conservatively modified variant" refers to a change that results in a substitution of an amino acid with another amino acid that can be classified as having similar characteristics. Examples of categories of conservatively defined amino acid groups are "charged / polar groups" including Glu (glutamic acid or E), Asp (aspartic acid or D), Asn (asparagine or N), Gln (glutamine or Q), Lys (lysine or K), Arg (arginine or R), and His (histidine or H), "aromatic groups" including Phe (phenylalanine or F), Tyr (tyrosine or Y), Trp (tryptophan or W), and (histidine or H), and "aliphatic groups" including Gly (glycine or G), Ala (alanine or A), Val (valine or V), Leu (leucine or L), Ile (isoleucine or I), Met (methionine or M), Ser (serine or S), Thr (threonine or T), and Cys (cysteine or C). Subgroups can also be identified within each group. For example, the group of charged or polar amino acids can be subdivided into subgroups including a "positive charge subgroup" including Lys, Arg, and His, a "negative charge subgroup" including Glu and Asp, and a "polar subgroup" including Asn and Gln. In another example, the aromatic or cyclic group can be subdivided into subgroups including a "nitrogen ring subgroup" including Pro, His, and Trp, and a "phenyl subgroup" including Phe and Tyr. Yet another further example is that the aliphatic group can be divided into subgroups such as an "aliphatic nonpolar subgroup" including Val, Leu, Gly, and Ala, and an "aliphatic slightly polar subgroup" including Met, Ser, Thr, and Cys. Examples of categories of conservative mutations are amino acid substitutions within the above subgroups, for example, but not limited to, Lys for Arg or Arg for Lys such that a positive charge can be maintained, Asp for Glu or Glu for Asp such that a negative charge can be maintained, Ser for Thr or Thr for Ser such that a free - OH can be maintained, and free - NH 2Contains Gln for Asn or Asn for Gln such that it can be maintained. In some embodiments, a hydrophobic amino acid is used, for example, in place of a naturally occurring hydrophobic amino acid at the active site, to maintain hydrophobicity.

[0034] The terms "identical" or percent "identity" in the context of two or more polypeptide sequences refers to two or more sequences or subsequences having the same or a specified percentage of amino acid residues, e.g., at least 60% identity, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or more, which when compared and aligned for maximum correspondence over a comparison window, or using a sequence comparison algorithm, or by manual alignment and visual inspection, are the same in a particular region.

[0035] In the sequence comparison of polypeptides, typically one amino acid sequence functions as a reference sequence and is compared to a candidate sequence. The alignment can be performed using various methods available to those of skill in the art, e.g., visual alignment or publicly available software that uses known algorithms to achieve maximum alignment. Such programs include the BLAST program, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.), or Megalign (DNASTAR). The parameters used for alignment to achieve maximum alignment can be determined by those of skill in the art. For the sequence comparison of polypeptide sequences for the purposes of this application, the BLASTP algorithm standard protein BLAST for aligning two protein sequences with default parameters is used.

[0036] The term "comprising" is intended to mean that the elements recited in the compositions and methods are included, but other elements are not excluded. When used to define compositions and methods, "consisting essentially of" refers to those specific materials or steps and those that do not materially affect the basic and novel characteristics (s) of the claimed invention. "Consisting of" means excluding other components in excess of trace amounts and the substantial method steps recited. Embodiments defined by each of these transitional terms are within the scope of the present invention.

[0037] The terms "polynucleotide", "nucleic acid", and "oligonucleotide" are used interchangeably and refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, ESTs or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. Polynucleotides can include modified nucleotides such as methylated nucleotides and their analogs. When present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The nucleotide sequence can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, for example, by conjugation with a labeling component. The term also refers to both double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention that is a polynucleotide includes both the double-stranded form and each of the two complementary single-stranded forms known or predicted to constitute the double-stranded form.

[0038] A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); when the polynucleotide is RNA, uracil (U) instead of thymine. Thus, the term "polynucleotide sequence" is an alphabetical representation of a polynucleotide molecule.

[0039] The term "knock-in" refers to a one-to-one substitution of DNA sequence information at a given locus, or the insertion of sequence information not found within the locus. Those skilled in the art will readily understand how to knock-in a target polynucleotide sequence at a specific locus in the genome using various genetic approaches, such as the CRISPR / Cas9 system, ZFNs, TALENs, transposon-mediated insertion.

[0040] The term "blood-brain barrier" or "BBB" refers to a highly selective semipermeable membrane barrier that separates circulating blood from the extracellular fluid of the brain in the central nervous system (CNS). The blood-brain barrier is formed by brain endothelial cells connected by tight junctions.

[0041] Transferrin receptor Transferrin receptor brings about the cellular uptake of iron through receptor-mediated endocytosis of the transferrin receptor occupied by the ligand. TfR exists in both humans and non-human species such as primates and rodents. Native human TfR (huTfR), Uniprot P02786, SEQ ID NO: 6 is a homodimeric type II transmembrane protein and has an extracellular domain including a cytoplasmic domain, a transmembrane region, and an apical domain and a transferrin-binding domain. Each monomer of huTfR has three structurally different domains, a protease-like domain close to the membrane, a helical domain responsible for all dimer contacts, and a membrane-distal apical domain (Lawrence et al., Science, 286 (1999), pp. 779-782). The HuTfR dimer has a molecular weight of approximately 190,000 daltons. The apical domain of huTfR having the sequence of SEQ ID NO: 1 (encoded by SEQ ID NO: 2) is not involved in the interaction between transferrin and TfR. This domain is presumed to provide a contact surface for other proteins to bind to TfR. The TfRs of native cynomolgus monkeys, native rhesus monkeys, and native chimpanzees are also well-known as mentioned, for example, by accession numbers XP_005545315, NP_001244232.1, and XP_003310238.1 respectively. The apical domains of the TfRs of native cynomolgus monkeys, native rhesus monkeys, and native chimpanzees share about 96%, 95%, and 98% sequence identity with the apical domain of native human TfR of SEQ ID NO: 1 respectively.

[0042] Native mouse TfR (mTfR), Uniprot Q62351, SEQ ID NO: 5 has about 77% amino acid sequence identity with huTfR. The apical domain of native mTfR is about 74% identical to the apical domain of native huTfR. mTfR contains three structurally different domains similar to its human counterpart. The complete gene sequence of mouse TfR including the annotated exons and introns can be found in the NCBI database (Gene ID: 22042). Mouse TfR is found on chromosome 16 (NCBI reference sequence NC_000082.6).

[0043] In one aspect, it includes a chimeric TfR polypeptide. In some embodiments, the chimeric TfR comprises a non-human mammalian transferrin binding site and a heterologous apical domain that shares, for example, at least 75%, at least 77%, at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity with the apical domain of huTfR, SEQ ID NO: 1. In some embodiments, the heterologous apical domain has the sequence of SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

[0044] The non-human mammalian transferrin binding site of the chimeric TfR enables specific binding of non-mammalian transferrin to the chimeric TfR. In some embodiments, the non-human mammalian transferrin binding site is a native transferrin binding site, such as the mouse transferrin receptor binding site.

[0045] In some embodiments, the chimeric TfR polypeptide comprises a native TfR polypeptide in which only the native apical domain is replaced by a heterologous apical domain. In some embodiments, the chimeric TfR polypeptide comprises a native TfR binding site and an apical binding domain that is heterologous to the native TfR binding site and that has, for example, at least one domain or region with a non-native amino acid sequence in addition to the apical domain.

[0046] In some embodiments, the chimeric TfR polypeptide has at least 80%, at least 85%, at least 88%, at least 92%, at least 95%, or at least 98% amino acid sequence identity with SEQ ID NO: 3. In one embodiment, the polynucleotide encoding the chimeric TfR polypeptide comprises exons and introns of the mouse transferrin receptor gene and a nucleic acid sequence encoding the huTfR apical domain. In one embodiment, for example, the non-human mammalian TfR apical domain is replaced with the huTfR apical domain coding sequence by replacing the corresponding exon of the non-human mammalian TfR gene with the huTfR apical domain sequence. In an exemplary embodiment, the non-human mammalian TfR gene is the mouse TfR gene. In one embodiment, the mTfR apical domain is replaced with the huTfR apical domain coding sequence, which is placed, for example, after the fourth exon of the mouse transferrin receptor gene to generate a chimeric TfR.

[0047] In some aspects, the invention provides an isolated nucleic acid comprising a nucleic acid sequence encoding any of the polypeptides comprising the chimeric TfR polypeptide described herein. In some embodiments, the region of the nucleic acid sequence encoding the heterologous apical domain of the chimeric TfR polypeptide shares at least 75%, at least 77%, at least 80%, at least 85%, at least 90%, or at least 95% nucleic acid sequence identity with the coding sequence of the apical domain of native huTfR, SEQ ID NO: 2.

[0048] In another aspect, there is provided a polynucleotide comprising a nucleotide sequence encoding the chimeric transferrin receptor described herein. The polynucleotide can be single-stranded or double-stranded. In some embodiments, the polynucleotide is DNA. In certain embodiments, the polynucleotide is cDNA. In some embodiments, the polynucleotide is RNA.

