Non-human animals containing the humanized ASGR1 locus

Abstract

To provide a non-human animal including a humanized ASGR1 locus.SOLUTION: Provided is a non-human animal including a humanized Asgr1 locus, and expresses humanized or chimera ASGR1 protein from the humanized Asgr1 locus. Also provided is a method for using the non-human animal (i.e., rodents, i.e., rat or mouse), a cell and tissue induced from the non-human animal, and a nucleotide (i.e., targeted vector, genome or the like) useful in producing the animal.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Reference to sequence listing submitted as a text file via EFS Web The sequence listing set forth in file 10362WO01_ST25.txt is 50 kilobytes, was created on June 26, 2018, and is incorporated herein by reference. [Background technology]

[0002] Many diseases are not adequately treated due to poor targeting of therapeutic molecules to the relevant tissues or organs. Delivery of bioactive agents to a subject is often hindered by the difficulty of the components reaching the target cells or tissues.

[0003] Cell-type-specific internalization effectors, such as cell-type-specific receptors like human ASGR1 that are indirectly or directly internalized by target cells, can be used to facilitate and enhance the delivery of therapeutic molecules to specific target cells in vivo. Similarly, these cell-type-specific internalization effectors are suitable for therapeutic purposes and can promote the internalization of target cell surface receptors or target soluble proteins in vivo. However, there is still a lack of suitable models for evaluating the efficacy of these delivery and therapeutic mechanisms in vivo. Summary of the Invention [Means for solving the problem]

[0004] Non-human animals are provided that contain a humanized Asgr1 locus and express a humanized or chimeric ASGR1 protein from the humanized Asgr1 locus, as well as methods of using the non-human animals (e.g., rodents, e.g., rats or mice), cells and tissues derived from the non-human animals, and nucleotides (e.g., targeting vectors, genomes, etc.) useful in producing the animals.

[0005] In one embodiment, a non-human animal is provided that comprises a genetically modified endogenous Asgr1 locus encoding a modified Asgr1 protein, wherein the modified Asgr1 protein comprises a cytoplasmic domain, a transmembrane domain, and an extracellular domain, wherein all or a portion of the extracellular domain is encoded by a segment of the endogenous Asgr1 locus that has been deleted and replaced with an orthologous human ASGR1 sequence. In some such non-human animals, the extracellular domain comprises a coiled-coil domain and a C-type lectin domain, wherein all or a portion of the C-type lectin domain is encoded by a segment of the endogenous Asgr1 locus that has been deleted and replaced with an orthologous human ASGR1 sequence. Optionally, the C-type lectin domain is the C-type lectin domain of human ASGR1. Optionally, the C-type lectin domain comprises the sequence set forth in SEQ ID NO: 28.

[0006] In some such animals, the extracellular domain comprises a coiled-coil domain and a C-type lectin domain, and all or part of the coiled-coil domain is encoded by a segment of the endogenous Asgr1 locus that has been deleted and replaced with an orthologous human ASGR1 sequence. Optionally, the coiled-coil domain is the coiled-coil domain of human ASGR1. Optionally, the coiled-coil domain comprises the sequence set forth in SEQ ID NO:27.

[0007] In some such animals, all or part of both the coiled-coil domain and the C-type lectin domain are encoded by segments of the endogenous Asgr1 locus that have been deleted and replaced with orthologous human ASGR1 sequences. Optionally, the C-type lectin domain is a C-type lectin domain of human ASGR1, and the coiled-coil domain is a coiled-coil domain of human ASGR1. Optionally, the C-type lectin domain comprises the sequence set forth in SEQ ID NO:28, and the coiled-coil domain comprises the sequence set forth in SEQ ID NO:27.

[0008] In some such animals, the orthologous human ASGR1 sequence comprises exons 3 to 8 of the human ASGR1 gene. Optionally, the orthologous human ASGR1 sequence encodes an ASGR1 protein segment comprising the sequence set forth in SEQ ID NO:31.

[0009] In some of such animals, all or part of the cytoplasmic domain is encoded by an endogenous non-human animal Asgr1 sequence. In some of such animals, all or part of the transmembrane domain is encoded by an endogenous non-human animal Asgr1 sequence. Optionally, all or part of both the cytoplasmic domain and the transmembrane domain are encoded by endogenous non-human animal Asgr1 sequences.

[0010] In another embodiment, non-human animal cells or tissues (e.g., non-human hepatocytes, non-human embryonic stem (ES) cells, or other pluripotent cells, e.g., germ cells) containing a humanized Asgr1 locus and in vitro compositions containing them are provided. In one embodiment, non-human animal cells are provided containing a genetically modified endogenous Asgr1 locus encoding a modified Asgr1 protein, wherein the modified Asgr1 protein comprises a cytoplasmic domain, a transmembrane domain, and an extracellular domain, all or a portion of which is encoded by a segment of the endogenous Asgr1 locus deleted and replaced with an orthologous human ASGR1 sequence. In some such non-human animal cells, the extracellular domain comprises a coiled-coil domain and a C-type lectin domain, all or a portion of which is encoded by a segment of the endogenous Asgr1 locus deleted and replaced with an orthologous human ASGR1 sequence. Optionally, the C-type lectin domain is the C-type lectin domain of human ASGR1. Optionally, the C-type lectin domain comprises the sequence set forth in SEQ ID NO:28.

[0011] In some of such non-human animal cells, the extracellular domain comprises a coiled-coil domain and a C-type lectin domain, and all or part of the coiled-coil domain is encoded by a segment of the endogenous Asgr1 locus that has been deleted and replaced with an orthologous human ASGR1 sequence. Optionally, the coiled-coil domain is the coiled-coil domain of human ASGR1. Optionally, the coiled-coil domain comprises the sequence set forth in SEQ ID NO:27.

[0012] In some such non-human animal cells, all or part of both the coiled-coil domain and the C-type lectin domain are encoded by segments of the endogenous Asgr1 locus that have been deleted and replaced with orthologous human ASGR1 sequences. Optionally, the C-type lectin domain is the C-type lectin domain of human ASGR1, and the coiled-coil domain is the coiled-coil domain of human ASGR1. Optionally, the C-type lectin domain comprises the sequence set forth in SEQ ID NO:28, and the coiled-coil domain comprises the sequence set forth in SEQ ID NO:27.

[0013] In some such non-human animal cells, the orthologous human ASGR1 sequence comprises exons 3 to 8 of the human ASGR1 gene. Optionally, the orthologous human ASGR1 sequence encodes an ASGR1 protein segment comprising the sequence set forth in SEQ ID NO:31.

[0014] In some of such non-human animal cells, all or a portion of the cytoplasmic domain is encoded by an endogenous non-human animal Asgr1 sequence. In some of such animal cells, all or a portion of the transmembrane domain is encoded by an endogenous non-human animal Asgr1 sequence. Optionally, all or a portion of both the cytoplasmic domain and the transmembrane domain are encoded by an endogenous non-human animal Asgr1 sequence. Encoded by animal Asgr1 sequence.

[0015] A portion of such animals and / or animal cells are heterozygous for the genetically modified endogenous Asgr1 locus. A portion of such animals and / or animal cells are homozygous for the genetically modified endogenous Asgr1 locus.

[0016] Such non-human animals and / or portions of animal cells are mammals and / or mammalian cells. Optionally, the mammal is a rodent. Optionally, the rodent is a rat or a mouse. Optionally, the rodent is a mouse. Optionally, all or a portion of the cytoplasmic domain is encoded by an endogenous mouse Asgr1 sequence. Optionally, the cytoplasmic domain comprises the sequence set forth in SEQ ID NO: 29. Optionally, all or a portion of the transmembrane domain is encoded by an endogenous mouse Asgr1 sequence. Optionally, the transmembrane domain comprises the sequence set forth in SEQ ID NO: 30. Optionally, all or a portion of both the cytoplasmic domain and the transmembrane domain are encoded by an endogenous mouse Asgr1 sequence. Optionally, the cytoplasmic domain comprises the sequence set forth in SEQ ID NO: 29 and the transmembrane domain comprises the sequence set forth in SEQ ID NO: 30. Optionally, the extracellular domain comprises a coiled-coil domain and a C-type lectin domain, wherein all or a portion of both the coiled-coil domain and the C-type lectin domain are encoded by segments of the endogenous Asgr1 locus that have been deleted and replaced with orthologous human ASGR1 sequences, and all or a portion of both the cytoplasmic domain and the transmembrane domain are encoded by endogenous mouse Asgr1 sequences. Optionally, the C-type lectin domain is the C-type lectin domain of human ASGR1, the coiled-coil domain is the coiled-coil domain of human ASGR1, the cytoplasmic domain is the cytoplasmic domain of mouse Asgr1, and the transmembrane domain is the transmembrane domain of mouse Asgr1. Optionally, the C-type lectin domain comprises the sequence set forth in SEQ ID NO:28, the coiled-coil domain comprises the sequence set forth in SEQ ID NO:27, the cytoplasmic domain comprises the sequence set forth in SEQ ID NO:29, and the transmembrane domain comprises the sequence set forth in SEQ ID NO:30. Optionally, the modified Asgr1 protein comprises the sequence set forth in SEQ ID NO:3.

[0017] In another embodiment, methods are provided for evaluating in vivo delivery of a therapeutic complex to the liver via human ASGR1-mediated internalization. Some such methods include (a) administering a therapeutic complex to any of the non-human animals described above, the therapeutic complex comprising a therapeutic molecule and an antigen-binding protein or ligand that specifically binds to human ASGR1, and (b) evaluating delivery of the therapeutic molecule to the liver of the non-human animal. Optionally, the therapeutic molecule is a lysosomal replacement protein or enzyme, or a nucleic acid encoding a lysosomal replacement protein or enzyme, and step (b) includes evaluating the presence or activity of the lysosomal replacement protein or enzyme in the liver of the non-human animal. Optionally, the therapeutic molecule is a nucleic acid encoding a therapeutic secreted protein, and step (b) includes evaluating the serum level or activity of the therapeutic secreted protein in the non-human animal.

[0018] In another aspect, methods are provided for assessing the in vivo efficacy of a therapeutic molecule that targets a liver cell surface protein or a soluble protein in the liver for internalization via human ASGR1, some of which include (a) administering a therapeutic molecule to any of the non-human animals described above, wherein the therapeutic molecule comprises a bispecific antigen-binding protein that specifically binds to a liver cell surface protein or a soluble protein and specifically binds to human ASGR1, and (b) assessing the cell surface level or activity of the liver cell surface protein in the liver of the non-human animal, or assessing the expression or activity of the soluble protein in the liver.

[0019] In another embodiment, the humanized Asgr1 locus, e.g., the humanized ASGR1 locus described herein, Nucleic acids, such as targeting vectors, are provided for generating endogenous non-human animal Asgr1 loci that express proteins or chimeric ASGR1 proteins, and / or non-human animal genomes that include the human Asgr1 loci described herein. Some of these nucleic acids include a sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, and combinations thereof. Some of these nucleic acids include a sequence that encodes the amino acid sequence set forth in SEQ ID NO: 3. For example, they include the sequence set forth in SEQ ID NO: 24.