[0049] Codon optimization In some embodiments, the coding sequence of the chimeric TfR, particularly the sequence encoding the huTfR apical domain, is codon-optimized to improve the expression of the chimeric TfR in mice. Methods of codon optimization are readily available, such as the Optimizer accessible from, for example, http: / / genomes.urv.es / OPTIMIZER, and the GeneGPS® Expression Optimization Technology of DNA 2.0 (Newark, California). In a preferred embodiment, the coding sequence is codon-optimized for expression in mice using the OptimumGene™ algorithm of GenScript (Piscataway, New Jersey).

[0050] Method for replacing the apical domain of a non-human mammalian transferrin receptor with a desired apical domain Non-human transgenic animals comprising the knock-in of a heterologous apical domain disclosed herein can be generated using a variety of methods, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), transposon-mediated systems, and CRISPR / Cas9 systems. These methods typically involve administering to a cell one or more polynucleotides encoding one or more nucleases, which mediate the modification of an endogenous gene by cleaving the DNA to generate 5' and 3' cleavage ends on the DNA strands. In the presence of a donor sequence flanked by left and right homologous arms substantially homologous to the sequences extending in the 5' direction from the 5' end and the sequences extending in the 3' direction from the 3' end, the donor is incorporated into the endogenous gene targeted by the nuclease via homology-directed repair (HDR). In some embodiments, the knock-in is performed using the CRISPR / Cas9 system. For example, a nucleic acid sequence encoding a heterologous apical domain is introduced into the endogenous TfR gene to generate a chimeric TfR, resulting in the replacement of the naturally occurring sequence encoding the apical domain while maintaining the overall structure of the gene.

[0051] CRISPR In some embodiments, the knock-in of the apical domain that is at least 80% identical to SEQ ID NO: 1 is carried out using the CRIPSR / Cas9 system. The CRISPR / Cas9 system includes a Cas9 protein and at least one or two ribonucleic acids that direct the Cas9 protein to a target motif within the apical domain of the transferrin receptor to be replaced and that can hybridize to this target motif. These ribonucleic acids are generally referred to as "single guide RNA" or "sgRNA". Next, the Cas9 protein cleaves the target motif, causing a double-strand break or a single-strand break. In the presence of donor DNA containing the huTfR apical domain coding sequence flanked by two homologous arms, the donor DNA is inserted into the target transferrin receptor DNA and the apical domain is replaced.

[0052] The Cas9 protein used in the present invention can be a naturally occurring Cas9 protein or a functional derivative thereof. A "functional derivative" of a native sequence polypeptide is a compound that has qualitative biological properties common to the native sequence polypeptide. "Functional derivatives" include, but are not limited to, fragments of the native sequence, as well as derivatives of the native sequence polypeptide and its fragments, provided that they have a biological activity common to the corresponding native sequence polypeptide. The biological activity contemplated herein is the ability of a functional derivative of Cas9 to hydrolyze a DNA substrate into fragments. Suitable functional derivatives of the Cas9 polypeptide or its fragments include, but are not limited to, variants, fusions, and covalent modifications of the Cas9 protein or its fragments.

[0053] In some embodiments, the Cas9 protein is from Streptococcus pyogenes. Cas9 contains two endonuclease domains, a RuvC-like domain that cleaves target DNA non-complementary to the sgRNA and an HNH nuclease domain that cleaves target DNA complementary to the sgRNA. The double-stranded endonuclease activity of Cas9 also requires that a short conserved sequence (2-5 nucleotides), known as the protospacer adjacent motif (PAM), follow immediately 3' of the target motif within the target sequence. In some embodiments, the PAM motif is the NGG motif. In an exemplary embodiment, the apical domain of a mouse is replaced by using a Cas9 protein directed by an sgRNA against the region between exons 4 and 9 of a mouse gene. Donor DNA is introduced into the reaction. The donor DNA contains a human apical domain coding sequence between a left homology arm homologous to a mouse TfR sequence starting upstream of exon 4 and a right homology arm homologous to a mouse TfR sequence starting within exon 9. In certain embodiments, 817 nucleotides starting upstream of exon 4 overlap with the mouse TfR sequence for the left homology arm, and 807 nucleotides starting within exon 9 overlap with the mouse TfR sequence for the right homology arm. As a result, a nucleotide sequence encoding a desired apical domain having an amino acid sequence at least 80% identical to SEQ ID NO: 1 can be inserted after the fourth mouse exon, and the inserted nucleotide sequence is adjacent at its 3' end to a properly following mouse exon. In some embodiments, the human apical domain coding sequence inserted into the mouse TfR gene is codon-optimized for mouse expression.

[0054] The sgRNA can be selected according to the specific CRISPR / Cas9 system used and the sequence of the target polynucleotide. In some embodiments, one or two ribonucleic acids are designed to hybridize to a target motif that is directly adjacent to a deoxyribonucleic acid motif recognized by the Cas9 protein. In some embodiments, each of the one or two ribonucleic acids is designed to hybridize to a target motif that is directly adjacent to a deoxyribonucleic acid motif recognized by the Cas9 protein, and the target motif is adjacent to the genomic sequence to be substituted. The guide RNA can be designed using readily available software such as, for example, http: / / crispr.mit.edu. Exemplary sgRNAs that can be used to generate chimeric TfR transgenic mice include SEQ ID NOs: 10-11.

[0055] The donor DNA disclosed herein comprises a nucleotide sequence encoding an amino acid sequence that is at least 75% identical to SEQ ID NO: 1. In some embodiments, the donor DNA comprises the sequence encoding SEQ ID NO: 1, or a sequence encoding an amino acid sequence that shares at least 75%, at least 77%, at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity with SEQ ID NO: 1. In some embodiments, the donor DNA comprises the nucleotide sequence of SEQ ID NO: 2, or a sequence that shares at least 60%, at least 70%, at least 77%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 2. The donor DNA disclosed herein further comprises a left homologous arm and a right homologous arm that are adjacent to the top domain coding sequence and are designed to overlap with the 5' and 3' exon sequences relative to the cleavage site by the Cas9 protein. The homologous arms can extend beyond the 5' and 3' exon sequences, and each homologous arm can be at least 20, 30, 40, 50, 100, or 150 nucleotides in length. Those skilled in the art can readily determine the optimal length of the homologous arms required for the experiment. In an exemplary embodiment, the left homologous arm of the donor DNA extends to nucleotides 1-817 of SEQ ID NO: 4, and the right homologous arm extends to nucleotides 1523-2329 of SEQ ID NO: 4. In some embodiments, the left homologous arm shares at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with nucleotides 1-817 of SEQ ID NO: 4. In some embodiments, the right homologous arm shares at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with nucleotides 1523-2329 of SEQ ID NO: 4.

[0056] In some embodiments, the sgRNA can also be selected to minimize hybridization with nucleic acid sequences other than the target polynucleotide sequence. In some embodiments, to minimize off-target effects of the CRISPR / Cas9 system, one or two ribonucleic acids are designed to hybridize to a target motif that contains at least two mismatches when compared to all other genomic nucleotide sequences in the cell. Those skilled in the art will understand that various techniques (e.g., bioinformatics analysis) can be used to select a suitable target motif for minimizing off-target effects. Methods of reducing gene expression using the CRISPR / Cas9 system are described in various publications, e.g., U.S. Patent Publication Nos. 2014 / 0170753 and 2016 / 0257974, the disclosures of which are incorporated herein by reference in their entirety.

[0057] Zinc finger nuclease (ZFN) In some embodiments, the chimeric TfR is generated by using ZFNs to knock-in the huTfR apical domain. ZFNs are fusion proteins that contain the non-specific cleavage domain (N) of the FokI endonuclease and a zinc finger protein (ZFP). Pairs of ZNFs are involved in recognizing specific loci of the target gene, with one recognizing the sequence upstream of the site to be modified and the other recognizing the sequence downstream of the site to be modified. The nuclease portion of the ZFN cleaves at a specific locus. Next, donor DNA as described above can be inserted at the specific locus. Methods of reducing gene expression using ZFNs are well known, e.g., as disclosed in U.S. Patent No. 9,045,763 and further in Durai et al., “Zinc Finger Nucleases: Custom-Designed Molecular Scissors for Genome Engineering of Plant and Mammalian cells,” Nucleic Acid Research, 33(18):5978-5990 (2005), the disclosures of which are incorporated herein by reference in their entirety.

[0058] Transcription activator-like effector nuclease (TALEN) In some embodiments, the chimeric TfR is generated by knocking in the huTfR apical domain together with TALEN. TALENs are similar to ZFNs in that they bind as a pair around a genomic site and direct the same non-specific nuclease, FokI, to cleave the genome at a specific site, but instead of recognizing three bases of DNA, each domain recognizes a single nucleotide. Methods of using ZFNs to reduce gene expression are well known, as disclosed, for example, in U.S. Patent No. 9,005,973, and further in Christian et al., “Targeting DNA Double-Strand Breaks with TAL Effector Nucleases,” Genetics, 186(2):757-761 (2010), the disclosure of which is hereby incorporated by reference in its entirety.

[0059] Host cells / transgenic animals expressing chimeric TFR In some embodiments, the present invention provides a host cell that expresses a chimeric TfR comprising, for example, a nucleic acid sequence encoding the above-described chimeric transferrin receptor. In some embodiments, the host cell is a non-human mammalian cell. Any of the above-described knock-in methods, i.e., using CRISPR, TALEN, zinc finger nuclease, can be used to replace the apical domain of the native transferrin receptor in the host cell with a heterologous apical domain having an amino acid sequence that is at least 80% identical to SEQ ID NO: 1. In some embodiments, the host cell is a eukaryote, for example, a mouse cell that is at least 80% identical to SEQ ID NO: 1. In some cases, the host cell is contacted with a donor DNA comprising an sgRNA and Cas9, and a nucleic acid sequence encoding a heterologous apical domain, the nucleic acid sequence being flanked by left and right homologous arms. The sgRNA and the homologous arms have sequences such that the heterologous apical domain coding sequence is inserted at a position within the genome and replaces the coding sequence of the apical domain of the host cell's native transferrin receptor. In some embodiments, the host cell is a cell derived from a non-primate mammal such as a mouse, rat, rabbit, cow, sheep, dog, cat, horse, pig, etc.

[0060] In some embodiments, a method of knocking in to embryonic stem (ES) cells is performed to produce ES cells that express a chimeric transferrin receptor polypeptide. The embryonic stem cells can then develop into progeny cells or non-human transgenic animals whose genome contains a nucleic acid encoding the chimeric transferrin receptor polypeptide. In some embodiments, the ES cells are introduced into blastocysts and transplanted into pseudopregnant females. In some cases, a founder male carrying the transgene can be selected and mated with a wild-type female to generate F1 heterozygous mice. Homozygous non-human animals can be subsequently generated from the breeding of the F1 generation heterozygous non-human animals. Methods of culturing ES cells and introducing a nucleotide sequence to target the genome of the ES cells to generate transgenic animals are well known, for example, as disclosed in Ramirez-Solis et al., “Gene targeting in mouse embryonic stem cells,” Methods Enzymol., 225:855-878 (1993), and U.S. Patent Publication No. 2013 / 0318643, the disclosures of which are incorporated herein by reference in their entirety. In some embodiments, embryonic stem cells from transgenic animals having the chimeric TfR of the present invention can be used as a source for providing progeny of the transgenic animals.

[0061] In some embodiments, the knock-in method is performed in single-cell non-human animals. In an exemplary embodiment, the sgRNA, Cas9, and donor polynucleotide comprise a top domain coding sequence that is at least 80% identical to SEQ ID NO: 1, the coding sequence is flanked by a left homologous arm and a right homologous arm, and is introduced into a single-cell embryo via pronuclear microinjection. The recipient embryo is then transferred to a pseudopregnant female. The sgRNA forms a complex with the Cas9 protein and targets the coding sequence of the top domain of the transferrin receptor of the non-human animal embryo. As a result, the top domain of the transferrin receptor of the non-human animal is cleaved and replaced with the transferrin receptor top domain coding sequence from the donor polynucleotide. In some cases, a founder male carrying the transgene can be selected and mated with a wild-type female to generate F1 heterozygous mice. Homozygous non-human animals can be subsequently generated from the breeding of the F1 generation heterozygous non-human animals. The transgenic animals disclosed herein can be rodents, such as mice or rats.

[0062] In an exemplary embodiment, transgenic animals, such as non-human transgenic animals, for example non-primate mammals, are generated by knocking in an apical domain having an amino acid sequence that is at least 80% identical to SEQ ID NO:1, which is partly due to the fact that they retain introns and the transferrin-binding domain of native TfR. These transgenic animals are generally healthy and exhibit a physiological state similar to that of wild-type mice of the same species. In one embodiment, all introns outside the apical domain of TfR are retained. For example, the expression level of TfR is similar to that of wild-type animals of the same species, and the expression level of transgenic mice is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% lower than, or 10%, 20%, 30%, 40%, 50%, 75%, 100%, 150%, 200%, 300%, or 500% higher than, that of wild-type mice. The number of red blood cells, hemoglobin level, and / or hematocrit level are also similar to those of wild-type animals of the same species, with a difference of less than 50%, for example less than 40%, less than 30%, less than 20%, or less than 10%. In a typical embodiment, the transgenic animals according to the invention retain selective BBB transport that allows uptake of nutrients and proteins, retain the ability to protect the CNS from toxins, and the presence of the transgene does not inhibit transferrin binding or FcRn binding to antibodies that bind to the following apical domain. Usually, cell transport via TfR in transgenic animals is also similar to that in these wild-type animals. The transgenic animals according to the invention are more suitable as a model for pharmacokinetic or pharmacodynamic studies of human BBB-permeable drugs than wild-type mice that completely lack human TfR or transgenic animal models that express the entire extracellular domain of huTfR (e.g., express the entire huTfR protein).

[0063] Although the present invention has been described with reference to mice as shown in the examples, it will be understood by those skilled in the art that other non-human mammals, such as rodents, rabbits, cows, sheep, dogs, cats, horses, pigs, camels, non-human primates, and other mammals can be similarly manipulated to express chimeric TfR, and these transgenic animals can also be used for the uses disclosed herein.

[0064] Apical domain-binding polypeptide As used herein, "apical domain-binding polypeptide" or "ADBP" refers to a polypeptide that binds to an apical domain having an amino acid sequence that is at least 80% identical to SEQ ID NO: 1. The ADBP can be an antibody or any polypeptide that can bind to the apical domain of huTfR of chimeric TfR. In some embodiments, the ADBP is an agent that is delivered through the blood-brain barrier. In some embodiments, the ADBP further comprises an effector molecule bound thereto, for example, by a covalent bond. The effector molecule can be a therapeutic agent, a labeling agent, or a diagnostic agent. In certain embodiments, the effector molecule is a polypeptide such as a therapeutic or diagnostic antibody, or a polypeptide having enzymatic or inhibitory activity against an enzyme or a signaling molecule. In certain embodiments, the effector molecule comprises a small molecule, RNA, DNA, or protein.

[0065] In some embodiments, the ADBP is a bispecific antibody, the apical domain-binding region is an antibody that recognizes the apical domain, the effector molecule is an antibody that recognizes a different antigen, such as an enzyme or a signaling molecule, and the binding of the effector moiety activates or inhibits the enzyme or the signaling molecule.

[0066] Screening for ADBP that binds to chimeric TfR Using the chimeric TfR disclosed herein, ADBPs that can bind to TfR can be screened. The screening method includes the steps of contacting a candidate ADBP with the chimeric TfR disclosed above and determining the amount of candidate ADBP that binds to the chimeric TfR. In some embodiments, the step of contacting the candidate ADBP with the chimeric TfR includes contacting the ADBP with a host cell expressing the chimeric TfR. In some cases, the step of contacting the candidate ADBP with the chimeric TfR includes contacting the ADBP with an endothelium expressing the chimeric TfR. In some embodiments, the endothelium is a BBB endothelium.

[0067] The interaction between the candidate ADBP and TfR can be measured using methods well known in the art, such as immunoassays or SPR. In some embodiments, the binding of the candidate ADBP to the chimeric TfR is measured by ELISA, the Biacore™ system, or co-immunoprecipitation.

[0068] Screening of ADBPs that can cross the BBB Non-human transgenic animals expressing the chimeric TfR described above can be used to characterize the ability of ADBPs to bind to the apical domain of the chimeric TfR and ultimately to cross the BBB.

[0069] Typically, to evaluate the ability of an ADBP to cross the BBB, preferably by intravenous injection, the ADBP is administered to a transgenic animal carrying the chimeric TfR disclosed herein. After a certain period of time, for example, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 60 minutes, at least 90 minutes, at least 120 minutes, at least 180 minutes, or at least 240 minutes, the transgenic animal is sacrificed and the brain tissue is analyzed to determine the presence of the ADBP. The presence of the ADBP can be determined by assaying for the presence of the ADBP and / or an effector molecule bound thereto. In some embodiments, the brain tissue is perfused with saline, such as PBS, and fixed prior to detection. The presence of the effector molecule within the sections can be detected using standard imaging methods, such as immunohistochemistry or immunofluorescence. A positive detection of the effector molecule within the brain tissue indicates that the effector molecule can cross the BBB. In some cases, determination of the presence of the ADBP in the brain includes performing a quantitative immunoassay. Assays using chimeric TfR transgenic mice to measure transport across the BBB are robust and can measure improvements greater than 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold in the uptake of the ADBP.

[0070] In some embodiments, in addition to using an imaging method or immunoassay to detect the presence of the ADBP in the brain, a method of detecting a change in the substrate of the effector molecule can also be used to evaluate the uptake of the effector molecule into the brain. In an exemplary embodiment, the ADBP comprises an effector molecule capable of inhibiting the enzymatic activity of an enzyme within the brain. In some embodiments, the uptake of the ADBP into the brain, i.e., the ability of the BBB to transport, can be measured by evaluating the enzymatic activity of an enzyme regulated by either the ADBP or an effector molecule bound thereto.

[0071] In some embodiments, the uptake of candidate ADBPs into the brain is measured within the brain. Plasma can also be monitored to evaluate the pharmacokinetic profile. After administration of the candidate effector molecule, an increase in the brain-to-plasma ratio compared to non-BBB permeable molecules indicates that the candidate ADBP can cross the BBB.