[0020] In certain embodiments, for example, the following items are provided: (Item 1) A non-human animal or genome of a non-human animal comprising a genetically modified endogenous Asgr1 locus encoding a modified Asgr1 protein, the modified Asgr1 protein comprising a cytoplasmic domain, a transmembrane domain, and an extracellular domain, wherein all or part of the extracellular domain is encoded by a segment of the endogenous Asgr1 locus that has been deleted and replaced with an orthologous human ASGR1 sequence; The non-human animal is a non-human animal or a non-human animal genome that expresses the modified Asgr1 protein. (Item 2) 2. The non-human animal or non-human animal genome of item 1, wherein the extracellular domain comprises a coiled-coil domain and a C-type lectin domain, and all or part of the C-type lectin domain is encoded by a segment of the endogenous Asgr1 locus that has been deleted and replaced with an orthologous human ASGR1 sequence. (Item 3) 3. The non-human animal or non-human animal genome according to item 2, wherein the C-type lectin domain is the C-type lectin domain of human ASGR1. (Item 4) 4. The non-human animal or non-human animal genome according to item 2 or 3, wherein the C-type lectin domain comprises the sequence set forth in SEQ ID NO: 28. (Item 5) 2. The non-human animal or non-human animal genome of item 1, wherein the extracellular domain comprises a coiled-coil domain and a C-type lectin domain, and all or part of the coiled-coil domain is encoded by a segment of the endogenous Asgr1 locus that has been deleted and replaced with an orthologous human ASGR1 sequence. (Item 6) 6. The non-human animal or non-human animal genome according to item 5, wherein the coiled-coil domain is the coiled-coil domain of human ASGR1. (Item 7) 7. The non-human animal or non-human animal genome according to item 5 or 6, wherein the coiled-coil domain comprises the sequence set forth in SEQ ID NO: 27. (Item 8) 8. The non-human animal or non-human animal genome according to any one of items 2 to 7, wherein all or part of both the coiled-coil domain and the C-type lectin domain are encoded by segments of the endogenous Asgr1 locus that have been deleted and replaced with orthologous human ASGR1 sequences. (Item 9) 9. The non-human animal or non-human animal genome of item 8, wherein the C-type lectin domain is the C-type lectin domain of human ASGR1 and the coiled-coil domain is the C-type lectin domain of human ASGR1. (Item 10) the C-type lectin domain comprises the sequence set forth in SEQ ID NO: 28, and the coiled coil 10. The non-human animal or non-human animal genome according to item 8 or 9, wherein the domain comprises the sequence set forth in SEQ ID NO: 27. (Item 11) 11. The non-human animal or non-human animal genome according to any one of items 1 to 10, wherein the orthologous human ASGR1 sequence comprises exons 3 to 8 of the human ASGR1 gene. (Item 12) 12. The non-human animal or non-human animal genome of item 11, wherein the orthologous human ASGR1 sequence encodes an ASGR1 protein segment comprising the sequence set forth in SEQ ID NO: 31. (Item 13) Item 14. The non-human animal or non-human animal genome according to any one of Items 1 to 12, wherein all or part of the cytoplasmic domain is encoded by an endogenous non-human animal Asgr1 sequence. Item 15. The non-human animal or non-human animal genome according to any one of Items 1 to 13, wherein all or part of the transmembrane domain is encoded by an endogenous non-human animal Asgr1 sequence. 15. The non-human animal or non-human animal genome according to item 13 or 14, wherein all or part of both the cytoplasmic domain and the transmembrane domain are encoded by endogenous non-human animal Asgr1 sequences. (Item 16) 16. The non-human animal or non-human animal genome of any one of items 1 to 15, wherein the non-human animal or non-human animal genome is heterozygous for the genetically modified endogenous Asgr1 locus. (Item 17) 16. The non-human animal or non-human animal genome of any one of items 1 to 15, wherein the non-human animal or non-human animal genome is homozygous for the genetically modified endogenous Asgr1 locus. (Item 18) 18. The non-human animal or non-human animal genome according to any one of items 1 to 17, wherein the non-human animal is a mammal or the non-human animal genome is a mammalian genome. (Item 19) 19. The non-human animal or non-human animal genome of item 18, wherein the non-human animal is a rodent or the non-human animal genome is a rodent genome. (Item 20) 20. The non-human animal or non-human animal genome according to item 19, wherein the non-human animal is a rat or a mouse, or the non-human animal genome is a rat genome or a mouse genome. (Item 21) 21. The non-human animal or non-human animal genome according to item 20, wherein the non-human animal is a mouse or the non-human animal genome is a mouse genome. (Item 22) 22. The non-human animal or non-human animal genome of item 21, wherein all or part of the cytoplasmic domain is encoded by an endogenous mouse Asgr1 sequence. (Item 23) 23. The non-human animal or non-human animal genome of item 22, wherein the cytoplasmic domain comprises the sequence set forth in SEQ ID NO: 29. (Item 24) 22. The non-human animal or non-human animal genome of item 21, wherein all or part of the transmembrane domain is encoded by an endogenous mouse Asgr1 sequence. (Item 25) 25. The non-human animal or non-human animal genome of item 24, wherein the transmembrane domain comprises the sequence set forth in SEQ ID NO: 30. (Item 26) 26. The non-human animal or non-human animal genome according to any one of items 22 to 25, wherein all or part of both the cytoplasmic domain and the transmembrane domain are encoded by endogenous mouse Asgr1 sequences. (Item 27) 27. The non-human animal or non-human animal genome of item 26, wherein the cytoplasmic domain comprises the sequence set forth in SEQ ID NO: 29 and the transmembrane domain comprises the sequence set forth in SEQ ID NO: 30. (Item 28) 28. The non-human animal or non-human animal genome according to any one of items 21 to 27, wherein the extracellular domain comprises a coiled-coil domain and a C-type lectin domain, and all or a portion of both the coiled-coil domain and the C-type lectin domain are encoded by segments of the endogenous Asgr1 locus that have been deleted and replaced with an orthologous human ASGR1 sequence, and all or a portion of both the cytoplasmic domain and the transmembrane domain are encoded by an endogenous mouse Asgr1 sequence. (Item 29) 29. The non-human animal or non-human animal genome according to any one of Items 21 to 28, wherein the C-type lectin domain is the C-type lectin domain of human ASGR1, the coiled-coil domain is the coiled-coil domain of human ASGR1, the cytoplasmic domain is the cytoplasmic domain of mouse Asgr1, and the transmembrane domain is the transmembrane domain of mouse Asgr1. (Item 30) 30. The non-human animal or non-human animal genome according to item 28 or 29, wherein the C-type lectin domain comprises the sequence set forth in SEQ ID NO: 28, the coiled-coil domain comprises the sequence set forth in SEQ ID NO: 27, the cytoplasmic domain comprises the sequence set forth in SEQ ID NO: 29, and the transmembrane domain comprises the sequence set forth in SEQ ID NO: 30. (Item 31) 31. The non-human animal or non-human animal genome of Item 30, wherein the modified Asgr1 protein comprises the sequence set forth in SEQ ID NO:3. (Item 32) 1. A method for assessing in vivo delivery of a therapeutic conjugate to the liver via human-ASGR1-mediated internalization, comprising: (a) administering to the non-human animal of any one of items 1 to 31 a therapeutic complex, the therapeutic complex comprising a therapeutic molecule and an antigen-binding protein or ligand that specifically binds to human ASGR1; and (b) assessing delivery of said therapeutic molecule to the liver of said non-human animal. (Item 33) 33. The method of claim 32, wherein the therapeutic molecule is a lysosomal replacement protein or enzyme or a nucleic acid encoding the lysosomal replacement protein or enzyme, and step (b) comprises assessing the presence or activity of the lysosomal replacement protein or enzyme in the liver of the non-human animal. (Item 34) 33. The method of claim 32, wherein the therapeutic molecule is a nucleic acid encoding a therapeutic secreted protein, and step (b) comprises assessing the serum level or activity of the therapeutic secreted protein in the non-human animal. (Item 35) Regarding human ASGR1-mediated internalization in vivo, liver cell surface proteins or 1. A method for assessing the effectiveness of a therapeutic molecule that targets a soluble protein in the liver, comprising: (a) administering to the non-human animal of any one of items 1 to 31 a therapeutic molecule, wherein the therapeutic molecule comprises a bispecific antigen-binding protein that specifically binds to the liver cell surface protein or the soluble protein and specifically binds to human ASGR1; and (b) assessing the cell surface level or activity of said liver cell surface protein in the liver of said non-human animal, or assessing the expression or activity of said soluble protein in the liver of said non-human animal. (Item 36) A non-human animal cell comprising a genetically modified endogenous Asgr1 locus encoding a modified Asgr1 protein, wherein the modified Asgr1 protein comprises a cytoplasmic domain, a transmembrane domain and an extracellular domain, and all or part of the extracellular domain is encoded by a segment of the endogenous Asgr1 locus that has been deleted and replaced with an orthologous human ASGR1 sequence. (Item 37) 37. The non-human animal cell according to Item 36, wherein the non-human animal cell is a hepatocyte. (Item 38) 37. The non-human animal cell according to item 36, wherein the non-human animal cell is a pluripotent cell. (Item 39) 39. The non-human animal cell according to Item 38, wherein the non-human animal cell is an ES cell. (Item 40) 39. The non-human animal cell according to item 38, wherein the non-human animal cell is a germ cell. (Item 41) A composition comprising the non-human animal cell according to any one of items 36 to 40. (Item 42) A nucleic acid comprising a sequence selected from the group comprising SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, and combinations thereof. (Item 43) 43. The nucleic acid of item 42, comprising the sequence set forth as SEQ ID NO: 24. (Item 44) 44. Nucleic acid according to item 42 or claim 43, characterized in that it encodes the amino acid sequence set out as SEQ ID NO: 3. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0021] [Figure 1]Figure 1A shows a schematic of the human ASGR1 locus. The eight exons are indicated by black boxes, and the untranslated regions (UTRs) are indicated by white boxes. The transmembrane domain, coiled-coil domain, and C-type lectin domain are shown at the top of the figure. Asterisks indicate the positions of the upstream primer (7302hTU) and downstream primer (7302hTD) for the gain-of-allele assay. The fragment inserted into the mouse Asgr1 locus for humanization is shown at the bottom.

[0022] Figure 1B shows a schematic of the mouse Asgr1 locus. The eight exons are indicated by black boxes, and the UTRs are indicated by white boxes. The transmembrane domain, coiled-coil domain, and C-type lectin domain are shown at the top of the figure. Asterisks indicate the positions of the upstream primer (7302 mTU) and downstream primer (7302 mTD) for loss-of-allele assays. The fragment that was deleted and replaced with the corresponding fragment from the human ASGR1 locus is shown at the bottom.

[0023] [Figure 2] Figure 2A shows a schematic of the large targeting vector for generating a humanized Asgr1 allele (7302 allele) containing a hygromycin-resistance self-deleting cassette. Mouse exons are indicated by gray boxes, human exons by black boxes, and UTRs by white boxes. The boundaries between the different regions (mouse / human, human / cassette, and cassette / mouse) are indicated by lines labeled A, B, and C, respectively, at the bottom of the figure.

[0024] Figure 2B shows a schematic of the cassette-deleted version of the humanized Asgr1 allele in Figure 2A. Mouse exons are shown in gray boxes, human exons are shown in black boxes, and UTRs are shown in white boxes. The boundaries between the different regions (5' mouse / human boundary, 3' human / mouse boundary) are indicated at the bottom of the figure by lines labeled A and D, respectively.

[0025] [Figure 3] Figure 3 shows an alignment of the human ASGR1 protein (hASGR1; SEQ ID NO: 1), mouse Asgr1 protein (mAsgr1; SEQ ID NO: 2), and humanized mouse Asgr1 protein (7302 humIn; SEQ ID NO: 3). The underlined residues are those encoded by the introduced human exons. The boxed residues constitute the transmembrane domain. The dotted line indicates the C-type lectin domain. The thick solid line indicates the coiled-coil region.

[0026] [Figure 4] FIG. 4 shows that humanized Asgr1 mice (Asgr1hu / hu) have plasma lipid profiles (including total cholesterol, triglycerides, LDL-C, and HDL-C) similar to those of wild-type (Asgr1+ / +) littermates.

[0027] [Figure 5] FIG. 5 shows that humanized Asgr1 mice (Asgr1hu / hu) have similar body weights and blood glucose levels to their wild-type littermates.

[0028] [Figure 6] Figure 6 shows that humanized Asgr1 protein in humanized Asgr1 mice (Asgr1hu / hu) colocalizes with mouse Asgr1 in the liver membrane. Transferrin receptor (TRFR) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as loading controls for the liver membrane and cytosolic fractions, respectively. N = 4 per group is shown.

[0029] [Figure 7] Figure 7 shows an alignment of the human ASGR1 protein (SEQ ID NO: 1), cynomolgus monkey (cyno) (SEQ ID NO: 39), mouse Asgr1 protein (SEQ ID NO: 2), and rat Asgr1 protein (SEQ ID NO: 41). The underlined residues are those encoded by the introduced human exons. There is 97.6% sequence identity between the human and cynomolgus monkey ASGR1 proteins (98.3% sequence identity in the extracellular domain), 77% sequence identity between the human and mouse ASGR1 proteins, and 78.4% sequence identity between the human and rat ASGR1 proteins.

[0030] [Figure 8A] Figures 8A-8C show immunofluorescence microscopy images of liver (A), spleen (B), or kidney (C) samples collected from C57BL / 6 mice transgenically engineered to express human ASGR1 in hepatocytes (i-iv), or wild-type C57BL / 6 mice (v-viii), 10 days after intravenous injection of 1 x 10 wild-type scAAV2-CMV-eGFP (i, v), saline (ii, vi), 1 x 10 scAAV2-N587myc-CMV-eGFP viral vector alone (iii, vii), or scAAV2-N587myc-CMV-eGFP viral vector with bispecific anti-myc-ASGR1 antibody (iv, viii). [Figure 8B] Same as above. [Figure 8C] Same as above.

[0031] [Figure 9-1]Figures 9A-9R show immunofluorescence microscopy images of liver samples collected from C57BL / 6 mice transgenically engineered to express human ASGR1 in hepatocytes (Figures 9D-9F, 9J-9L, 9P-9R), or wild-type C57BL / 6 mice (Figures 9A-9C, 9G-9I, 9M-9O) 4 weeks after intravenous injection with 2.18 x 10 wild-type ssAAV2-CAGG-eGFP (Figures 9B, 9C, 9E, 9F), saline (Figures 9A, 9D), 2.18 x 10 ssAAV2-N587myc-CAGG-eGFP viral vector alone (Figures 9G-9I, 9J-9L), or ssAAV2-N587myc-CAGG-eGFP viral vector with bispecific anti-myc-ASGR1 antibody (Figures 9M-9O, 9P-9R). Each image represents one mouse. [Figure 9-2] Same as above. [Figure 9-3] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0032] definition The terms "protein," "polypeptide," and "peptide" are used interchangeably herein and include polymeric forms of amino acids of any length, including coded and non-coded amino acids, and chemically or biochemically modified or derivatized amino acids. The term also includes modified polymers, such as, for example, polypeptides with modified peptide backbones. The term "domain" refers to any portion of a protein or polypeptide having a particular function or structure.

[0033] Proteins are said to have an "N-terminus" and a "C-terminus." The term "N-terminus" refers to the beginning of a protein or polypeptide and is terminated by an amino acid having a free amine group (-NH2). The term "C-terminus" refers to the end of an amino acid chain (protein or polypeptide) and is terminated by a free carboxyl group (-COOH).

[0034] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein and include polymeric forms of nucleotides of any length, containing ribonucleotides, deoxyribonucleotides, or analogs or modified forms thereof. This term includes single-, double-, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers that contain purine, pyrimidine, or other naturally occurring, chemically, biochemically modified, non-naturally occurring, or derivatized nucleotide bases.

[0035] Nucleic acids are said to have "5' ends" and "3' ends" when mononucleotides are reacted to form oligonucleotides such that the 5' phosphate of the pentose ring of one mononucleotide is linked via a phosphodiester bond to the 3' oxygen of its adjacent mononucleotide in a certain orientation. An end of an oligonucleotide is called the "5' end" if its 5' phosphate is not linked to the 3' oxygen of the pentose ring of the mononucleotide. An end of an oligonucleotide is called the "3' end" if its 3' oxygen is not linked to the 5' phosphate of the pentose ring of another mononucleotide. A nucleic acid sequence, even an internal portion of a larger oligonucleotide, may be said to have 5' and 3' ends. In either a linear or circular DNA molecule, distinct elements are referred to as "upstream" or "downstream" 5' or 3' elements.

[0036] The term "genomically integrated" refers to a nucleic acid that has been introduced into a cell such that the nucleotide sequence is integrated into the genome of the cell and can be inherited by its progeny. Any protocol can be used for stable integration of a nucleic acid into the genome of a cell.

[0037] The term "targeting vector" refers to a recombinant nucleic acid that can be introduced by homologous recombination, non-homologous-end-joining-mediated ligation, or any other recombinant means into a target location within a cellular genome.

[0038] The term "viral vector" refers to a recombinant nucleic acid that contains at least one element of viral origin and contains elements sufficient or permissive for packaging into a viral vector particle. The vectors and / or particles can be used for the transfer of DNA, RNA, or other nucleic acids either in vitro or in vivo. Many forms of viral vectors are known.

[0039] The term "wild-type" includes entities having a structure and / or activity that is present under a "normal" state or situation (as opposed to mutant, diseased, altered, etc.). Wild-type genes and polypeptides often exist in multiple alternative forms (e.g., alleles).

[0040] The term "endogenous" refers to a nucleic acid sequence that is naturally present in a cell or a non-human animal. For example, an endogenous Asgr1 sequence of a non-human animal refers to a native Asgr1 sequence that is naturally present at the Asgr1 locus in the non-human animal.

[0041] An "exogenous" molecule or sequence includes a molecule or sequence that is not normally present in a cell in that form. Normal presence includes presence in relation to a particular developmental stage and environmental conditions of the cell. An exogenous molecule or sequence can include, for example, a mutant version of a corresponding endogenous sequence in the cell, e.g., a humanized version of an endogenous sequence, or can include a sequence that corresponds to an endogenous sequence in the cell but in a different form (i.e., not in a chromosome). In contrast, an endogenous molecule or sequence includes a molecule or sequence that is normally present in that form in a particular cell at a particular developmental stage under particular environmental conditions.

[0042] The term "heterologous" when used with respect to a nucleic acid or protein indicates that the nucleic acid or protein comprises at least two moieties that are not naturally found together in the same molecule. For example, the term "heterologous" when used with respect to a nucleic acid moiety or protein moiety indicates that the nucleic acid or protein comprises two or more subsequences that are not naturally found in the same relationship to each other (e.g., not linked together). As an example, a "heterologous" region of a nucleic acid vector can be a segment of nucleic acid within another nucleic acid molecule that is not naturally associated with that molecule, or linked to another nucleic acid molecule that is not naturally associated with that molecule. For example, a heterologous region of a nucleic acid vector can comprise a coding sequence that is flanked by sequences that are not naturally associated with the coding sequence. Similarly, a "heterologous" region of a protein can be a segment of amino acids within another peptide molecule that is not naturally associated with that molecule, or linked to another peptide molecule that is not naturally associated with that molecule (e.g., a fusion protein or tagged protein). Similarly, a nucleic acid or protein can comprise a heterologous label or a heterologous secretion or localization sequence.

[0043] "Codon optimization" takes advantage of codon degeneracy, exemplified by the multiplicity of combinations of three-base pairs of codons that specify amino acids. Generally, expression in a particular host cell is improved by replacing at least one codon in the native sequence with a codon that is more frequently or most frequently used in the host cell's genes, while maintaining the native amino acid sequence. This includes the process of modifying a nucleic acid sequence to enhance its expression. For example, a nucleic acid encoding a Cas9 protein can be modified to substitute codons that are more frequently used compared to the native nucleic acid sequence in a given prokaryotic or eukaryotic cell, including bacterial cells, yeast cells, human cells, non-human cells, mammalian cells, rodent cells, mouse cells, rat cells, hamster cells, or any other host cell. Codon usage tables are readily available, for example, from the "Codon Usage Database." These tables can be adapted in a number of ways. See Nakamura et al. (2000) Nucleic Acids Research 28:292, which is incorporated herein by reference in its entirety for all purposes. Computer algorithms are also available for codon optimization of a particular sequence for expression in a particular host (see, e.g., Gene Forge).

[0044] The term "locus" refers to a specific location of a gene (or key sequence), a DNA sequence, a sequence encoding a polypeptide, or a location on a chromosome in the genome of an organism. For example, "Asgr1 locus" can refer to the specific location of the Asgr1 gene, the Asgr1 DNA sequence, the sequence encoding Asgr1, or the location of Asgr1 on a chromosome in the genome of an organism that is specified for the location where such a sequence is present. The "Asgr1 locus" can include regulatory elements of the Asgr1 gene, including, for example, an enhancer, a promoter, a 5' and / or a 3' untranslated region (UTR), or a combination thereof.

[0045] The term "gene" refers to a DNA sequence in a chromosome that encodes a product (e.g., an RNA product and / or a polypeptide product), including coding regions interrupted by non-coding introns and sequences adjacent to the coding region at both the 5' and 3' ends, such that the gene corresponds to the full-length mRNA (including 5' and 3' untranslated sequences). The term "gene" also includes other non-coding sequences, including regulatory sequences (e.g., promoters, enhancers, and transcription factor binding sites), polyadenylation signals, internal ribosome entry sites, silencers, insulating sequences, and matrix attachment regions. These sequences may be close to the coding region of the gene (e.g., within 10 kb) or may be distant. They influence the level or rate of gene transcription and translation.

[0046] The term "allele" refers to a variant form of a gene. Some genes have different forms that are at the same location on a chromosome, or at the same locus. Diploid organisms have two alleles at each locus. Each pair of alleles represents a genotype at a particular locus. A genotype is described as homozygous when there are two identical alleles at a particular locus, or heterozygous when the two alleles are different.

[0047] A "promoter" is a regulatory region of DNA, typically containing a TATA box, which can provide instructions to RNA polymerase II to initiate RNA synthesis at the appropriate transcription start site for a particular polynucleotide sequence. Promoters may also contain other regions that influence the rate of transcription initiation. The promoter sequences disclosed herein regulate transcription of an operably linked polynucleotide. The promoters may be active in one or more of the cell types disclosed herein (e.g., eukaryotic cells, non-human mammalian cells, human cells, rodent cells, pluripotent cells, one-cell stage embryos, differentiated cells, or combinations thereof). The promoters may be, for example, constitutively active, conditional, inducible, temporally restricted (e.g., developmentally regulated), or spatially restricted (e.g., cell-specific or tissue-specific). Examples of promoters can be found, for example, in International Patent Publication No. WO 2013 / 176772, which is incorporated by reference in its entirety for all purposes.

[0048] "Operable linkage" or "operably linked" includes the juxtaposition of two or more components (e.g., a promoter and another sequence element) such that both components function normally and at least one of the components mediates a function affecting at least one of the other components. For example, a promoter can be operably linked to a coding sequence if the promoter controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. Operable linkage can include sequences that are contiguous with each other, or sequences that act in trans (e.g., regulatory sequences can act at a distance to control transcription of the coding sequence).

[0049] The term "variant" refers to a nucleotide sequence that differs (eg, by one nucleotide) from the most prevalent sequence in a population, or a protein sequence that differs (eg, by one amino acid) from the most prevalent sequence in a population.

[0050] The term "fragment," when referring to a protein, means a protein that is shorter or has fewer amino acids than the full-length protein. The term "fragment," when referring to a nucleic acid, means a nucleic acid that is shorter or has fewer nucleotides than the full-length nucleic acid. A fragment can be, for example, an N-terminal fragment (i.e., the C-terminal portion of the protein has been removed), a C-terminal fragment (i.e., the N-terminal portion of the protein has been removed), or an internal fragment.

[0051] "Sequence identity" or "identity," in the context of two polynucleotide or polypeptide sequences, refers to residues in the two sequences that are identical when aligned for maximum correspondence over a specified comparison window. When percentage sequence identity is used with respect to proteins, non-identical residue positions often differ by conservative amino acid substitutions, in which an amino acid residue is replaced with another having similar chemical properties (e.g., charge or hydrophobicity), thus leaving the functional properties of the molecule unchanged. When sequences differ by conservative substitutions, the percentage of sequence identity may be adjusted upward to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity." Means for making this adjustment are known. Typically, such means include ranking conservative substitutions as partial rather than full-length mismatches, thereby increasing the percentage sequence identity. Thus, for example, identical amino acids are assigned a score of 1, non-conservative substitutions are assigned a score of zero, and conservative substitutions are assigned a score between zero and 1. Conservative substitution scores are calculated, for example, as implemented in the PC / GENE program (Intelligenetics, Mountain View, Calif.).

[0052] "Percentage of sequence identity" includes values ​​determined by comparing two optimally aligned (highest number of perfectly matched residues) sequences over a comparison window, where the portion of the polynucleotide sequence in the comparison window may contain additions or deletions (i.e., gaps) relative to the reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences to determine the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to determine the percentage of sequence identity. Unless otherwise specified (e.g., the shorter sequence includes linked heterologous sequences), the comparison window is the entire length of the shorter of the two sequences being compared.

[0053] Unless otherwise stated, sequence identity / similarity values ​​include values ​​obtained using GAP Version 10, or any equivalent program, using the following parameters: GAP Weight of 50 and Length Weight of 3, and for nucleotide sequences using the nwsgapdna.cmp scoring matrix. % identity and % similarity for amino acid sequences using a GAP Weight of 8 and a Length Weight of 2, and the BLOSUM62 scoring matrix. "Equivalent programs" include any sequence comparison programs that will generate alignments and percentage sequence identity for any two sequences of interest that have identical nucleotide or amino acid residue matches when compared to a corresponding alignment generated by GAP Version 10.

[0054] The term "conservative amino acid substitution" refers to the substitution of an amino acid normally present in a sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue, such as isoleucine, valine, or leucine, for another non-polar residue. Similarly, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another, such as arginine for lysine, glutamine for asparagine, or glycine for serine. Further examples of conservative substitutions include the substitution of a basic residue, such as lysine, arginine, or histidine, for another basic residue, or the substitution of one acidic residue, such as aspartic acid or glutamic acid, for another acidic residue. Examples of non-conservative substitutions include the substitution of a non-polar (hydrophobic) amino acid residue, such as isoleucine, valine, leucine, alanine, or methionine, for a polar (hydrophilic) residue, such as cysteine, glutamine, glutamic acid, or lysine, or the substitution of a polar residue for a non-polar residue. Common amino acid classes are summarized below. [Table A]

[0055] A "homologous" sequence (e.g., a nucleic acid sequence) includes a sequence that is either identical or substantially homologous to a known reference sequence, including, for example, a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a known reference sequence. Homologous sequences include, for example, orthologous and paralogous sequences. Homologous genes may include genes that have evolved from a common ancestral DNA sequence, for example, typically through speciation events (orthologous genes) or through gene duplication events (paralogous genes). "Orthologous" genes include genes from different species that evolved from a common ancestral gene through speciation. Orthologs typically retain the same function during evolution. "Paralogue" genes include genes that are related by intragenomic duplication. Paralogs may develop new functions during evolution.

[0056] The term "in vitro" includes an artificial environment and refers to processes or reactions that occur within an artificial environment (e.g., a test tube). The term "in vivo" includes a natural environment (e.g., a cell or organism or body) and refers to processes or reactions that occur within a natural environment. The term "ex vivo" includes cells removed from an individual's body and refers to processes or reactions that occur within those cells.

[0057] The term "reporter gene" refers to a nucleic acid having a sequence encoding a gene product (typically an enzyme) that is easily and quantitatively assayed when a construct containing the reporter gene sequence operably linked to a heterologous promoter and / or enhancer element is introduced into a cell that contains (or can be made to contain) the factors necessary for activation of the promoter and / or enhancer element. Examples of reporter genes include, but are not limited to, the gene encoding β-galactosidase (lacZ), the bacterial chloramphenicol acetyltransferase (cat) gene, the firefly luciferase gene, the gene encoding beta-glucuronidase (GUS), and genes encoding fluorescent proteins. "Reporter protein" refers to the protein encoded by the reporter gene.

[0058] The term "fluorescent reporter protein," as used herein, refers to a reporter protein that is detectable based on fluorescence, where the fluorescence may be derived directly from the reporter protein, from the activity of the reporter protein on a fluorogenic substrate, or from the protein having binding affinity for a fluorescently tagged compound. Fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, eGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, and ZsGreenl), yellow fluorescent proteins (e.g., YFP, eYFP, Citrine, Venus, YPet, PhiYFP, and ZsYellowl), blue fluorescent proteins (e.g., BFP, eBFP, eBFP2, Azurite, mKalamal, GFPuv, Sapphire, and T-sapphire), cyan fluorescent proteins (e.g., CFP, eCFP, Cerulean, CyPet, AmCyanl, and Mido). Examples of suitable fluorescent proteins include RFP, mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611, mRaspberry, mStrawberry, and Jred), orange fluorescent proteins (e.g., mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, and tdTomato), and any other suitable fluorescent proteins whose presence in cells can be detected by flow cytometry.

[0059] The term "recombination" includes any process of exchange of genetic information between two polynucleotides and can occur by any mechanism. Recombination in response to double-strand breaks (DSBs) primarily involves two conservative DNA repair processes: Repair pathway: occurs via non-homologous end joining (NHEJ) and homologous recombination (HR). See Kasparek & Humphrey (2011) Seminars in Cell & Dev. Biol. 22:886-897, which is incorporated by reference in its entirety for all purposes. Similarly, repair of a target nucleic acid mediated by an exogenous donor nucleic acid can include any process of exchange of genetic information between two polynucleotides.

[0060] NHEJ involves the repair of double-strand breaks in nucleic acids by direct ligation of the broken ends to each other or to an exogenous sequence, without the need for a homologous template. Ligation of non-contiguous sequences by NHEJ often results in deletions, insertions, or translocations near the double-strand break. For example, NHEJ can also result in targeted integration of an exogenous donor nucleic acid through direct ligation of the broken ends to the exogenous donor nucleic acid (i.e., NHEJ-based incorporation). Such NHEJ-mediated targeted integration may be preferable for the insertion of exogenous donor nucleic acids in cases where the homology-directed repair (HDR) pathway is not readily available (e.g., non-dividing cells, primary cells, and cells where homology-based DNA repair is rarely performed). Additionally, in contrast to homology-directed repair, knowledge of large regions of sequence identity adjacent to the cleavage site is not required, which may be beneficial when attempting targeted insertion into organisms with genomes with limited knowledge of the genome sequence. Integration can proceed via blunt-end ligation between the exogenous donor nucleic acid and the cleaved genomic sequence, or via ligation of cohesive ends (i.e., ends with 5' or 3' overhangs) using an exogenous donor nucleic acid flanked by overhangs compatible with the ends generated by the nuclease agent in the cleaved genomic sequence. See, for example, US2011 / 020722, WO2014 / 033644, WO2014 / 089290, and Maresca et al. (2013) Genome Res. 23(3):539-546, each of which is incorporated herein by reference in its entirety for all purposes. If blunt ends are ligated, excision of the target and / or donor may be required to generate regions of microhomology necessary for fragment joining, which may result in undesirable changes in the target sequence.