[0072] In some cases, a non-human transgenic animal comprising a polynucleotide encoding a chimeric TfR can be mated with a non-human transgenic animal engineered to exhibit a particular disease phenotype. In some cases, the non-human transgenic animal is a transgenic mouse that can be mated with various mouse models, such as the ALS mouse model as described in U.S. Patent No. 8,476,485, the AD mouse models as described in U.S. Patent Nos. 5,898,094 and 6,175,057, the TSPO mouse model as described in U.S. Patent Publication No. 2016 / 0050895, and the autism spectrum disorder (ASD) mouse model as described in U.S. Patent Publication No. 2014 / 0041062. The entire contents of these aforementioned patents and patent applications are incorporated herein by reference. In some cases, the hybrid mice generated by such mating can be used to evaluate both the distribution of ADBPs containing effector molecules in the brain and the effectiveness of ADBPs or effector molecules in the treatment of brain diseases.

[0073] Kit In some embodiments, a kit is provided that includes the chimeric transferrin receptor polynucleotide or polypeptide described herein, or cells that express such a polypeptide. In some embodiments, the kit is for use in screening for the above-described ADBPs.

[0074] In some embodiments, the kit further includes a buffer and a container that can be used in an assay to detect binding between the chimeric TfR polypeptide and the candidate ADBP. In some embodiments, the kit further includes instructional materials (e.g., instructions for using the kit to administer a composition across the blood-brain barrier) that include instructions (i.e., protocols) for performing the methods described herein. Typically, the instructions include, but are not limited to, a document or a printed matter. Any medium that can store such instructions and communicate them to an end user is contemplated by the present invention. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD-ROMs), etc. Such media may include the address of an Internet site that provides such instructions.

Examples

[0075] The following examples are for illustrative purposes only and should not be construed as limiting the claimed invention. Similarly, there are various alternative techniques and procedures available to those of ordinary skill in the art that can enable the intended invention to be carried out successfully.

[0076] Example 1: Generation and Characterization of huTfR Mice Methods for generating knock-in / knock-out mice have been published in the literature and are well known to those skilled in the art. Briefly, C57Bl6 mice were used to generate knock-ins of the human apical TfR mouse line via pronuclear microinjection into single-cell embryos, followed by embryo transfer into pseudopregnant females. Specifically, Cas9, sgRNA, SEQ ID NOs: 10-11, and donor DNA, SEQ ID NO: 4 were introduced into the embryos. The donor DNA contained a human apical domain coding sequence, SEQ ID NO: 2, codon-optimized for expression in mice. The apical domain coding sequence was flanked by a left homologous arm (nucleotides 1-817 of SEQ ID NO: 4) and a right homologous arm (nucleotides 1523-2329 of SEQ ID NO: 4). The donor sequence was designed in this way such that the apical domain was inserted after the 4th mouse exon and the 9th mouse exon was directly adjacent to its 3' end. Founder males from the progeny of the embryo-transferred females were mated with wild-type females to generate F1 heterozygous mice. Subsequently, homozygous mice were generated from the breeding of F1 generation heterozygous mice.

[0077] Example 2: Generation of Tools for Monitoring Antibody Uptake into the Brain Tool antibodies targeting human TfR or human / mouse BACE1 were generated by transforming Expi293 or ExpiCHO cells with expression plasmids containing DNA encoding the heavy and light chains and using protocols well known to those skilled in the art. Bispecific antibodies were generated using the "knobs-into-holes" technology, where the knob and hole half-antibodies were expressed separately and conjugated using published methods. Antibodies were first purified by protein A and then by size exclusion chromatography. The antibodies generated for these studies are as follows: Anti-TfR: A human IgG1 antibody that binds to the human TfR apical domain. Anti-BACE1: A human IgG1 antibody that binds to human BACE1 and cross-reacts with mouse BACE1. This antibody inhibits the enzymatic activity of BACE1. Anti-TfR / BACE1: A human IgG1 knobs-into-holes bispecific antibody that binds to the human TfR apical domain and human and mouse BACE1. The knob half-antibody has the variable domain from an anti-BACE1 antibody, and the hole half-antibody has the variable domain from an anti-TfR antibody.

[0078] Example 3: huTfR 頂端+ / - and huTfR 頂端+ / + Blood analysis of mice Blood was collected from wild-type C57Bl6, huTfR 頂端+ / - , huTfR 頂端+ / + mice (n = 3 / group), and standard complete blood count (CBC) analysis was performed. No genotype-specific differences were observed in all erythrocyte parameters, including total erythrocytes, hemoglobin, and hematocrit levels (Figure 1).

[0079] Example 4: huTfR 頂端+ / - and huTfR 頂端+ / + Brain localization of TFR-targeting antibodies in mice In this example, anti-TfR antibodies were generated to evaluate the uptake of TfR-targeting therapeutics into the brain in huTfR 頂端+ / - mice. huTfR 頂端+ / - mice or wild-type C57Bl6 were injected intravenously with 5 mg / kg of anti-TfR antibody. One hour later, the mice were sacrificed and perfused with PBS. The half brains were fixed in 4% PFA overnight and then stored in 30% sucrose. Sagittal brain sections (35 μm) were cut using a microtome, blocked with 5% BSA + 0.3% Triton X-100, and then fluorescently secondary stained with Alexa488 anti-huIgG1 (1:500). Brain images were taken using a Zeiss wide-field microscope with a 20x objective lens. Significant vascular staining was observed in huTfR 頂端+ / - mice, indicating strong binding of the human apical-specific anti-TfR on the brain endothelial cells of the BBB where TfR is highly expressed (Figure 2). In contrast, very little staining was observed in wild-type mice.

[0080] To confirm TfR-specific BBB transport, anti-BACE1 antibody and anti-TfR / BACE1 bispecific antibody were tested using a similar approach as above. huTfR 頂端+ / + Mice were injected intravenously with either 50 mg / kg of one of the antibodies. After 24 hours, the mice were perfused with PBS, huTfR 頂端+ / - As described above for mice, the hemibrains were processed and stained. Extensive brain parenchymal staining was observed with anti-TfR / BACE1, but no staining was observed with anti-BACE1, indicating that the TfR apical domain-binding polypeptide is required for transcytosis of the BBB in these mice (Figure 3).

[0081] Example 5: huTfR 頂端+ / + PK / PD of antibodies in mouse brain and plasma In this example, huTfR 頂端+ / + Mice were injected intravenously with either 50 mg / kg of anti-BACE1 antibody or anti-TfR / BACE1 bispecific antibody. After 24 hours, blood was collected via cardiac puncture and the mice were perfused with PBS. Brain tissues were homogenized in lysis buffer containing 1% NP-40 in 10-fold tissue weight of PBS. Blood was collected into EDTA tubes to prevent clotting and centrifuged at 14,000 rpm for 7 minutes to separate plasma. Antibody concentrations in mouse plasma and brain lysates were quantified using a common human IgG assay (MSD human IgG kit #K150JLD) according to the manufacturer's instructions. Briefly, pre-coated plates were blocked with MSD Blocker A for 30 minutes. Plasma samples were diluted 1:10,000 using a Hamilton Nimbus liquid handler and added in duplicate to the blocked plates. Brain samples were homogenized in 1% NP40 lysis buffer and the lysates were diluted 1:10 for PK analysis. The dosing solution was also analyzed on the same plate to confirm the exact dose administered. A standard curve of IgG from 0.78 to 200 ng / mL was fitted using 4-parameter logistic regression.

[0082] After 24 hours, the plasma levels of anti-TfR / BACE1 were lower than those of anti-BACE1, presumably due to the clearance of this antibody via binding to huTfR expressed peripherally (Figure 4A). In the brain, an approximately 28-fold increase in the concentration of anti-TfR / BACE1 compared to anti-BACE1 was observed (Figure 4B). The significant accumulation of anti-TfR / BACE1 is due to transcytosis via TfR at the BBB, and this result demonstrates that huTfR 頂端 mice are a tool for measuring the BBB uptake of the human TfR apical domain-binding polypeptide. 頂端+ / + Inhibition of BACE1 cleavage of amyloid precursor protein (APP) was used as a pharmacodynamic readout of antibody activity in plasma and brain. Brain tissue was homogenized in 5 M guanidine-HCl at 10 times the tissue weight and then diluted 1:10 in 0.25% casein buffer in PBS. Mouse Aβ40 levels in plasma and brain lysates were measured using sandwich ELISA. 384-well MaxiSorp plates were coated overnight with a polyclonal capture antibody (Millipore #ABN240) specific for the C-terminus of the Aβ40 peptide. Casein-diluted guanidine brain lysates were further diluted 1:2 in the ELISA plate and added simultaneously with the detection antibody, biotinylated M3.2. Plasma was analyzed at a 1:5 dilution. Samples were incubated overnight at 4 °C before adding streptavidin-HRP, followed by the TMB substrate. Standard curves, 0.78 - 50 pg / mL of msAβ40, were fitted using four-parameter logistic regression.