[0061] Recombination can also occur through homology-guided repair (HDR) or homologous recombination (HR). HDR or HR involves a form of nucleic acid repair that may require nucleotide sequence homology, using a "donor" molecule as a template for repair of a "target" molecule (i.e., a molecule that has undergone a double-strand break), inducing the transfer of genetic information from the donor to the target. Without wishing to be bound by any particular theory, such transfer can involve mismatch repair of heteroduplex DNA formed between the broken target and the donor and / or synthesis-dependent strand annealing and / or related processes, in which the donor is used to resynthesize genetic information, which becomes part of the target. In some instances, the donor polynucleotide, a portion of the donor polynucleotide, a copy of the donor polynucleotide, or a portion of a copy of the donor polynucleotide is integrated into the target DNA. See Wang et al. (2013) Cell 153:910-918; Mandalos et al. (2012) PLOS ONE 7:e45768:1-9; and Wang et al. (2013) Nat Biotechnol. 31:530-532, each of which is incorporated by reference in its entirety and for all purposes.

[0062] The term "antigen-binding protein" includes any protein that binds to an antigen. Examples of antigen-binding proteins include antibodies, antigen-binding fragments of antibodies, multispecific antibodies (e.g., bispecific antibodies), scFV, bis-scFV, diabodies, triabodies, tetrabodies, V-NAR, VHH, VL, F(ab), F(ab)2, DVD (double variable domain), and the like. Examples of suitable antibodies include bispecific T-cell engagers (BiTEs), single variable domain antigen binding proteins (SVDs), bispecific T-cell engagers (BiTEs), or Davisbodies (U.S. Patent No. 8,586,713, herein incorporated by reference in its entirety).

[0063] The term "multispecific" or "bispecific" in reference to an antigen-binding protein means that the protein recognizes different epitopes, either on the same antigen or on different antigens. A multispecific antigen-binding protein can be a single multifunctional polypeptide or a multimeric complex of two or more polypeptides covalently or noncovalently associated with each other. For example, an antibody or fragment thereof can be operatively linked (e.g., by chemical coupling, genetic fusion, noncovalent association, or otherwise) to one or more other molecular entities, such as proteins or fragments thereof, to produce a bispecific or multispecific antigen-binding molecule having a second binding specificity.

[0064] The term "antigen" refers to any substance, either a whole molecule or a domain within a molecule, that is capable of eliciting the production of antibodies with binding specificity to that substance. The term antigen also includes substances that do not elicit antibody production in wild-type host organisms due to self-recognition, but that can elicit such a response in host animals with appropriate genetic manipulations that disrupt immune tolerance.

[0065] The term "epitope" refers to a site on an antigen to which an antigen-binding protein (e.g., an antibody) binds. Epitopes can be formed from contiguous amino acids or from noncontiguous amino acids juxtaposed by tertiary folding of one or more proteins. Epitopes formed from contiguous amino acids (also known as linear epitopes) typically are retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding (also known as conformational epitopes) are typically lost upon treatment with denaturing solvents. Typically, an epitope contains at least three, more usually at least five, or 8-10 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of an epitope include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, "Epitope Mapping Protocols," in "Methods in Molecular Biology," Vol. 66, Glenn E. Morris, Ed. (1996), which is incorporated herein by reference in its entirety and for all purposes.

[0066] The antibody paratopes described herein generally comprise, at a minimum, the complementarity determining regions (CDRs) that specifically recognize the heterologous epitope (eg, the CDR3 regions of the heavy and / or light chain variable domains).

[0067] The term "antibody" includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable domain and a heavy chain constant region (C H The heavy chain constant region comprises C H 1. C H 2, and C H Each light chain contains three domains: a light chain variable domain and a light chain constant region (C L). The heavy and light chain variable domains can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each heavy and light chain variable domain contains three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs may also be abbreviated as HCDR1, HCDR2, and HCDR3, and light chain CDRs may also be abbreviated as LCDR1, LCDR2, and LCDR3). The term "high affinity" antibody refers to an antibody that has approximately 10 -9 M or less (for example, about 1 × 10 -9 M, 1 x 10 -10 M, 1 x 10 -11 M, or approximately 1 x 10 -12 M)'s K D In one embodiment, K D is measured by surface plasmon resonance, e.g., BIACORE™, and in another embodiment, K D is measured by ELISA.

[0068] The term "bispecific antibody" includes antibodies that can selectively bind two or more epitopes. Bispecific antibodies typically contain two different heavy chains, each of which specifically binds to a different epitope, i.e., either on two different molecules (e.g., two different antigens) or on the same molecule (e.g., the same antigen). When a bispecific antibody can selectively bind two different epitopes (a first epitope and a second epitope), the affinity of the first heavy chain for the first epitope is generally at least one to two, or even three or four orders of magnitude lower than the affinity of the first heavy chain for the second epitope, or vice versa. The epitopes recognized by a bispecific antibody can be on the same target or on different targets (e.g., on the same protein or on different proteins). Bispecific antibodies can be generated, for example, by combining heavy chains that recognize different epitopes of the same antigen. For example, nucleic acid sequences encoding heavy chain variable sequences that recognize different epitopes of the same antigen can be fused to nucleic acid sequences encoding different heavy chain constant regions, and these sequences can be expressed in cells that express immunoglobulin light chains. A typical bispecific antibody has two heavy chains, each having three heavy chain CDRs followed (N- to C-terminus) by a CH1 domain, a hinge, a CH2 domain, and a CH3 domain, and an immunoglobulin light chain that does not confer antigen-binding specificity but is capable of associating with each heavy chain, or is capable of associating with each heavy chain and binding one or more epitopes bound by the heavy chain antigen-binding region, or is capable of associating with each heavy chain and enabling one or both heavy chains to bind one or both epitopes.

[0069] The term "heavy chain" or "immunoglobulin heavy chain" includes immunoglobulin heavy chain sequences (such as immunoglobulin heavy chain constant region sequences) from any organism. Unless otherwise specified, a heavy chain variable domain contains three heavy chain CDRs and four FR regions. Fragments of heavy chains include CDRs, CDRs and FRs, and combinations thereof. A typical heavy chain contains (from N- to C-terminus) the variable domain followed by a CH 1 domain, hinge, C H 2 domain, and C H A functional fragment of a heavy chain may have three domains that specifically recognize an epitope (e.g., a K D The heavy chain variable domain is encoded by a variable region nucleotide sequence and generally corresponds to the V present in germline cells. H , D H , and J. H From the segment repertoire, V H , D H , and J. H The sequences, locations, and nomenclature of V, D, and J heavy chain segments from various organisms can be found in the IMGT database, which is accessible via the Internet on the World Wide Web (www) at the URL "imgt.org".

[0070] The term "light chain" includes immunoglobulin light chain sequences from any organism, and includes human kappa (κ) and lambda (λ) light chains, as well as surrogate light chains, and VpreB, unless otherwise specified. A light chain variable domain typically includes three light chain CDRs and four framework (FR) regions, unless otherwise specified. Generally, a full-length light chain includes, from the amino terminus to the carboxyl terminus, a variable domain including FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and a light chain constant region amino acid sequence. The light chain variable domain is encoded by a light chain variable region nucleotide sequence and generally includes a light chain V and J gene segment derived from a repertoire of light chain V and J gene segments present in germline cells. L and light chain J LLight chains include light chain V gene segments and light chain J gene segments. The sequences, locations, and nomenclature of light chain V gene segments and light chain J gene segments of various organisms can be found in the IMGT database, which is accessible via the Internet on the World Wide Web (www) at URL "imgt.org." Light chains include, for example, light chains that do not selectively bind either a first epitope or a second epitope that selectively binds to an epitope-binding protein in which the light chain is included. Light chains also include light chains that bind and recognize or assist heavy chains while binding and recognizing one or more epitopes that selectively bind to an epitope-binding protein in which the light chain is included.

[0071] As used herein, the term "complementarity-determining region" or "CDR" includes an amino acid sequence encoded by the nucleic acid sequence of an organism's immunoglobulin gene, which amino acid sequence is normally (i.e., in wild-type animals) located between two framework regions in the variable region of the light or heavy chain of an immunoglobulin molecule (e.g., an antibody or T cell receptor). CDRs can be encoded, for example, by germline sequences or rearranged sequences, e.g., by naive or mature B cells or T cells. CDRs can be somatically mutated (e.g., different from the sequence encoded in the animal germline), humanized, and / or modified with amino acid substitutions, additions, or deletions. In some situations (e.g., for CDR3), a CDR can be encoded by two or more sequences (e.g., germline sequences), which are not contiguous (e.g., in an unrearranged nucleic acid sequence) but are contiguous in the nucleic acid sequence of a B cell, e.g., as a result of splicing or joining of sequences (e.g., VDJ rearrangement to form a heavy chain CDR3).

[0072] The specific binding of the antigen-binding protein to the target antigen is at least 10 6 , 10 7 , 10 8 , 10 9 , or 10 10 M -1Specific binding includes binding with an affinity of at least one target. Specific binding is more detectable and distinguishable from nonspecific binding that occurs to at least one unrelated target. Specific binding can be the result of bonds formed between specific functional groups or specific spatial fits (e.g., lock and key types), while nonspecific binding is usually the result of van der Waals forces. However, specific binding does not necessarily mean that an antigen-binding protein binds to only one target.

[0073] A composition or method that "comprises" or "contains" one or more recited elements may include other elements not specifically recited. For example, a composition that "comprises" or "contains" a protein may contain that protein alone or in combination with other components. The transitional phrase "consisting essentially of" means that the claim shall be construed to include the specific elements recited in that claim and elements that do not materially affect the basic and novel characteristics of the claimed invention. Thus, when used in the claims of the present invention, the term "consisting essentially of" is not intended to be construed as the equivalent of "comprising."

[0074] "Optional" or "optionally" means that the described event or circumstance may or may not occur, and that the description includes examples where the said event or circumstance occurs and examples where it does not occur.

[0075] Designations of ranges of values ​​include every integer within the range or all integers defining the range, and all sub-ranges defined by the integers within the range.

[0076] Unless otherwise clear from the context, the term "about" encompasses values ​​within the standard error of measurement error (eg, SEM) of the specified value.

[0077] The term "and / or" means the inclusion of one or more of the associated listed items. It refers to and encompasses any and all possible combinations, as well as the absence of a combination when interpreted alternatively ("or").

[0078] The term "or" refers to any one member of a particular list and also includes any combination of members of that list.

[0079] The singular forms of articles "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a protein" or "at least one protein" can include a plurality of proteins, including mixtures thereof.

[0080] Statistically significant means p≦0.05.

[0081] I. Overview Disclosed herein are non-human animal cells and animals containing a humanized Asgr1 locus, as well as methods for using such non-human animal cells and animals. Non-human animal cells or animals containing a humanized Asgr1 locus express a human ASGR1 protein or a chimeric Asgr1 protein containing one or more fragments of the human ASGR1 protein (e.g., all or part of the human ASGR1 extracellular domain). As a liver-specific receptor with highly efficient endocytosis, human ASGR1 can be used for liver-specific delivery of therapeutic agents, such as antibodies, small molecules (as part of antibody-drug conjugates), and DNA. However, antigen-binding proteins or bispecific antigen-binding proteins that specifically bind to human ASGR1 often do not bind to orthologous non-human animal Asgr1 proteins, such as mouse Asgr1, due to sequence differences between human ASGR1 and non-human animal Asgr1. For example, antibodies raised against the human ASGR1 extracellular domain do not bind to mouse Asgr1 orthologs (data not shown). Because of this, the in vivo efficacy of human ASGR1-mediated delivery or therapeutic mechanisms cannot be efficiently evaluated in wild-type non-human animals with an unmodified, endogenous (i.e., native) Asgr1 locus. Humanized Asgr1 non-human animals (e.g., humanized Asgr1 mice) can be used to validate liver-specific delivery of various therapeutic agents via human ASGR1-mediated internalization using a number of different methods. For example, non-human animals containing a humanized Asgr1 locus can be used to evaluate the in vivo efficacy of human ASGR1-mediated delivery of therapeutic molecules or therapeutic complexes to the liver. Similarly, non-human animals containing a humanized Asgr1 locus can be used to evaluate the efficacy of therapeutic molecules or therapeutic complexes that act via human ASGR1-mediated mechanisms.

[0082] II. Non-human animals containing a humanized Asgr1 locus The cells and non-human animals disclosed herein contain a humanized Asgr1 locus. The cells or non-human animals containing the humanized Asgr1 locus express either a human ASGR1 protein or a partially humanized chimeric Asgr1 protein in which one or more fragments of a native Asgr1 protein have been replaced with corresponding fragments from human ASGR1 (e.g., all or part of the extracellular domain).

[0083] A. Asialoglycoprotein receptor 1 (ASGR1) The cells and non-human animals described herein contain a humanized Asgr1 locus. Asialoglycoprotein receptor 1 (C-type lectin domain family 4 member H1, hepatic lectin H1, HL-1, ASGP-R 1, ASGPR 1, ASGR1) is the major subunit of the asialoglycoprotein receptor (ASGPR or ASGR) encoded by the ASGR1 (CLEC4H1) gene. ASGPR is a hetero-oligomeric protein expressed primarily on the cell surface of hepatocytes, and is present in approximately 1-5 x 10 5 binding sites / cell and binds desialylated glycoproteins ASGPR is a well-characterized C-type hepatic lectin that is primarily expressed on the sinusoidal surface of hepatocytes. It plays a role in the selective binding and internalization of galactose- and N-acetylgalactosamine-terminated glycoproteins via receptor-mediated endocytosis by liver parenchymal cells. It contains two proteins, asialoglycoprotein receptor 1 and 2 (ASGR1 and ASGR2), encoded by the ASGR1 and ASGR2 genes. Both subunits are type II single-pass proteins that broadly contain an N-terminal cytoplasmic domain, a single transmembrane domain, and a C-terminal extracellular carbohydrate-recognition domain (CRD). ASGR1 contains an N-terminal cytoplasmic domain (approximately 40 amino acids), a single-pass transmembrane domain (approximately 20 amino acids), an extracellular coiled-coil (oligomerization) region (approximately 80 amino acids), and a functional C-type (calcium-dependent) carbohydrate-recognition domain (C-type lectin domain) (approximately 140 amino acids). CRDs bind to glycoproteins with terminal galactose or N-acetylgalactosamine (GalNac) motifs and have low affinity for desialylated glycoproteins in their monomeric state.

[0084] The genes encoding ASGR1 and ASGR2 (ASGR1 and ASGR2, respectively) are located approximately 58.6 kilobases (kb) apart on the short arm of chromosome 17. Although these genes are evolutionarily related, their structural organization is quite different. ASGR1 contains eight exons and is approximately 6 kb long. ASGR2 contains nine exons and is approximately 13.5 kb long.

[0085] An exemplary coding sequence for human ASGR1 has been assigned NCBI accession number NM_001671 (SEQ ID NO: 5). An exemplary coding sequence for mouse Asgr1 has been assigned NCBI accession number NM_009714 (SEQ ID NO: 4). An exemplary human ASGR1 protein has been assigned UniProt accession number P07306 (SEQ ID NO: 1). An exemplary mouse Asgr1 protein has been assigned UniProt accession number P34927 (SEQ ID NO: 2). A mouse Asgr1 protein having a humanized coiled-coil domain and a C-type lectin domain is set forth in SEQ ID NO: 3. An exemplary rat Asgr1 protein has been assigned UniProt accession number P02706. An exemplary orangutan Asgr1 protein has been assigned UniProt accession number Q5RBQ8.

[0086] B. Humanized Asgr1 locus The humanized Asgr1 locus may be an Asgr1 locus in which the entire Asgr1 gene has been replaced with the corresponding orthologous human ASGR1 sequence, or an Asgr1 locus in which a portion of the Asgr1 gene has been replaced with the corresponding orthologous human ASGR1 sequence (i.e., humanized). Optionally, the corresponding orthologous human ASGR1 sequence is modified to optimize codons based on codon usage in the non-human animal. The replaced (i.e., humanized) region can include, for example, an exon, a non-coding region such as an intron, an untranslated region, or a regulatory region (e.g., a promoter, enhancer, or transcriptional repressor-binding element), or any combination thereof. In one example, exons corresponding to one, two, three, four, five, six, seven, or all eight exons of the human ASGR1 gene can be humanized. For example, exons corresponding to exons 3 to 8 of the human ASGR1 gene can be humanized. Alternatively, the region of Asgr1 encoding the epitope recognized by the anti-human ASGR1 antigen-binding protein can be humanized. As another example, one or more or all of the N-terminal cytoplasmic domain, the transmembrane domain, the coiled-coil domain, or the C-type lectin domain can be humanized. For example, all or part of the region of the Asgr1 locus encoding the coiled-coil domain can be humanized. Alternatively, all or a portion of the region of the Asgr1 locus encoding the C-terminal lectin domain can be humanized, all or a portion of the region of the Asgr1 locus encoding the transmembrane domain can be humanized, and / or all or a portion of the region of the Asgr1 locus encoding the cytoplasmic domain can be humanized. In one example, only all or a portion of the region of the Asgr1 locus encoding the coiled-coil domain can be humanized, only all or a portion of the region of the Asgr1 locus encoding the C-type lectin domain can be humanized, or only all or a portion of the region of the Asgr1 locus encoding the extracellular domain (i.e., the coiled-coil domain and the C-type lectin domain) can be humanized. For example, the regions of the Asgr1 locus encoding the coiled-coil domain and the C-type lectin domain can be humanized, thereby producing a chimeric Asgr1 protein having an endogenous N-terminal cytoplasmic domain, an endogenous transmembrane domain, a humanized coiled-coil domain, and a humanized C-type lectin domain. Similarly, introns corresponding to one, two, three, four, five, six, or all seven introns of the human ASGR1 gene can be humanized. The adjacent untranslated regions, including regulatory sequences, can also be humanized. For example, the 5' untranslated region (UTR), the 3' UTR, or both the 5' UTR and the 3' UTR can be humanized, or the 5' UTR, the 3' UTR, or both the 5' UTR and the 3' UTR can remain endogenous. In one specific example, the 3' UTR is humanized, while the 5' UTR remains endogenous. Depending on the degree of replacement with orthologous sequences, regulatory sequences, such as promoters, can be endogenous or can be provided by substituted human orthologous sequences. For example, a humanized Asgr1 locus can include an endogenous non-human animal Asgr1 promoter.

[0087] The Asgr1 protein encoded by the humanized Asgr1 locus may contain one or more domains derived from the human ASGR1 protein. For example, the Asgr1 protein may contain one or more, or all, of the human ASGR1 coiled-coil domain, the human ASGR1 C-type lectin domain, the human ASGR1 transmembrane domain, and the human ASGR1 cytoplasmic domain. For example, the Asgr1 protein may contain only the human ASGR1 coiled-coil domain, only the human ASGR1 C-type lectin domain, or only the human ASGR1 extracellular domain (i.e., the coiled-coil domain and the C-type lectin domain). Optionally, the Asgr1 protein encoded by the humanized Asgr1 locus may further contain one or more domains derived from an endogenous (i.e., naturally occurring) non-human animal Asgr1 protein. For example, the Asgr1 protein encoded by the humanized Asgr1 locus may include a coiled-coil domain derived from the human ASGR1 protein, a C-type lectin domain derived from the human ASGR1 protein, an N-terminal cytoplasmic domain derived from an endogenous (i.e., native) non-human animal Asgr1 protein, and a transmembrane domain derived from an endogenous (i.e., native) non-human animal Asgr1 protein. The domain derived from the human ASGR1 protein may be encoded by a fully humanized sequence (i.e., the entire sequence encoding the domain is replaced with an orthologous human ASGR1 sequence) or a partially humanized sequence (i.e., a portion of the sequence encoding the domain is replaced with an orthologous human ASGR1 sequence, and the remaining endogenous (i.e., native) sequence encoding the domain encodes the same amino acids as the orthologous human ASGR1 sequence, thereby making the encoded domain identical to that in the human ASGR1 protein).

[0088] As an example, the Asgr1 protein encoded by the humanized Asgr1 locus may comprise a coiled-coil domain of human ASGR1. Optionally, the coiled-coil domain of human ASGR1 comprises, consists essentially of, or consists of a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 27, and the Asgr1 protein maintains the activity of native Asgr1 (i.e., galactose-terminated and N-terminally cleaved via receptor-mediated endocytosis). (The Asgr1 protein encoded by the humanized Asgr1 locus retains the ability to selectively bind to and internalize acetylgalactosamine-terminal glycoproteins.) As another example, the Asgr1 protein encoded by the humanized Asgr1 locus may contain the C-type lectin domain of human ASGR1. Optionally, the C-type lectin domain of human ASGR1 comprises, consists essentially of, or consists of a sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:28, and the Asgr1 protein retains the activity of native Asgr1. For example, a region of the Asgr1 protein derived from human ASGR1 may comprise, consist essentially of, or consist of a sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:31, and the Asgr1 protein retains the activity of native Asgr1. As another example, the Asgr1 protein encoded by the humanized Asgr1 locus may comprise an endogenous non-human animal Asgr1 cytoplasmic domain (e.g., a mouse Asgr1 cytoplasmic domain). Optionally, the cytoplasmic domain of the non-human animal Asgr1 comprises, consists essentially of, or consists of a sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 29, and the Asgr1 protein maintains the activity of native Asgr1. As another example, the Asgr1 protein encoded by the humanized Asgr1 locus may comprise an endogenous non-human animal Asgr1 transmembrane domain (e.g., a mouse Asgr1 transmembrane domain). Optionally, the transmembrane domain of the non-human animal Asgr1 comprises, consists essentially of, or consists of a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 30, and the Asgr1 protein maintains the activity of native Asgr1.For example, the Asgr1 protein encoded by the humanized Asgr1 locus may comprise, consist essentially of, or consist of a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 3, and the Asgr1 protein maintains the activity of native Asgr1.

[0089] Optionally, the humanized Asgr1 locus may contain other elements. Examples of such elements include a selection cassette, a reporter gene, a recombinase recognition site, or other elements. Alternatively, the humanized Asgr1 locus may lack other elements (e.g., it may lack a selection marker or selection cassette). Examples of suitable reporter genes and reporter proteins are disclosed elsewhere herein. Examples of suitable selection markers include neomycin phosphotransferase (neomycin phosphotransferase). r ), hygromycin B phosphotransferase (hyg r ), puromycin-N-acetyltransferase (puro r ), blasticidin S deaminase (bsr r ), xanthine / guanine phosphoribosyltransferase (gpt), or herpes simplex virus thymidine kinase (HSV-k). Examples of recombinases include Cre, Flp, and Dre recombinase. An example of a Cre recombinase gene is Crei, in which the two exons encoding Cre recombinase are separated by an intron, preventing expression in prokaryotic cells. Such recombinases may further contain a nuclear localization signal (e.g., NLS-Crei) to promote nuclear localization. Recombinase recognition sites contain nucleotide sequences recognized by site-specific recombinases and can serve as substrates for recombination events. Examples of recombinase recognition sites include FRT, FRT11, FRT71, attp, att, rox, and lox sites, such as loXp, lox511, lox2272, lox66, lox71, loxM2, and lox5171.

[0090] Other elements, such as reporter genes or selection cassettes, may be auto-excision cassettes flanked by recombinase recognition sites. See, e.g., US8,697,851 and US2013 / 0312129, each of which is incorporated herein by reference in its entirety. The auto-excision cassette is incorporated herein for purposes of illustration. As an example, the auto-excision cassette contains a Crei gene (comprising two exons encoding Cre recombinase, separated by an intron) operably linked to the mouse Prm1 promoter and a neomycin resistance gene operably linked to the human ubiquitin promoter. By employing the Prm1 promoter, the auto-excision cassette can be specifically deleted in the male germ cells of F0 animals. The polynucleotide encoding the selection marker may be operably linked to a promoter active in the target cells. Examples of promoters are described elsewhere herein. As another specific example, the auto-excision selection cassette may contain a hygromycin resistance gene coding sequence operably linked to one or more promoters (e.g., both the human ubiquitin promoter and the EM7 promoter), followed by a polyadenylation signal, followed by a Crei coding sequence operably linked to one or more promoters (e.g., the mPrm1 promoter), followed by another polyadenylation signal, in which case the entire cassette is flanked by loxP sites.

[0091] The humanized Asgr1 locus may be a conditional allele. For example, the conditional allele may be a multifunctional allele as described in US2011 / 0104799, which is incorporated herein by reference in its entirety for all purposes. For example, the conditional allele may include (a) an actuating sequence in the sense orientation relative to transcription of the target gene; (b) a drug selection cassette (DSC) in the sense or antisense orientation; (c) a nucleotide sequence of interest (NSI) in the antisense orientation; and (d) a conditional inversion module (utilizing a COIN, exon-splitting intron, and reversible gene trap-like module) in the reverse orientation. See, for example, US2011 / 0104799. The conditional allele may further include a recombinable unit that recombines upon exposure to a first recombinase to form a conditional allele that (i) lacks the actuating sequence and DSC, and (ii) includes an NSI in the sense orientation and a COIN in the antisense orientation. See, for example, US2011 / 0104799.

[0092] One exemplary humanized Asgr1 locus (e.g., a humanized mouse Asgr1 locus) is one in which coding exons 3-8 are replaced with the corresponding human sequences. These exons encode the coiled-coil domain and C-type lectin domain of Asgr1. Optionally, the humanized sequence may extend past the stop codon to the 3' UTR and optionally into the sequence immediately downstream of the 3' UTR. Optionally, the intron portion upstream of coding exon 3 is also humanized. See Figures 2A and 2B and SEQ ID NOs: 21 and 24.

[0093] C. Non-human cells and animals containing a humanized Asgr1 locus Non-human animal cells and animals are provided that contain the humanized Asgr1 locus described elsewhere herein. The cells and non-human animals may be heterozygous or homozygous for the humanized Asgr1 locus. Diploid organisms have two alleles at each locus. Each pair of alleles represents a genotype at a particular locus. A genotype is described as homozygous when there are two identical alleles at a particular locus, and heterozygous when the two alleles are different.

[0094] The non-human animal cells provided herein may be, for example, any non-human cell that contains an Asgr1 locus or genomic locus homologous or orthologous to the human ASGR1 locus. The cell may be, for example, a eukaryotic cell, including a fungal cell (e.g., yeast), a plant cell, an animal cell, a mammalian cell, a non-human mammalian cell, and a human cell. The term "animal" includes mammals, fish, and birds. The mammalian cell may be, for example, a non-human mammalian cell, a rodent cell, a rat cell, a mouse cell, or a hamster cell. Other non-human mammals include, for example, non-human primates such as monkeys, apes, orangutans, Examples of animals include cats, dogs, rabbits, horses, bulls, deer, bison, and livestock (e.g., bovine species such as cows and steers; ovine species such as sheep and goats; and porcine species such as pigs and boars). Examples of birds include chickens, turkeys, ostriches, geese, ducks, and the like. Domesticated animals and agricultural animals are also included. The term "non-human" excludes humans.

[0095] Cells may also be in any type of undifferentiated or differentiated state. For example, cells may be totipotent cells, pluripotent cells (e.g., human pluripotent cells or non-human pluripotent cells such as mouse embryonic stem (ES) cells or rat ES cells), or non-pluripotent cells. Totipotent cells include undifferentiated cells that can give rise to any cell type, while pluripotent cells include undifferentiated cells that retain the ability to develop into multiple differentiated cell types. Such pluripotent and / or totipotent cells may be, for example, ES cells or ES-like cells, such as induced pluripotent stem (iPS) cells. ES cells include embryonically derived totipotent or pluripotent cells that, when introduced into an embryo, can contribute to any tissue of the developing embryo. ES cells can be derived from the inner cell mass of a blastocyst and can differentiate into cells of any of the three vertebrate germ layers (endoderm, ectoderm, and mesoderm).

[0096] The cells provided herein may be germ cells (e.g., sperm or oocytes). The cells may be mitotically competent or mitotically inactive, meiotically competent or meiotically inactive. Similarly, the cells may be primary somatic cells or non-primary somatic cells. Somatic cells include any cells that are not gametes, germ cells, mother cells, or undifferentiated stem cells. For example, the cells may be liver cells, such as hepatoblasts or hepatocytes.