[0083]

[0084] Compared to anti-BACE1, anti-TfR / BACE1 treatment was huTfR 頂端+ / + ​Increasing the reduction of mouse A beta, indicating that the involvement of the BACE1 target in the brain is achieved using anti-TfR / BACE1 (Figure 4C). Plasma A beta was reduced to the same extent with both anti-TfR / BACE1 and anti-BACE1 compared to untreated wild-type mice (Figure 4D). These data support the use of huTfR in studies involving targets that require uptake into the brain via human TfR, particularly for the evaluation of human TfR apical domain-binding polypeptides. 頂端+ / + Support the use of mice.

[0085] Example 6: huTfR 頂端+ / + Expression of TfR in mice To determine whether TfR expression levels vary in huTfR 頂端+ / + mice, brains and various peripheral tissues were isolated from wild-type and huTfR 頂端+ / + mice. Brains, livers, lungs, and kidneys were harvested from mice after perfusion with PBS. Tissues were homogenized in lysis buffer containing 1% NP-40 in 10 volumes of tissue weight PBS. Samples were run on Western blots, and TfR expression levels were determined using a TfR antibody (1:2000; Thermofisher #13-6800) that recognizes the intracellular portion of TfR and cross-reacts with both wild-type and huTfR 頂端+ / + . Quantification of TfR expression was expressed as the ratio to actin (1:5000; Abcam 8227). Figures 5A - 5D show that TfR expression in huTfR 頂端+ / + mice is very similar to that in the brains (Figure 5A), livers (Figure 5B), kidneys (Figure 5C), and lungs (Figure 5D) of wild-type mice.

[0086] It is understood that the examples and embodiments described herein are for illustrative purposes only, and various modifications or changes contemplated thereby are suggested to those skilled in the art and should be included within the spirit and scope of this application and the appended claims. All publications, sequence accession numbers, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0087] Table of exemplary arrays Accession number 1: Protein sequence of human apical domain insert TIFF2025087857000001.tif11136 Accession number 2: DNA sequence of human apical domain insert TIFF2025087857000002.tif36135 Accession number 3: Chimeric TfR sequence expressed in transgenic mice (italicized part represents the cytoplasmic domain, bold part represents the transmembrane domain, gray part represents the extracellular domain, and bold and underlined part represents the apical domain) TIFF2025087857000003.tif53136 Accession number 4: Sequence of the complete donor DNA (left homologous arm: 1 - 817, right homologous arm: 1523 - 2329, human apical domain: 941 - 1492, codon-optimized sequence: 821 - 1522) TIFF2025087857000004.tif52136TIFF2025087857000005.tif102136 Accession number 5: Mouse TfR protein sequence (Uniprot Q62351) (italicized part represents the cytoplasmic domain, bold part represents the transmembrane domain, gray part represents the extracellular domain, and bold and underlined part represents the apical domain) TIFF2025087857000006.tif53136 Accession number 6: Human TfR protein sequence (Uniprot P02786) (italicized part represents the cytoplasmic domain, bold part represents the transmembrane domain, gray part represents the extracellular domain, and bold and underlined part represents the apical domain) TIFF2025087857000007.tif53136 Accession number 7: Apical domain of Macaca mulatta (rhesus monkey) TfR (NCBI reference sequence NP_001244232.1), which has 95% identity with the apical domain of native human TfR. TIFF2025087857000008.tif11136 Accession number 8: the apical domain of chimpanzee TfR (NCBI Reference Sequence XP_003310238.1), which is 98% identical to the apical domain of native human TfR. TIFF2025087857000009.tif11136 Accession number 9: the apical domain of cynomolgous monkey (macaca fascicularis) TfR (NCBI Reference Sequence XP_005545315), which is 96% identical to the apical domain of native human TfR. TIFF2025087857000010.tif11136 Accession number 10: TIFF2025087857000011.tif3128 Accession number 11: TIFF2025087857000012.tif3128