[0097] Suitable cells provided herein also include primary cells. Primary cells include cells or cell cultures directly isolated from organisms, organs or tissues. Primary cells include cells that are neither transformed nor immortalized. Primary cells include any cells obtained from organisms, organs or tissues that have not previously been subcultured in tissue culture, or have previously been subcultured in tissue culture but cannot be subcultured indefinitely in tissue culture. Such cells can be isolated by conventional techniques and include, for example, hepatocytes.

[0098] Other suitable cells provided herein include immortalized cells.Immortalized cells include cells derived from multicellular organisms that do not normally proliferate indefinitely, but can avoid normal cell aging through mutation or change, and instead continue to divide.Such mutation or change can occur naturally or be intentionally induced.A specific example of an immortalized cell line is the HepG2 human liver cancer cell line.Various types of immortalized cells are known.Immortalized cells or primary cells typically include cells used for cell culture or cells used for expressing recombinant genes or recombinant proteins.

[0099] The cells provided herein also include one-cell stage embryos (i.e., fertilized oocytes or eggs). Such one-cell stage embryos may be from any genetic background (e.g., for mice, BALB / c, C57BL / 6, 129, or combinations thereof), may be fresh or frozen, and may be derived from natural mating or in vitro fertilization.

[0100] The cells provided herein can be normal, healthy cells, or can be diseased or mutated cells.

[0101] Non-human animals comprising the humanized Asgr1 locus described herein can be produced by methods described elsewhere herein. The term "animal" includes mammals, fish, and birds. Non-human mammals include, for example, non-human primates such as monkeys, apes, orangutans, cats, dogs, horses, bulls, deer, bison, sheep, rabbits, rodents (e.g., mice, rats, hamsters, and guinea pigs), and livestock (e.g., bovine species such as cows and steers; ovine species such as sheep and goats; and porcine species such as pigs and boars). Birds include, for example, chickens, turkeys, ostriches, geese, and ducks. Domesticated animals and agricultural animals are also included. The term "non-human animal" excludes humans. Preferred non-human animals include rodents such as mice and rats.

[0102] Non-human animals may be from any genetic background. For example, suitable mice may be from the 129 strain, C57BL / 6 strain, a 129 / C57BL / 6 hybrid, a BALB / c strain, or a Swiss Webster strain. Examples of 129 strains include 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / Svlm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, and 129T2. See, e.g., Festing et al. (1999) Mammalian Genome 10:836, which is incorporated herein by reference in its entirety for all purposes. Examples of C57BL strains include C57BL / A, C57BL / An, C57BL / GrFa, C57BL / Kal_wN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr, and C57BL / Ola. Suitable mice may be derived from a hybrid of the aforementioned 129 strain and the aforementioned C57BL / 6 strain (e.g., 50% 129 and 50% C57BL / 6). Similarly, suitable mice may be derived from a hybrid of the aforementioned 129 strain and the aforementioned BL / 6 strain (e.g., 129S6 (129 / SvEvTac) strain).

[0103] Similarly, rats may be from any rat strain, including, for example, the ACI rat strain, the Dark Agouti (DA) rat strain, the Wistar rat strain, the LEA rat strain, the Sprague Dawley (SD) rat strain, or a Fischer rat strain, such as Fisher F344 or Fisher F6. Rats may also be obtained from strains derived from crossbreeding two or more of the above strains. For example, suitable rats may be from the DA strain or the ACI strain. The ACI rat strain has a black agouti color, a white belly and paws, and an RT1 av1The Dark Agouti (DA) rat strain has agouti coat color and is characterized by the RT1 haplotype. The strain is available from various sources, including Harlan Laboratories. av1 The rat is characterized by a haplotype. Such rats are available from various sources, including Charles River and Harlan Laboratories. Some suitable rats may be derived from inbred rat strains. See, for example, US2014 / 0235933, which is incorporated herein by reference in its entirety for all purposes.

[0104] III. Methods for using non-human animals containing a humanized Asgr1 locus to evaluate the in vivo efficacy of human ASGR1-mediated delivery of therapeutic complexes to the liver and to evaluate the action of therapeutic molecules via human ASGR1-mediated mechanisms. Various methods are provided for using non-human animals comprising a humanized Asgr1 locus, as described elsewhere herein, to evaluate the in vivo efficacy of therapeutic molecules or human ASGR1-mediated delivery of therapeutic molecules to the liver, and for evaluating human ASGR1-mediated mechanisms. This would more accurately reflect human ASGR1-mediated delivery or human ASGR1-mediated therapeutic mechanisms than non-human animals with a non-humanized Asgr1 locus. For example, the method can assess the delivery of a therapeutic complex to the liver via human ASGR1-mediated internalization in vivo, comprising administering a therapeutic complex to a non-human animal containing a humanized Asgr1 locus, the therapeutic complex comprising a therapeutic molecule and an antigen-binding protein or ligand that specifically binds to human ASGR1, and then assessing the delivery of the therapeutic molecule to the liver of the non-human animal. For another example, the method can assess the in vivo efficacy of a therapeutic molecule or therapeutic complex designed to act via human ASGR1-mediated internalization, e.g., a therapeutic complex designed to internalize a target liver cell surface protein via human ASGR1.

[0105] A. Methods for assessing the in vivo efficacy of delivery of therapeutic molecules to the liver via human ASGR1-mediated internalization Various methods are provided for using a non-human animal comprising a humanized Asgr1 locus, as described elsewhere herein, to evaluate the in vivo efficacy of delivering a therapeutic molecule to the liver via human ASGR1-mediated internalization. For example, such methods may include (a) administering a therapeutic complex to a non-human animal comprising a humanized Asgr1 locus, the therapeutic complex comprising a therapeutic molecule and an antigen-binding protein or ligand that specifically binds to human ASGR1, and (b) evaluating delivery of the therapeutic molecule to the liver of the non-human animal.

[0106] Therapeutic molecules can be any biological or chemical agent used in the treatment or prevention of any disease or disorder. For example, therapeutic molecules can be nucleic acids encoding therapeutic nucleic acids (e.g., CRISPR / Cas guide RNA, short hairpin RNA (shNA), or small interfering RNA (siRNA)) or therapeutic proteins (e.g., Cas proteins such as Cas9 proteins, replacement enzymes, secreted therapeutic proteins, etc.). Alternatively, therapeutic molecules can be therapeutic proteins, therapeutic antibodies or therapeutic antigen-binding proteins, or any other therapeutic macromolecules or small molecules.

[0107] The therapeutic molecule and the human ASGR1 antigen-binding protein or ligand may be conjugated together by any means. For example, the therapeutic molecule and the human ASGR1 antigen-binding protein or ligand may be linked via a direct covalent bond or via a linker, such as a peptide linker or a chemical linker. The therapeutic molecule and the human ASGR1 antigen-binding protein or ligand may be conjugated together by forming a bond between specific functional groups or a specific spatial fit (e.g., lock and key type). As a specific example, the human ASGR1 antigen-binding protein may be a bispecific antigen-binding protein that also specifically binds to the therapeutic molecule.

[0108] Administration of the therapeutic conjugate may be by any means and by any route of administration disclosed in detail elsewhere herein. Means of delivery and routes of administration of therapeutic conjugates and therapeutic molecules are disclosed in detail elsewhere herein.

[0109] Human ASGR1 antigen-binding proteins or human ASGR1 ligands that specifically bind to human ASGR1 may also be used. The human ASGR1 protein is a transmembrane protein that mediates the endocytosis of certain glycoproteins in the liver, and therefore, molecules complexed with human ASGR1 can be internalized along with human ASGR1. Examples of suitable antigen-binding proteins include receptor fusion molecules, trap molecules, receptor-Fc fusion molecules, antibodies, Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, single-chain Fv (scFv) molecules, dAb fragments, isolated complementarity-determining regions (CDRs), CDR3 peptides, constrained FR3-CDR3-FR4 peptides, domain-specific antibodies, single-domain antibodies, and domain-deleted antibodies. Examples of antigen-binding proteins include chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies, monovalent nanobodies, bivalent nanobodies, small modular immunopharmaceuticals (SMIPs), camelid antibodies (VHH heavy chain homodimeric antibodies), and shark variable IgNAR domains. In one specific example, the antigen-binding protein is a bispecific antibody that binds to human ASGR1 and a therapeutic molecule (e.g., a replacement protein or enzyme, or a delivery vehicle such as AAV). Alternatively, a ligand or ligand moiety that specifically interacts with human ASGR1 (e.g., asialoorosomucoid (ASOR) or Beta-GalNac, or a receptor-binding moiety for ASGR1) may be used.

[0110] Delivery of a therapeutic molecule to the liver of a non-human animal may be assessed by any known means. For example, the presence of the therapeutic molecule in the liver may be assessed. For example, if the therapeutic molecule is a therapeutic protein, the presence of the therapeutic protein in the liver of the non-human animal may be assessed using a known assay for protein detection. Similarly, if the therapeutic molecule is a nucleic acid encoding a therapeutic protein, known assays may be used to assess the expression of the nucleic acid (e.g., mRNA expression or protein expression) in the liver of the non-human animal. If the encoded therapeutic protein is a secreted protein, known assays may be used to measure the serum level of the therapeutic protein or to assess the activity of the secreted therapeutic protein in its intended target cell type, tissue type, or organ. The activity of a therapeutic molecule in the liver of a non-human animal may be assessed by known assays depending on the intended function of the therapeutic molecule. For example, if a genome editing agent, such as CRISPR / Cas, is introduced, known assays may be used to assess genome editing at specific target genome loci.

[0111] In certain examples, the therapeutic complex may comprise a viral vector composition (e.g., a liver-specific viral vector composition). Such viral vector compositions designed to target human ASGR1 may have their intrinsic tropism reduced or abolished in order to target the liver. For example, such a modified viral vector complex may comprise (i) a modified viral vector comprising a therapeutic nucleic acid or a nucleic acid encoding a therapeutic protein, where the modified viral vector has its intrinsic tropism abolished or abolished and comprises a heterologous epitope, and (ii) a retargeting moiety comprising: (1) a paratope of an antigen-binding protein (e.g., an antibody) that specifically binds to the heterologous epitope; and (2) a targeting ligand that specifically binds to human ASGR1.

[0112] In one example of how this can be achieved, a protein tagging system such as SpyCatcher-SpyTag can be used to covalently link antibodies to the viral surface. Alternatively, a bispecific antigen-binding protein can be used (e.g., a bispecific antibody in which one arm of the antibody binds to the virus and the other arm mediates binding to human ASGR1). In a particular example, the redirecting moiety is a bispecific antigen-binding protein (e.g., a bispecific antibody) comprising first and second antigen-binding domains, where the first antigen-binding domain comprises a paratope that specifically binds to a heterologous epitope inserted / displayed in a recombinant human viral capsid protein, and the second antigen-binding domain specifically binds to human ASGR1.

[0113] An example of a suitable heterologous epitope is a Myc tag. For example, a Myc tag may be inserted after N587 of AAV2. Such insertion disables the original ligand-binding activity of AAV2 and allows the modified AAV to be recognized by an anti-Myc antibody. Then, a bispecific antibody that specifically binds to both Myc and human ASGR1 can be used to retarget AAV (e.g., AAV2 N587 Myc) to hepatocytes expressing human ASGR1 (or humanized Asgr1).

[0114] In another example, the therapeutic conjugate comprises a lysosomal replacement enzyme or protein or a nucleic acid encoding a lysosomal replacement enzyme or protein. Lysosomal storage diseases are a group of rare disorders affecting the degradation of various substrates in lysosomes, including sphingolipids, mucopolysaccharides, glycoproteins, glycogen, and oligosaccharides, the accumulation of which in diseased cells can lead to cell death. Organs affected by lysosomal storage diseases include the liver. The etiology of these diseases is thought to be the accumulation of incomplete degradation products in lysosomes, usually due to loss of protein function. Lysosomal storage diseases are generally caused by loss-of-function or attenuated variants in proteins that normally degrade or regulate the degradation of lysosomal contents. Examples of lysosomal storage diseases are provided in WO2017 / 100467, which is incorporated herein by reference in its entirety for all purposes. For example, one of the most common lysosomal storage diseases is Pompe disease. Pompe disease is caused by a defective lysosomal enzyme, alpha-glucosidase (GAA), which results in insufficient processing of glycogen within the lysosomes. The accumulation of glycogen within the lysosomes occurs primarily in skeletal, cardiac, and hepatic tissues.

[0115] One option for treating lysosomal storage diseases is enzyme or protein replacement therapy. Replacement enzymes or proteins can be efficiently delivered to the lysosomes of specific target cells by associating them with a therapeutic conjugate containing an antigen-binding protein or ligand that specifically binds to human ASGR1. See WO2017 / 100467, which is incorporated herein by reference in its entirety for all purposes. Such a therapeutic conjugate can be administered to a subject, where it can enter the lysosomes of the subject's target cells and provide enzymatic activity to replace the enzymatic activity associated with lysosomal storage diseases. Human ASGR1 protein is a transmembrane protein that mediates the endocytosis and lysosomal degradation of glycoproteins with exposed terminal galactose or N-acetylgalactosamine residues in the liver. Therefore, proteins that bind to and are internalized with human ASGR1 are targeted to lysosomes. Therefore, therapeutic agents can be targeted to lysosomes (e.g., lysosomes in the liver) using delivery methods that target therapeutic molecules or therapeutic conjugates to human ASGR1.

[0116] Thus, some of the methods provided herein are methods for assessing the delivery of a therapeutic complex containing a lysosomal replacement protein or enzyme to the liver via human ASGR1 in vivo. For example, such methods may include: (a) administering a therapeutic molecule or therapeutic complex to a non-human animal as otherwise described herein, the therapeutic molecule or therapeutic complex comprising a lysosomal replacement protein or enzyme (or a nucleic acid encoding the lysosomal replacement protein or enzyme) and an antigen binding protein that specifically binds to human ASGR1, where ASGR1 mediates cell binding and uptake into the lysosomal compartment; and (b) assessing the presence or activity of the lysosomal replacement protein or enzyme in the liver of the non-human animal. The activity of the replaced protein or enzyme in the liver of the non-human animal may be assessed by known assays for the specific replaced protein or enzyme, or by measuring the degradation of various affected substrates in lysosomes using known assays.