[0088] Sequence information SEQUENCE LISTING <110> Denali Therapeutics Inc. <120> TRANSFERRIN RECEPTOR TRANSGENIC MODELS <150> US 62 / 460,692 <151> 2017-02-17 <150> US 62 / 543,559 <151> 2017-08-10 <150> US 62 / 543,658 <151> 2017-08-10 <150> US 62 / 583,314 <151> 2017-11-08 <160> 11 <170> PatentIn version 3.5 <210> 1 <211> 184 <212> PRT <213> Homo sapiens <400> 1 Ala Gln Asn Ser Val Ile Ile Val Asp Lys Asn Gly Arg Leu Val Tyr 1 5 10 15 Leu Val Glu Asn Pro Gly Gly Tyr Val Ala Tyr Ser Lys Ala Ala Thr 20 25 30 Val Thr Gly Lys Leu Val His Ala Asn Phe Gly Thr Lys Lys Asp Phe 35 40 45 Glu Asp Leu Tyr Thr Pro Val Asn Gly Ser Ile Val Ile Val Arg Ala 50 55 60 Gly Lys Ile Thr Phe Ala Glu Lys Val Ala Asn Ala Glu Ser Leu Asn 65 70 75 80 Ala Ile Gly Val Leu Ile Tyr Met Asp Gln Thr Lys Phe Pro Ile Val 85 90 95 Asn Ala Glu Leu Ser Phe Phe Gly His Ala His Leu Gly Thr Gly Asp 100 105 110 Pro Tyr Thr Pro Gly Phe Pro Ser Phe Asn His Thr Gln Phe Pro Pro 115 120 125 Ser Arg Ser Ser Gly Leu Pro Asn Ile Pro Val Gln Thr Ile Ser Arg 130 135 140 Ala Ala Ala Glu Lys Leu Phe Gly Asn Met Glu Gly Asp Cys Pro Ser 145 150 155 160 Asp Trp Lys Thr Asp Ser Thr Cys Arg Met Val Thr Ser Glu Ser Lys 165 170 175 Asn Val Lys Leu Thr Val Ser Asn 180 <210> 2 <211> 552 <212> DNA <213> Homo sapiens <400> 2 gctcagaact ccgtgatcat cgtggataag aacggccggc tggtgtacct ggtggagaac 60 cctggcggat acgtggctta ctctaaggcc gctaccgtga caggcaagct ggtgcacgcc 120 aacttcggaa ccaagaagga ctttgaggat ctgtacacac cagtgaacgg ctctatcgtg 180 atcgtgcgcg ctggaaagat caccttcgcc gagaaggtgg ctaacgccga gagcctgaac 240 gccatcggcg tgctgatcta catggatcag acaaagtttc ccatcgtgaa cgctgagctg 300 tctttctttg gacacgctca cctgggcacc ggagacccat acacacccgg attccctagc 360 tttaaccaca cccagttccc cccttccagg tctagcggac tgccaaacat ccccgtgcag 420 acaatcagca gagccgctgc cgagaagctg tttggcaaca tggagggaga ctgcccctcc 480 gattggaaga ccgactctac atgtaggatg gtgacctccg agtcaaaaaa tgtcaaactc 540 accgtgtcca at 552 <210> 3 <211> 763 <212> PRT <213> Artificial Sequence <220> <223> synthetic chimeric transferrin receptor (TfR) polypeptide sequence <220> <221> DOMAIN <222> (1)..(67) <223> Cytoplasmic Domain <220> <221> DOMAIN <222> (68)..(88) <223> Transmembrane Domain <220> <221> DOMAIN <222> (89)..(763) <223> Extracellular Domain <220> <221> DOMAIN <222> (198)..(381) <223> Apical Domain <400> 3 Met Met Asp Gln Ala Arg Ser Ala Phe Ser Asn Leu Phe Gly Gly Glu 1 5 10 15 Pro Leu Ser Tyr Thr Arg Phe Ser Leu Ala Arg Gln Val Asp Gly Asp 20 25 30 Asn Ser His Val Glu Met Lys Leu Ala Ala Asp Glu Glu Glu Asn Ala 35 40 45 Asp Asn Asn Met Lys Ala Ser Val Arg Lys Pro Lys Arg Phe Asn Gly 50 55 60 Arg Leu Cys Phe Ala Ala Ile Ala Leu Val Ile Phe Phe Leu Ile Gly 65 70 75 80 Phe Met Ser Gly Tyr Leu Gly Tyr Cys Lys Arg Val Glu Gln Lys Glu 85 90 95 Glu Cys Val Lys Leu Ala Glu Thr Glu Glu Thr Asp Lys Ser Glu Thr 100 105 110 Met Glu Thr Glu Asp Val Pro Thr Ser Ser Arg Leu Tyr Trp Ala Asp 115 120 125 Leu Lys Thr Leu Leu Ser Glu Lys Leu Asn Ser Ile Glu Phe Ala Asp 130 135 140 Thr Ile Lys Gln Leu Ser Gln Asn Thr Tyr Thr Pro Arg Glu Ala Gly 145 150 155 160 Ser Gln Lys Asp Glu Ser Leu Ala Tyr Tyr Ile Glu Asn Gln Phe His 165 170 175 Glu Phe Lys Phe Ser Lys Val Trp Arg Asp Glu His Tyr Val Lys Ile 180 185 190 Gln Val Lys Ser Ser Ala Gln Asn Ser Val Ile Ile Val Asp Lys Asn 195 200 205 Gly Arg Leu Val Tyr Leu Val Glu Asn Pro Gly Gly Tyr Val Ala Tyr 210 215 220 Ser Lys Ala Ala Thr Val Thr Gly Lys Leu Val His Ala Asn Phe Gly 225 230 235 240 Thr Lys Lys Asp Phe Glu Asp Leu Tyr Thr Pro Val Asn Gly Ser Ile 245 250 255 Val Ile Val Arg Ala Gly Lys Ile Thr Phe Ala Glu Lys Val Ala Asn 260 265 270 Ala Glu Ser Leu Asn Ala Ile Gly Val Leu Ile Tyr Met Asp Gln Thr 275 280 285 Lys Phe Pro Ile Val Asn Ala Glu Leu Ser Phe Phe Gly His Ala His 290 295 300 Leu Gly Thr Gly Asp Pro Tyr Thr Pro Gly Phe Pro Ser Phe Asn His 305 310 315 320 Thr Gln Phe Pro Pro Ser Arg Ser Ser Gly Leu Pro Asn Ile Pro Val 325 330 335 Gln Thr Ile Ser Arg Ala Ala Ala Glu Lys Leu Phe Gly Asn Met Glu 340 345 350 Gly Asp Cys Pro Ser Asp Trp Lys Thr Asp Ser Thr Cys Arg Met Val 355 360 365 Thr Ser Glu Ser Lys Asn Val Lys Leu Thr Val Ser Asn Val Leu Lys 370 375 380 Glu Arg Arg Ile Leu Asn Ile Phe Gly Val Ile Lys Gly Tyr Glu Glu 385 390 395 400 Pro Asp Arg Tyr Val Val Val Gly Ala Gln Arg Asp Ala Leu Gly Ala 405 410 415 Gly Val Ala Ala Lys Ser Ser Val Gly Thr Gly Leu Leu Leu Lys Leu 420 425 430 Ala Gln Val Phe Ser Asp Met Ile Ser Lys Asp Gly Phe Arg Pro Ser 435 440 445 Arg Ser Ile Ile Phe Ala Ser Trp Thr Ala Gly Asp Phe Gly Ala Val 450 455 460 Gly Ala Thr Glu Trp Leu Glu Gly Tyr Leu Ser Ser Leu His Leu Lys 465 470 475 480 Ala Phe Thr Tyr Ile Asn Leu Asp Lys Val Val Leu Gly Thr Ser Asn 485 490 495 Phe Lys Val Ser Ala Ser Pro Leu Leu Tyr Thr Leu Met Gly Lys Ile 500 505 510 Met Gln Asp Val Lys His Pro Val Asp Gly Lys Ser Leu Tyr Arg Asp 515 520 525 Ser Asn Trp Ile Ser Lys Val Glu Lys Leu Ser Phe Asp Asn Ala Ala 530 535 540 Tyr Pro Phe Leu Ala Tyr Ser Gly Ile Pro Ala Val Ser Phe Cys Phe 545 550 555 560 Cys Glu Asp Ala Asp Tyr Pro Tyr Leu Gly Thr Arg Leu Asp Thr Tyr 565 570 575 Glu Ala Leu Thr Gln Lys Val Pro Gln Leu Asn Gln Met Val Arg Thr 580 585 590 Ala Ala Glu Val Ala Gly Gln Leu Ile Ile Lys Leu Thr His Asp Val 595 600 605 Glu Leu Asn Leu Asp Tyr Glu Met Tyr Asn Ser Lys Leu Leu Ser Phe 610 615 620 Met Lys Asp Leu Asn Gln Phe Lys Thr Asp Ile Arg Asp Met Gly Leu 625 630 635 640 Ser Leu Gln Trp Leu Tyr Ser Ala Arg Gly Asp Tyr Phe Arg Ala Thr 645 650 655 Ser Arg Leu Thr Thr Asp Phe His Asn Ala Glu Lys Thr Asn Arg Phe 660 665 670 Val Met Arg Glu Ile Asn Asp Arg Ile Met Lys Val Glu Tyr His Phe 675 680 685 Leu Ser Pro Tyr Val Ser Pro Arg Glu Ser Pro Phe Arg His Ile Phe 690 695 700 Trp Gly Ser Gly Ser His Thr Leu Ser Ala Leu Val Glu Asn Leu Lys 705 710 715 720 Leu Arg Gln Lys Asn Ile Thr Ala Phe Asn Glu Thr Leu Phe Arg Asn 725 730 735 Gln Leu Ala Leu Ala Thr Trp Thr Ile Gln Gly Val Ala Asn Ala Leu 740 745 750 Ser Gly Asp Ile Trp Asn Ile Asp Asn Glu Phe 755 760 <210> 4 <211> 2329 <212> DNA <213> Artificial Sequence <220> <223> synthetic donor DNA sequence <220> <221> misc_feature <222> (1)..(817) <223> left homology arm <220> <221> misc_feature <222> (821)..(1522) <223> codon-optimized sequence <220> <221> misc_feature <222> (941)..(1492) <223> human apical domain <220> <221> misc_feature <222> (1523)..(2329) <223> righ homology arm <400> 4 ctatacagat atataaggat ggggcttttt ttttttaatt tttaaaaaag atttgtttat 60 tattatatgt aagtacactg tagctgtctt cagacactcc agaagagggc atcagatctc 120 attacagatg gttgtgagct accatgtggt cactgggatt tgaactcagg accttcagaa 180 gagcagtcag tgctcttaac tgataagtta ataataagtt aactgataag gtaataaagg 240 tcccctatga aaagggttca gacccaaaga gtcagagatc cacaggttga gaacctcctg 300 ccctaaatct tgttgctctc cttattcaag accactcctg ttgcagttgc tcttaagcat 360 gagtatgctc ccttctgaaa gtctccatag cagccatctc tccagcccca gagtgaggct 420 tttaaaggaa tcttcatgat aaatagaatt tttaaaaaag taactgaagt tacttaaggt 480 gttaaggtac attttattcc ctcagtaact ggttaatcta gcagttttga gtcatacttc 540 atttatcttg actttgaaga gtaagatatt aaaacaattt gcttgatcct tgaagtaagt 600 atttaaatag acattttaat gcagactttt tttagttgac tggtggtgtt gcacgtggtc 660 aatccaagta ctcatgggag gcagaggcag gaggatctct ctctagacca gcctggtcta 720 tagagcaagt tccaggacag ccagggctac acagaaacct tgtttcaaac aagactttta 780 tccttccagg cagctgagcc agaatacata cactcctagg gaagctggtt cacagaagga 840 cgaatccctg gcatactaca tcgagaatca gtttcacgag ttcaagttta gcaaagtctg 900 gagagatgag cactacgtga agatccaggt gaagagctcc gctcagaact ccgtgatcat 960 cgtggataag aacggccggc tggtgtacct ggtggagaac cctggcggat acgtggctta 1020 ctctaaggcc gctaccgtga caggcaagct ggtgcacgcc aacttcggaa ccaagaagga 1080 ctttgaggat ctgtacacac cagtgaacgg ctctatcgtg atcgtgcgcg ctggaaagat 1140 caccttcgcc gagaaggtgg ctaacgccga gagcctgaac gccatcggcg tgctgatcta 1200 catggatcag acaaagtttc ccatcgtgaa cgctgagctg tctttctttg gacacgctca 1260 cctgggcacc ggagacccat acacacccgg attccctagc tttaaccaca cccagttccc 1320 cccttccagg tctagcggac tgccaaacat ccccgtgcag acaatcagca gagccgctgc 1380 cgagaagctg tttggcaaca tggagggaga ctgcccctcc gattggaaga ccgactctac 1440 atgtaggatg gtgacctccg agtcaaaaaa tgtcaaactc accgtgtcca atgtgctgaa 1500 agaacgacgc atcctgaata tctttggagt tattaaaggt tatgaggaac caggtaaaga 1560 cctgctttgt actttttcac tttactgttt tgcttactgt agataggtct agtgcaggaa 1620 ggagaaggat gctagcttgg catgaactgc tatatcttgt ttgtcctaat gtgaactttg 1680 taatatatgt gtatataaca cataatatgg ccatgtaagt gtatggagag gccagagtta 1740 agtattaaat atctttctgt aatcatttaa aattttacat atgaaggtca gtgaacagat 1800 tgaaggagtt ttgtccaggt gggacttgga tctaaatttt ttacaatgcc tggcagcaaa 1860 caccttttta atcaactgag ctgtctcccc aaataaagtg aatgtgatat cagcttgtgg 1920 ataatttttt tttgttgctt tgataagtgg ttttcttaca ggatcacata ccagttctgt 1980 ccatagcatt aaacaaacat aactgtcatg cagtagatta atgtgcaggg cacatccaac 2040 agtcacattt attaatagga caaaaagttg gaccttatat gtagcacacc tataattcca 2100 gtgctaggaa gatccgggta ggagatcctt agttcggtgc tacttagtga gggtttgttt 2160 caaaaaacaa aagctatgat ggtgtgttgc cttttttctt ttagaccgtt atgttgtagt 2220 aggagcccag agagacgctt tgggtgctgg tgttgcggcg aagtccagtg tgggaacagg 2280 tcttctgttg aaacttgccc aagtattctc agatatgatt tcaaaaggt 2329 <210> 5 <211> 763 <212> PRT <213> Mus musculus <220> <221> DOMAIN <222> (1)..(67) <223> cytoplasmic domain <220> <221> DOMAIN <222> (68)..(88) <223> transmembrane domain <220> <221> DOMAIN <222> (89)..(763) <223> extracellular domain <220> <221> DOMAIN <222> (199)..(381) <223> apical domain <400> 5 Met Met Asp Gln Ala Arg Ser Ala Phe Ser Asn Leu Phe Gly Gly Glu 1 5 10 15 Pro Leu Ser Tyr Thr Arg Phe Ser Leu Ala Arg Gln Val Asp Gly Asp 20 25 30 Asn Ser His Val Glu Met Lys Leu Ala Ala Asp Glu Glu Glu Asn Ala 35 40 45 Asp Asn Asn Met Lys Ala Ser Val Arg Lys Pro Lys Arg Phe Asn Gly 50 55 60 Arg Leu Cys Phe Ala Ala Ile Ala Leu Val Ile Phe Phe Leu Ile Gly 65 70 75 80 Phe Met Ser Gly Tyr Leu Gly Tyr Cys Lys Arg Val Glu Gln Lys Glu 85 90 95 Glu Cys Val Lys Leu Ala Glu Thr Glu Glu Thr Asp Lys Ser Glu Thr 100 105 110 Met Glu Thr Glu Asp Val Pro Thr Ser Ser Arg Leu Tyr Trp Ala Asp 115 120 125 Leu Lys Thr Leu Leu Ser Glu Lys Leu Asn Ser Ile Glu Phe Ala Asp 130 135 140 Thr Ile Lys Gln Leu Ser Gln Asn Thr Tyr Thr Pro Arg Glu Ala Gly 145 150 155 160 Ser Gln Lys Asp Glu Ser Leu Ala Tyr Tyr Ile Glu Asn Gln Phe His 165 170 175 Glu Phe Lys Phe Ser Lys Val Trp Arg Asp Glu His Tyr Val Lys Ile 180 185 190 Gln Val Lys Ser Ser Ile Gly Gln Asn Met Val Thr Ile Val Gln Ser 195 200 205 Asn Gly Asn Leu Asp Pro Val Glu Ser Pro Glu Gly Tyr Val Ala Phe 210 215 220 Ser Lys Pro Thr Glu Val Ser Gly Lys Leu Val His Ala Asn Phe Gly 225 230 235 240 Thr Lys Lys Asp Phe Glu Glu Leu Ser Tyr Ser Val Asn Gly Ser Leu 245 250 255 Val Ile Val Arg Ala Gly Glu Ile Thr Phe Ala Glu Lys Val Ala Asn 260 265 270 Ala Gln Ser Phe Asn Ala Ile Gly Val Leu Ile Tyr Met Asp Lys Asn 275 280 285 Lys Phe Pro Val Val Glu Ala Asp Leu Ala Leu Phe Gly His Ala His 290 295 300 Leu Gly Thr Gly Asp Pro Tyr Thr Pro Gly Phe Pro Ser Phe Asn His 305 310 315 320 Thr Gln Phe Pro Pro Ser Gln Ser Ser Gly Leu Pro Asn Ile Pro Val 325 330 335 Gln Thr Ile Ser Arg Ala Ala Ala Glu Lys Leu Phe Gly Lys Met Glu 340 345 350 Gly Ser Cys Pro Ala Arg Trp Asn Ile Asp Ser Ser Cys Lys Leu Glu 355 360 365 Leu Ser Gln Asn Gln Asn Val Lys Leu Ile Val Lys Asn Val Leu Lys 370 375 380 Glu Arg Arg Ile Leu Asn Ile Phe Gly Val Ile Lys Gly Tyr Glu Glu 385 390 395 400 Pro Asp Arg Tyr Val Val Val Gly Ala Gln Arg Asp Ala Leu Gly Ala 405 410 415 Gly Val Ala Ala Lys Ser Ser Val Gly Thr Gly Leu Leu Leu Lys Leu 420 425 430 Ala Gln Val Phe Ser Asp Met Ile Ser Lys Asp Gly Phe Arg Pro Ser 435 440 445 Arg Ser Ile Ile Phe Ala Ser Trp Thr Ala Gly Asp Phe Gly Ala Val 450 455 460 Gly Ala Thr Glu Trp Leu Glu Gly Tyr Leu Ser Ser Leu His Leu Lys 465 470 475 480 Ala Phe Thr Tyr Ile Asn Leu Asp Lys Val Val Leu Gly Thr Ser Asn 485 490 495 Phe Lys Val Ser Ala Ser Pro Leu Leu Tyr Thr Leu Met Gly Lys Ile 500 505 510 Met Gln Asp Val Lys His Pro Val Asp Gly Lys Ser Leu Tyr Arg Asp 515 520 525 Ser Asn Trp Ile Ser Lys Val Glu Lys Leu Ser Phe Asp Asn Ala Ala 530 535 540 Tyr Pro Phe Leu Ala Tyr Ser Gly Ile Pro Ala Val Ser Phe Cys Phe 545 550 555 560 Cys Glu Asp Ala Asp Tyr Pro Tyr Leu Gly Thr Arg Leu Asp Thr Tyr 565 570 575 Glu Ala Leu Thr Gln Lys Val Pro Gln Leu Asn Gln Met Val Arg Thr 580 585 590 Ala Ala Glu Val Ala Gly Gln Leu Ile Ile Lys Leu Thr His Asp Val 595 600 605 Glu Leu Asn Leu Asp Tyr Glu Met Tyr Asn Ser Lys Leu Leu Ser Phe 610 615 620 Met Lys Asp Leu Asn Gln Phe Lys Thr Asp Ile Arg Asp Met Gly Leu 625 630 635 640 Ser Leu Gln Trp Leu Tyr Ser Ala Arg Gly Asp Tyr Phe Arg Ala Thr 645 650 655 Ser Arg Leu Thr Thr Asp Phe His Asn Ala Glu Lys Thr Asn Arg Phe 660 665 670 Val Met Arg Glu Ile Asn Asp Arg Ile Met Lys Val Glu Tyr His Phe 675 680 685 Leu Ser Pro Tyr Val Ser Pro Arg Glu Ser Pro Phe Arg His Ile Phe 690 695 700 Trp Gly Ser Gly Ser His Thr Leu Ser Ala Leu Val Glu Asn Leu Lys 705 710 715 720 Leu Arg Gln Lys Asn Ile Thr Ala Phe Asn Glu Thr Leu Phe Arg Asn 725 730 735 Gln Leu Ala Leu Ala Thr Trp Thr Ile Gln Gly Val Ala Asn Ala Leu 740 745 750 Ser Gly Asp Ile Trp Asn Ile Asp Asn Glu Phe 755 760 <210> 6 <211> 760 <212> PRT <213> Homo sapiens <220> <221> DOMAIN <222> (1)..(67) <223> cytoplasmic domain <220> <221> DOMAIN <222> (68)..(89) <223> transmembrane domain <220> <221> DOMAIN <222> (90)..(760) <223> extracellular domain <220> <221> DOMAIN <222> (196)..