[0117] As another example, the therapeutic molecule may be a nucleic acid encoding a therapeutic secreted protein. The liver plays a major role in the production of proteins secreted into the blood, including major plasma proteins, hemostatic and fibrinolytic factors, carrier proteins, hormones, prohormones, and apolipoproteins. The human ASGR1 protein is a transmembrane protein that mediates the endocytosis of specific glycoproteins in the liver. Therefore, molecules complexed with human ASGR1 are internalized together with human ASGR1. Thus, nucleic acids encoding therapeutic secreted proteins can be targeted to the liver by delivering the nucleic acid in a complex that targets human ASGR1. The nucleic acid may be internalized via ASGR1, and the liver may produce and secrete the therapeutic protein.

[0118] Thus, some of the methods provided herein are methods for assessing the delivery of a nucleic acid encoding a therapeutic secreted protein to the liver via human ASGR1 in vivo. For example, such methods may include (a) administering to a non-human animal as otherwise described herein a nucleic acid (e.g., DNA) encoding the therapeutic secreted protein, wherein the nucleic acid is delivered in a therapeutic complex that specifically binds to human ASGR1 and ASGR1 mediates internalization of the complex; and (b) assessing the secretion level (e.g., serum level) or activity of the therapeutic secreted protein in the non-human animal.

[0119] The production and secretion of a therapeutic protein may be evaluated by any known means. For example, the expression of the introduced nucleic acid may be evaluated by measuring the level of the encoded mRNA in the liver of a non-human animal, or by measuring the level of the encoded therapeutic protein in the liver of a non-human animal, using known assays. The secretion of a therapeutic protein may be evaluated by measuring the serum level of the encoded therapeutic protein in a non-human animal, using known assays. Furthermore, if the secreted therapeutic protein acts on a specific cell type, tissue, or organ, the activity of the secreted therapeutic protein may be evaluated in the target cell type, tissue, or organ.

[0120] B. Methods for assessing the in vivo efficacy of therapeutic conjugates for internalization of target liver cell surface proteins or target soluble proteins in the liver via human ASGR1-mediated internalization Various methods are provided for evaluating the in vivo efficacy of therapeutic conjugates designed to internalize target liver cell surface proteins or target soluble proteins in the liver via human ASGR1, using non-human animals containing the humanized Asgr1 locus described elsewhere herein. Therapeutic treatments often require the inactivation or blocking of one or more target molecules acting on or near cells. For example, antibody-based therapies often function by binding to specific antigens or soluble ligands expressed on the surface of cells, thereby interfering with the antigen's normal biological activity. This type of therapeutic typically works by blocking the interaction between cytokines and their receptors to attenuate or inhibit cell signaling. However, in certain contexts, it may be therapeutically beneficial to inactivate or inhibit the activity of a target molecule in a manner that does not necessarily involve blocking its physical interaction with another component. One way in which such non-blocking attenuation of a target molecule can be achieved is by reducing its concentration extracellularly or at the cell surface. For example, a target molecule can be attenuated or inactivated by promoting or implementing physical binding between the target molecule and an internalization effector protein, such as ASGR1. This can be achieved, for example, through the use of a multispecific (e.g., bispecific) antigen-binding molecule comprising a first antigen-binding domain and a second antigen-binding domain. Each antigen-binding domain binds to a different molecule. The first domain specifically binds to the target molecule, and the second domain specifically binds to ASGR1. Through this type of physical intermolecular binding, the target molecule can be internalized into the cell along with ASGR1 and processed by the cell's internal degradative machinery, or otherwise attenuated, sequestered, or inactivated. See, e.g., WO2013 / 138400 and US2013 / 0243775, each of which is incorporated herein by reference in its entirety for all purposes.

[0121] The human ASGR1 protein is a transmembrane protein that mediates the endocytosis and lysosomal degradation of glycoproteins with exposed terminal galactose or N-acetylgalactosamine residues in the liver. Therefore, cell surface proteins complexed with human ASGR1 are internalized along with human ASGR1, and target cell surface proteins or target soluble proteins are re-distributed to the degradation fraction, or the target cell surface proteins or target soluble proteins are sequestered in the internal fraction or exosomes.

[0122] Thus, provided herein are methods for evaluating the in vivo efficacy of a therapeutic complex designed to internalize a target liver cell surface protein or a target soluble protein in the liver via human ASGR1, using a non-human animal comprising a humanized Asgr1 locus, as described elsewhere herein. For example, such methods include (a) administering to a non-human animal, as described elsewhere herein, a therapeutic complex comprising a bispecific antigen-binding protein that specifically binds to a target cell surface protein or a target soluble protein and specifically binds to human ASGR1, where ASGR1 mediates internalization of the target cell surface protein or the target soluble protein, and (b) assessing the cell surface level or activity of the target liver cell surface protein in the liver of the non-human animal, or assessing the expression level or activity of the target soluble protein in the liver of the non-human animal. Administration of the therapeutic complex may be by any suitable means, as described elsewhere herein.

[0123] Target cell surface protein can include any cell surface protein that is expressed in liver.Target soluble protein can include any soluble protein that is expressed in liver.The cell surface level or activity of target liver cell surface protein in the liver of non-human animals can be evaluated by known assays for measuring the cell surface level of receptors or other proteins.Similarly, the level of soluble protein can be evaluated by known assays.

[0124] C. Administration of Molecules to Non-Human Animals The methods described herein involve introducing various molecules (therapeutic molecules or therapeutic complexes) into non-human animals, including nucleic acids, proteins, or protein complexes. Introduction can be achieved by any means. For example, the molecules may be introduced into the non-human animal via, for example, vector delivery, particle-mediated delivery, exosome-mediated delivery, lipid nanoparticle-mediated delivery, cell-penetrating peptide-mediated delivery, or implantable device-mediated delivery. Specific examples include introducing the molecules into cells or non-human animals in carriers such as poly(lactic acid) (PLA) microspheres, poly(D,L-lactic-coglycolic acid) (PLGA) microspheres, liposomes, micelles, reverse micelles, cochleate lipids, or lipid microtubules. Some specific examples of delivery to non-human animals include hydrodynamic delivery, viral-mediated delivery (e.g., adeno-associated virus (AAV)-mediated delivery), and lipid nanoparticle-mediated delivery.

[0125] The introduction of nucleic acids, proteins, or other components into non-human animals may be achieved by hydrodynamic delivery (HDD). Hydrodynamic delivery has emerged as a perfect method for intracellular DNA delivery in vivo. For gene delivery to parenchymal cells, only the essential DNA sequence needs to be injected through selected blood vessels, thereby eliminating the safety concerns associated with current viral and synthetic vectors. When injected into the bloodstream, DNA can reach cells in various tissues accessible to blood. Hydrodynamic delivery utilizes the force generated by rapidly injecting a large volume of solution into the incompressible blood of the circulatory system, thereby overcoming the physical barriers of the endothelium and cell membranes that prevent large, membrane-impermeable compounds from entering parenchymal cells. This method has been shown to be efficient for the delivery of RNA, proteins, and other small molecules in addition to DNA. These compounds are useful for effective in vivo intracellular delivery. See, e.g., Bonamassa et al. (2011) Pharm. Res. 28(4):694-701, which is incorporated herein by reference in its entirety for all purposes.

[0126] Nucleic acid introduction can also be achieved by virus-mediated delivery, such as AAV-mediated delivery or lentivirus-mediated delivery. Other exemplary viruses / viral vectors include retroviruses, adenoviruses, vaccinia viruses, poxviruses, and herpes simplex viruses. Viruses can infect dividing cells, non-dividing cells, or both dividing and non-dividing cells. Viruses can integrate into the host genome. Alternatively, they do not integrate into the host genome. Such viruses can be engineered to reduce immunity. Viruses can be replication-competent or replication-deficient (e.g., defective in one or more genes required for additional rounds of virion replication and / or packaging). Viruses can produce transient expression, long-term expression (e.g., for at least 1 week, 2 weeks, 1 month, 2 months, or 3 months), or persistent expression (e.g., for Cas9 and / or gRNA). Exemplary virus titers (e.g., AAV titers) range from 10 to 1000 ng / ml. 12 , 10 13 , 10 14 , 10 15 , and 10 16 vector genomes / mL.

[0127] The ssDNA AAV genome consists of two open reading frames, Rep and Cap, flanked by two inverted terminal repeats, allowing synthesis of a complementary DNA strand. When constructing an AAV transfer plasmid, the transgene is placed between the two ITRs, and Rep and Cap may be supplied in trans. In addition to Rep and Cap, AAV may require a helper plasmid containing genes from adenovirus. These genes (E4, E2a, and VA) mediate AAV replication. For example, the transfer plasmid, Rep / Cap, and helper plasmid may be transfected into HEK293 cells containing the adenovirus E1 gene to produce infectious AAV particles. Alternatively, Rep, Cap, and adenovirus helper genes may be combined into a single plasmid. Similar packaging cells and methods may be used for other viruses, such as retroviruses.

[0128] Several AAV serotypes have been identified. These serotypes infect different cell types (i.e., have different tropisms), allowing preferential transduction of specific cell types. Serotypes specific to liver tissue include AAV7, AAV8, and AAV9, with AAV8 being particularly prominent.

[0129] Tropism can be further improved through pseudotyping. Pseudotyping is a method of mixing capsids and genomes from different viral serotypes. Pseudotyped viruses can be used to improve transduction efficiency and change tropism. Hybrid capsids derived from different serotypes can also be used to change the tropism of viruses. For example, AAV-DJ contains hybrid capsids from eight serotypes and exhibits high infectivity in a wide range of cell types in vivo. The serotype of AAV can also be modified through mutation. Other pseudotyped / modified AAV variants include AAV2 / 1, AAV2 / 6, AAV2 / 7, AAV2 / 8, AAV2 / 9, AAV2.5, AAV8.2, and AAV / SASTG.

[0130] Self-complementary AAV (scAAV) variants may be used to expedite transgene expression. Transgene expression can be delayed because AAV relies on the cell's DNA replication machinery to synthesize the complementary strand of the AAV single-stranded DNA genome. To address this delay, scAAVs containing complementary sequences that can spontaneously anneal upon infection are used. may be used, thereby eliminating the requirement for host cell DNA synthesis.

[0131] To enhance packaging capacity, long transgenes can be split between two AAV transfer plasmids. One plasmid contains a 3' splice donor and the second contains a 5' splice acceptor. Upon coinfection of cells, these viruses form concatemers that can be spliced ​​together to express the full-length transgene. This allows for expression of longer transgenes, but at a lower efficiency. A similar method for enhancing capacity utilizes homologous recombination. For example, the transgene is split between two transfer plasmids, but due to the substantial sequence overlap, coexpression induces homologous recombination and expression of the full-length transgene.

[0132] The introduction of nucleic acids and proteins may be achieved via lipid nanoparticle (LNP)-mediated delivery. For example, LNP-mediated delivery may be used to deliver guide RNA in the form of RNA. Delivery via such methods results in the transient presence of guide RNA, improving clearance of biodegradable lipids, improving tolerability, and improving immunogenicity. Lipid formulations can protect biomolecules from degradation while also improving cellular uptake. Lipid nanoparticles are particles containing multiple lipid molecules physically associated with each other through intermolecular forces. These lipid nanoparticles include microspheres (e.g., unilamellar and multilamellar vesicles such as liposomes), the dispersed phase in emulsions, micelles, or the internal phase in suspensions. Such lipid nanoparticles may be used to encapsulate one or more nucleic acids or proteins for delivery. Formulations containing cationic lipids are useful, for example, for the delivery of polyanions such as nucleic acids. Other lipids that may be included are neutral lipids (i.e., uncharged or amphipathic lipids), anionic lipids, helper lipids to enhance transfection, and stealth lipids to extend the length of time the nanoparticles can persist in vivo. Examples of suitable cationic lipids, neutral lipids, anionic lipids, helper lipids, and stealth lipids can be found in WO2016 / 010840A1, which is incorporated herein by reference in its entirety for all purposes. Exemplary lipid nanoparticles can include a cationic lipid and one or more other components. In one example, the other components can include a helper lipid, such as cholesterol. In another example, the other components can include a helper lipid, such as cholesterol, and a neutral lipid, such as DSPC. In another example, the other components can include a helper lipid, such as cholesterol, any neutral lipid, such as DSPC, and a stealth lipid, such as S010, S024, S027, S031, or S033.

[0133] A delivery mode may be selected that reduces immunogenicity. Different modes may confer different pharmacodynamic or pharmacokinetic properties to the subject to which the molecule is delivered. For example, different modes may have different tissue distribution, different half-lives, or different temporal distribution. Some delivery modes (e.g., delivery of nucleic acid vectors that persist in cells by autonomous replication or genomic integration) result in more sustained expression and presence of the molecule, while other delivery modes are transient and less persistent (e.g., delivery of RNA or proteins). Delivering components in a more transient manner, such as as RNA or protein, can ensure that the components are present and active for only a short period of time, reducing immunogenicity.

[0134] In vivo administration may be by any suitable route, including, for example, parenteral, intravenous, subcutaneous, intraarterial, or intraperitoneal. Systemic administration modes include parenteral routes, such as intravenous, intraarterial, subcutaneous, and intraperitoneal routes. A specific example is intravenous infusion. Local administration modes may also be used.

[0135] The composition containing the nucleic acid or protein may contain one or more physiologically and pharmaceutically acceptable The composition may be formulated using any suitable carrier, diluent, excipient, or adjuvant. The formulation may depend on the selected route of administration. The term "pharmaceutically acceptable" means that the carrier, diluent, excipient, or adjuvant is compatible with the other ingredients of the formulation and is not substantially deleterious to the recipient.

[0136] The frequency of administration and the number of doses may depend, inter alia, on the half-life and route of administration of the component being introduced. The introduction of nucleic acid or protein into a non-human animal may be performed once or multiple times over a period of time. For example, the introduction may be performed at least twice over a period of time, at least three times over a period of time, at least four times over a period of time, at least five times over a period of time, at least six times over a period of time, at least seven times over a period of time, at least eight times over a period of time, at least nine times over a period of time, at least ten times over a period of time, at least eleven times over a period of time, at least 12 times over a period of time, at least 13 times over a period of time, at least 14 times over a period of time, at least 15 times over a period of time, at least 16 times over a period of time, at least 17 times over a period of time, at least 18 times over a period of time, at least 19 times over a period of time, or at least 20 times over a period of time.