(379) <223> apical domain <400> 6 Met Met Asp Gln Ala Arg Ser Ala Phe Ser Asn Leu Phe Gly Gly Glu 1 5 10 15 Pro Leu Ser Tyr Thr Arg Phe Ser Leu Ala Arg Gln Val Asp Gly Asp 20 25 30 Asn Ser His Val Glu Met Lys Leu Ala Val Asp Glu Glu Glu Asn Ala 35 40 45 Asp Asn Asn Thr Lys Ala Asn Val Thr Lys Pro Lys Arg Cys Ser Gly 50 55 60 Ser Ile Cys Tyr Gly Thr Ile Ala Val Ile Val Phe Phe Leu Ile Gly 65 70 75 80 Phe Met Ile Gly Tyr Leu Gly Tyr Cys Lys Gly Val Glu Pro Lys Thr 85 90 95 Glu Cys Glu Arg Leu Ala Gly Thr Glu Ser Pro Val Arg Glu Glu Pro 100 105 110 Gly Glu Asp Phe Pro Ala Ala Arg Arg Leu Tyr Trp Asp Asp Leu Lys 115 120 125 Arg Lys Leu Ser Glu Lys Leu Asp Ser Thr Asp Phe Thr Gly Thr Ile 130 135 140 Lys Leu Leu Asn Glu Asn Ser Tyr Val Pro Arg Glu Ala Gly Ser Gln 145 150 155 160 Lys Asp Glu Asn Leu Ala Leu Tyr Val Glu Asn Gln Phe Arg Glu Phe 165 170 175 Lys Leu Ser Lys Val Trp Arg Asp Gln His Phe Val Lys Ile Gln Val 180 185 190 Lys Asp Ser Ala Gln Asn Ser Val Ile Ile Val Asp Lys Asn Gly Arg 195 200 205 Leu Val Tyr Leu Val Glu Asn Pro Gly Gly Tyr Val Ala Tyr Ser Lys 210 215 220 Ala Ala Thr Val Thr Gly Lys Leu Val His Ala Asn Phe Gly Thr Lys 225 230 235 240 Lys Asp Phe Glu Asp Leu Tyr Thr Pro Val Asn Gly Ser Ile Val Ile 245 250 255 Val Arg Ala Gly Lys Ile Thr Phe Ala Glu Lys Val Ala Asn Ala Glu 260 265 270 Ser Leu Asn Ala Ile Gly Val Leu Ile Tyr Met Asp Gln Thr Lys Phe 275 280 285 Pro Ile Val Asn Ala Glu Leu Ser Phe Phe Gly His Ala His Leu Gly 290 295 300 Thr Gly Asp Pro Tyr Thr Pro Gly Phe Pro Ser Phe Asn His Thr Gln 305 310 315 320 Phe Pro Pro Ser Arg Ser Ser Gly Leu Pro Asn Ile Pro Val Gln Thr 325 330 335 Ile Ser Arg Ala Ala Ala Glu Lys Leu Phe Gly Asn Met Glu Gly Asp 340 345 350 Cys Pro Ser Asp Trp Lys Thr Asp Ser Thr Cys Arg Met Val Thr Ser 355 360 365 Glu Ser Lys Asn Val Lys Leu Thr Val Ser Asn Val Leu Lys Glu Ile 370 375 380 Lys Ile Leu Asn Ile Phe Gly Val Ile Lys Gly Phe Val Glu Pro Asp 385 390 395 400 His Tyr Val Val Val Gly Ala Gln Arg Asp Ala Trp Gly Pro Gly Ala 405 410 415 Ala Lys Ser Gly Val Gly Thr Ala Leu Leu Leu Lys Leu Ala Gln Met 420 425 430 Phe Ser Asp Met Val Leu Lys Asp Gly Phe Gln Pro Ser Arg Ser Ile 435 440 445 Ile Phe Ala Ser Trp Ser Ala Gly Asp Phe Gly Ser Val Gly Ala Thr 450 455 460 Glu Trp Leu Glu Gly Tyr Leu Ser Ser Leu His Leu Lys Ala Phe Thr 465 470 475 480 Tyr Ile Asn Leu Asp Lys Ala Val Leu Gly Thr Ser Asn Phe Lys Val 485 490 495 Ser Ala Ser Pro Leu Leu Tyr Thr Leu Ile Glu Lys Thr Met Gln Asn 500 505 510 Val Lys His Pro Val Thr Gly Gln Phe Leu Tyr Gln Asp Ser Asn Trp 515 520 525 Ala Ser Lys Val Glu Lys Leu Thr Leu Asp Asn Ala Ala Phe Pro Phe 530 535 540 Leu Ala Tyr Ser Gly Ile Pro Ala Val Ser Phe Cys Phe Cys Glu Asp 545 550 555 560 Thr Asp Tyr Pro Tyr Leu Gly Thr Thr Met Asp Thr Tyr Lys Glu Leu 565 570 575 Ile Glu Arg Ile Pro Glu Leu Asn Lys Val Ala Arg Ala Ala Ala Glu 580 585 590 Val Ala Gly Gln Phe Val Ile Lys Leu Thr His Asp Val Glu Leu Asn 595 600 605 Leu Asp Tyr Glu Arg Tyr Asn Ser Gln Leu Leu Ser Phe Val Arg Asp 610 615 620 Leu Asn Gln Tyr Arg Ala Asp Ile Lys Glu Met Gly Leu Ser Leu Gln 625 630 635 640 Trp Leu Tyr Ser Ala Arg Gly Asp Phe Phe Arg Ala Thr Ser Arg Leu 645 650 655 Thr Thr Asp Phe Gly Asn Ala Glu Lys Thr Asp Arg Phe Val Met Lys 660 665 670 Lys Leu Asn Asp Arg Val Met Arg Val Glu Tyr His Phe Leu Ser Pro 675 680 685 Tyr Val Ser Pro Lys Glu Ser Pro Phe Arg His Val Phe Trp Gly Ser 690 695 700 Gly Ser His Thr Leu Pro Ala Leu Leu Glu Asn Leu Lys Leu Arg Lys 705 710 715 720 Gln Asn Asn Gly Ala Phe Asn Glu Thr Leu Phe Arg Asn Gln Leu Ala 725 730 735 Leu Ala Thr Trp Thr Ile Gln Gly Ala Ala Asn Ala Leu Ser Gly Asp 740 745 750 Val Trp Asp Ile Asp Asn Glu Phe 755 760 <210> 7 <211> 184 <212> PRT <213> Macaca mulatta <400> 7 Ala Gln Asn Ser Val Ile Ile Val Asp Lys Asn Gly Gly Leu Val Tyr 1 5 10 15 Leu Val Glu Asn Pro Gly Gly Tyr Val Ala Tyr Ser Lys Ala Ala Thr 20 25 30 Val Thr Gly Lys Leu Val His Ala Asn Phe Gly Thr Lys Lys Asp Phe 35 40 45 Glu Asp Leu Asp Ser Pro Val Asn Gly Ser Ile Val Ile Val Arg Ala 50 55 60 Gly Lys Ile Thr Phe Ala Glu Lys Val Ala Asn Ala Glu Ser Leu Asn 65 70 75 80 Ala Ile Gly Val Leu Ile Tyr Met Asp Gln Thr Lys Phe Pro Ile Val 85 90 95 Lys Ala Asp Leu Ser Phe Phe Gly His Ala His Leu Gly Thr Gly Asp 100 105 110 Pro Tyr Thr Pro Gly Phe Pro Ser Phe Asn His Thr Gln Phe Pro Pro 115 120 125 Ser Gln Ser Ser Gly Leu Pro Asn Ile Pro Val Gln Thr Ile Ser Arg 130 135 140 Ala Ala Ala Glu Lys Leu Phe Gly Asn Met Glu Gly Asp Cys Pro Ser 145 150 155 160 Asp Trp Lys Thr Asp Ser Thr Cys Lys Met Val Thr Ser Glu Asn Lys 165 170 175 Ser Val Lys Leu Thr Val Ser Asn 180 <210> 8 <211> 184 <212> PRT <213> Pan troglodytes <400> 8 Ala Gln Asn Ser Val Ile Ile Val Asp Lys Asn Gly Ser Leu Val Tyr 1 5 10 15 Leu Val Glu Asn Pro Gly Gly Tyr Val Ala Tyr Ser Lys Ala Ala Thr 20 25 30 Val Thr Gly Lys Leu Val His Ala Asn Phe Gly Thr Lys Lys Asp Phe 35 40 45 Glu Asp Leu His Thr Pro Val Asn Gly Ser Ile Val Ile Val Arg Ala 50 55 60 Gly Lys Ile Thr Phe Ala Glu Lys Val Ala Asn Ala Glu Ser Leu Asn 65 70 75 80 Ala Ile Gly Val Leu Ile Tyr Met Asp Gln Thr Lys Phe Pro Ile Val 85 90 95 Asn Ala Glu Leu Ser Phe Phe Gly His Ala His Leu Gly Thr Gly Asp 100 105 110 Pro Tyr Thr Pro Gly Phe Pro Ser Phe Asn His Thr Gln Phe Pro Pro 115 120 125 Ser Arg Ser Ser Gly Leu Pro Asn Ile Pro Val Gln Thr Val Ser Arg 130 135 140 Ala Ala Ala Glu Lys Leu Phe Gly Asn Met Glu Gly Asp Cys Pro Ser 145 150 155 160 Asp Trp Lys Thr Asp Ser Thr Cys Arg Met Val Thr Ser Glu Ser Lys 165 170 175 Asn Val Lys Leu Thr Val Ser Asn 180 <210> 9 <211> 184 <212> PRT <213> Macaca fascicularis <400> 9 Ala Gln Asn Ser Val Ile Ile Val Asp Lys Asn Gly Gly Leu Val Tyr 1 5 10 15 Leu Val Glu Asn Pro Gly Gly Tyr Val Ala Tyr Ser Lys Ala Ala Thr 20 25 30 Val Thr Gly Lys Leu Val His Ala Asn Phe Gly Thr Lys Lys Asp Phe 35 40 45 Glu Asp Leu Asp Ser Pro Val Asn Gly Ser Ile Val Ile Val Arg Ala 50 55 60 Gly Lys Ile Thr Phe Ala Glu Lys Val Ala Asn Ala Glu Ser Leu Asn 65 70 75 80 Ala Ile Gly Val Leu Ile Tyr Met Asp Gln Thr Lys Phe Pro Ile Val 85 90 95 Lys Ala Asp Leu Ser Phe Phe Gly His Ala His Leu Gly Thr Gly Asp 100 105 110 Pro Tyr Thr Pro Gly Phe Pro Ser Phe Asn His Thr Gln Phe Pro Pro 115 120 125 Ser Gln Ser Ser Gly Leu Pro Asn Ile Pro Val Gln Thr Ile Ser Arg 130 135 140 Ala Ala Ala Glu Lys Leu Phe Gly Asn Met Glu Gly Asp Cys Pro Ser 145 150 155 160 Asp Trp Lys Thr Asp Ser Thr Cys Lys Met Val Thr Ser Glu Asn Lys 165 170 175 Ser Val Lys Leu Thr Val Ser Asn 180 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> synthetic sgRNA sequence <400> 10 gaatacatac actcctcgtg agg 23 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> synthetic sgRNA sequence <400> 11 agaagaatac ttaacatctt tgg 23

Claims

[Claim 1] The invention described herein.

Images