[0137] IV. METHODS FOR GENERATING NON-HUMAN ANIMALS COMPRISING A HUMANIZED Asgr1 LOCUS Various methods are provided for producing non-human animals comprising the humanized Asgr1 locus described elsewhere herein. Any convenient method or protocol for producing genetically modified organisms is suitable for producing such genetically modified non-human animals. For example, Cho et al. (2009) Current Protocols in Cell Biology 42:19.11:19.11.1-19.11.22 and Gama Sosa See, e.g., S. et al. (2010) Brain Struct. Funct. 214(2-3):91-109, each of which is incorporated herein by reference in its entirety and for all purposes. Such genetically modified non-human animals may be generated, for example, via targeted gene knock-in at the Asgr1 locus.

[0138] For example, a method for producing a non-human animal comprising a humanized Asgr1 locus may include: (1) modifying the genome of a pluripotent cell to comprise a humanized Asgr1 locus; (2) identifying or selecting a genetically modified pluripotent cell comprising a humanized Asgr1 locus; (3) introducing the genetically modified pluripotent cell into an embryo of a non-human animal host; and (4) implanting and gestation of the host embryo in a surrogate mother. Optionally, the host embryo comprising the modified pluripotent cell (e.g., a non-human ES cell) may be incubated to the blastocyst stage and then implanted and gestation in a surrogate mother to produce an F0 non-human animal. The surrogate mother may then produce an F0 generation non-human animal comprising a humanized Asgr1 locus.

[0139] The method may further include identifying a cell or animal that has the modified target genomic locus. A variety of methods may be used to identify cells and animals that have the targeted genetic modification.

[0140] The screening step may include, for example, a quantitative assay to evaluate the allelic variation (MOA) of the parent chromosome. For example, the quantitative assay may be performed via quantitative PCR, such as real-time PCR (qPCR). The real-time PCR may utilize a first primer set that recognizes the target locus and a second primer set that recognizes a non-target reference locus. The primer set may include a fluorescent probe that recognizes the amplified sequence.

[0141] Other examples of suitable quantitative assays include fluorescence-mediated in situ hybridization (FISH), comparative genomic hybridization, isothermal DNA amplification, immobilized probes, and genomic DNA amplification. Examples of suitable methods include quantitative hybridization to ribosomal DNA, INVADER® probes, TAQMAN® molecular beacon probes, or ECLIPSE™ probe technology (see, e.g., US 2005 / 0144655, which is incorporated herein by reference in its entirety and for all purposes).

[0142] An example of a suitable pluripotent cell is an embryonic stem (ES) cell (e.g., a mouse ES cell or a rat ES cell). The modified pluripotent cell may be generated, for example, via recombination by (a) introducing into a cell one or more targeting vectors containing an insert nucleic acid flanked by 5' and 3' homology arms corresponding to 5' and 3' target sites, the insert nucleic acid including a humanized Asgr1 locus, and (b) identifying at least one cell containing the insert nucleic acid integrated into its genome at the target genomic locus. Alternatively, the modified pluripotent cells may be produced by (a) introducing into a cell (i) a nuclease agent that induces a nick or double-strand break at a recognition site within a target genomic locus, and (ii) one or more targeting vectors that include an insert nucleic acid flanked by 5' and 3' homology arms corresponding to the target site located 5' and 3' to the recognition site, the insert nucleic acid including a humanized Asgr1 locus, and (c) identifying at least one cell that contains a modification (e.g., integration of the insert nucleic acid) at the target genomic locus. Any nuclease agent that induces a nick or double-strand break within the desired recognition site can be used. Examples of suitable nucleases include Transcription Activator-Like Effector Nuclease (TALEN), zinc finger nuclease (ZFN), meganuclease, and Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) / CRISPR-associated (Cas) system or components of such system (such as CRISPR / Cas9).See, for example, US2013 / 0309670 and US2015 / 0159175.Each of them is incorporated herein by reference in its entirety for all purposes.

[0143] Donor cells can be introduced into host embryos at any stage, such as at the blastocyst stage or pre-morula stage (i.e., 4-cell stage or 8-cell stage). Offspring can be generated that can transmit genetic modification through germline. See, for example, U.S. Patent No. 7,294,754, which is incorporated herein by reference in its entirety for all purposes.

[0144] Alternatively, the method for producing a non-human animal as described elsewhere herein may include (1) modifying the genome of a one-cell stage embryo to contain a humanized Asgr1 locus using the methods described above for modifying pluripotent cells; (2) selecting a genetically modified embryo; and (3) implanting the genetically modified embryo into a surrogate mother and allowing it to gestate. Offspring capable of transmitting the genetic modification through the germline are produced.

[0145] Nuclear transfer techniques can also be used to generate non-human mammalian animals. Briefly, nuclear transfer methods may include: (1) enucleating an oocyte or providing an enucleated oocyte; (2) isolating or providing a donor cell or donor nucleus to be combined with the enucleated oocyte; (3) injecting the cell or nucleus into an enucleated oocyte to form a reconstituted cell; (4) implanting the reconstituted cell into the uterus of an animal to form an embryo; and (5) allowing the embryo to develop. In such methods, oocytes are typically harvested from dead animals, but may also be isolated from either the oviducts and / or ovaries of live animals. Oocytes may be matured in a variety of known media prior to enucleation. Enucleation of oocytes can be performed in many known ways. Injecting a donor cell or donor nucleus into an enucleated oocyte to form a reconstituted cell involves removing the donor cell from the zona pellucida prior to fusion. Fusion may be induced by applying a DC electric pulse across the contact / fusion surface (electrofusion), by exposing the cells to a fusion-promoting chemical, such as polyethylene glycol, or by using an inactivated virus, such as Sendai virus. The reconstituted cells may be activated by electrical and / or non-electrical means before, during, and / or after fusion of the nuclear donor and recipient oocytes. Activation methods include electrical pulses, chemical shock, sperm penetration, increasing divalent cation levels in the oocyte, and reducing the phosphorylation of cellular proteins in the oocyte (by kinase inhibitors). Activated reconstituted cells or embryos may be cultured in a known medium and then implanted into the uterus of an animal. See, e.g., US2008 / 0092249, WO1999 / 005266, US2004 / 0177390, WO2008 / 017234, and U.S. Patent No. 7,612,250. each of which is incorporated herein by reference in its entirety and for all purposes.

[0146] The various methods presented herein enable the generation of genetically modified non-human F0 animals, where the cells of the genetically modified F0 animal contain a humanized Asgr1 locus. It is recognized that the number of cells in an F0 animal containing a humanized Asgr1 locus will vary depending on the method used to generate the F0 animal. Introducing donor ES cells from a corresponding organism (e.g., an 8-cell stage mouse embryo) into a pre-morula stage embryo, for example, via the VELOCIMOUSE® method, increases the percentage of the F0 animal's cell population containing cells with a nucleotide sequence of interest containing a targeted genetic modification. For example, at least 50%, 60%, 65%, 70%, 75%, 85%, 86%, 87%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the non-human F0 animal's cellular contribution may contain a cell population with the targeted modification.

[0147] The cells of the genetically modified F0 animal may be heterozygous for the humanized Asgr1 locus, or may be homozygous for the humanized Asgr1 locus.

[0148] All patent applications, websites, other publications, accession numbers, etc., cited above and below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item was specifically and individually indicated to be incorporated by reference. Where alternative versions of a sequence are associated with accession numbers at different times, the version associated with the accession number as of the effective filing date of this application is intended. The effective filing date is intended to be the earlier of the actual filing date or the filing date of the priority application referencing the accession number, as appropriate. Similarly, where alternative versions of publications, websites, etc. are published at different times, the version published closest to the effective filing date of this application is intended unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the invention can be used in combination with any other, unless specifically indicated otherwise. The invention has been described in detail in the figures and examples for purposes of clarity and understanding, but it will be apparent that certain changes and modifications can be made within the scope of the appended claims.

[0149] A brief description of arrays The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases and three-letter codes for amino acids. The nucleotide sequences follow the standard convention of beginning at the 5'-end of the sequence and proceeding to the 3'-end (i.e., left to right on each line). Only one strand of each nucleotide sequence is shown, but it is understood that any reference to the presented strand also includes the complementary strand. The amino acid sequences begin at the amino-terminus of the sequence and proceed to the carboxy-terminus ( i.e., from left to right on each line) following standard conventions.

[0150] Table 1. Sequence description [Table 1-1] [Table 1-2] [Example]

[0151] Example 1 Generation of mice containing a humanized Asgr1 locus The asialoglycoprotein receptor (Ashwell Receptor, ASGR) is a liver membrane-bound, predominantly glycoprotein receptor (approximately 33 kDa) belonging to the C-type class of lectin receptors. ASGR binds to glycoproteins with terminal galactose or N-acetylgalactosamine (GalNac) motifs. Desialylated glycoproteins can be removed from the circulation by receptor-mediated endocytosis. While the ligand undergoes lysosomal degradation, ASGR is recycled back to the cell surface. This receptor is a hetero-oligomer of two subunits, ASGR1 and ASGR2 (H1 and H2). As a liver-specific receptor with highly efficient endocytosis, ASGR1 can be utilized for liver-specific delivery of therapeutic agents, such as antibodies, small molecules (as part of antibody-drug conjugates), and DNA. However, our antibodies and bispecific antibodies against the human ASGR1 extracellular domain do not bind to the mouse Asgr1 ortholog (Biacore data not shown). Therefore, we have utilized a number of different methods to generate humanized Asgr1 mice for use in testing liver-specific delivery of various therapeutic agents.

[0152] A large targeting vector was constructed with a 5' homology arm containing 24 kb from bMQ-69B11 and a 3' homology arm containing 67 kb from bMQ-69B11, replacing the coding exons 3-8 of mouse Asgr1 (through the stop codon and 3' UTR, up to the sequence just downstream of the 3' UTR) with the corresponding human ASGR1 sequence. The encoded Asgr1 protein contained the mouse Asgr1 transmembrane domain, followed by the human coiled-coil domain and C-type lectin domain (see Figures 2A and 3). To generate mutant alleles, CRISPR / Cas9 components were introduced into F1H4 mouse embryonic stem cells along with the large targeting vector. Loss-of-allele assays were performed using the primers and probes listed in Table 2 to detect loss of the endogenous mouse allele, and gain-of-allele assays were performed using the primers and probes listed in Table 3 to detect gain of the humanized allele. Allele loss assays and allele gain assays are described, for example, in US 2014 / 0178879; US 2016 / 0145646; WO 2016 / 081923; and Frendewey et al. (2010) Methods Enzymol. 476:2 95-307, each of which is incorporated herein by reference in its entirety and for all purposes.

[0153] Table 2. Mouse TAQMAN® allele loss assay [Table 2]

[0154] Table 3. Human TAQMAN® Allele Gain Assay [Table 3]

[0155] F0 mice were then generated using the VELOCIMOUSE® method. See, e.g., US 7,576,259; US 7,659,442; US 7,294,754; US 2008 / 007800; and Poueymirou et al. (2007) Nature Biotech. 25(1):91-99, each of which is incorporated herein by reference in its entirety and for all purposes. The intronic portion upstream of coding exon 3 was also humanized. A total of 1863 bp of mouse Asgr1 sequence was replaced with 3907 bp of human ASGR1 sequence. Additionally, a loxP-hUb1-em7-hygromycin resistance gene-polyadenylation signal-mPrm1-Crei-polyadenylation signal-loxP cassette (5218 bp) was inserted downstream of the human 3'UTR. The cassette was immediately preceded by approximately 190 bp of 3' human sequence 3' of the 3' UTR. The resulting partially humanized mouse Asgr1 allele containing the hygromycin resistance auto-deletion cassette is set forth in SEQ ID NO: 21 (designated the 7302 allele). In Figure 2A, the sequence border regions A, B, and C are set forth as SEQ ID NOs: 18, 19, and 20, respectively. A comparison of the human ASGR1 protein (SEQ ID NO: 1), mouse Asgr1 protein (SEQ ID NO: 2), and partially humanized mouse Asgr1 protein (SEQ ID NO: 3) is shown in Figure 3.

[0156] Upon removal of the auto-deletion cassette with Cre recombinase, a loxP and cloning site (77 bp) remains downstream of the human 3'UTR, with approximately 190 bp of 3' human sequence following the 3'UTR immediately preceding the remaining loxP. See Figure 2B. The resulting partially humanized mouse, cassette-deleted Asgr1 allele, is set forth in SEQ ID NO: 24 (referred to as the 7303 allele). In Figure 2B, sequence border regions A and C are denoted by the 7303 allele. They are set forth in SEQ ID NOs: 22 and 23, respectively.

[0157] Example 2: Validation of mice containing a humanized Asgr1 locus Humanized Asgr1 mice (Asgr1) as a valid model hu / hu ) to verify the Asgr1hu / hu Mice were phenotyped and their phenotypes were compared with those of wild-type littermates. hu / hu Mice showed no differences in plasma lipid levels (total cholesterol, triglycerides, HDL-C, LDL-C) compared to wild-type littermates. See Figure 4. Similarly, Asgr1 hu / hu Mice showed no difference in body weight or blood glucose levels compared to wild-type littermates (see Figure 5). Human ASGR1 protein, like mouse Asgr1, was co-localized in the liver membrane (see Figure 6). In conclusion, Asgr1 hu / hu The mice express human ASGR1 protein on liver membranes and have normal plasma lipid profiles.

[0158] method Evaluation of circulating lipid levels in Asgr1-humanized mice. Male Asgr1-humanized mice (Asgr1 hum / hum ) and their wild-type littermates ( Asgr1 + / + Plasma was collected from 11-week-old rats in a non-fasting state and analyzed for serum lipids (triglycerides (TG), total cholesterol, low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C)) using an ADVIA® Chemistry XPT System (Siemens). N = 8 rats / group, 11 weeks of age. Data are presented as mean ± SEM for each group.

[0159] Blood Glucose Assessment Blood glucose was measured from the tail tip using an Accu-Chek glucometer (Roche) in fasted (16 h) and fed (non-fasted) states.

[0160] Western blot analysis of Asgr1. Whole livers were harvested from Asgr1-humanized and WT mice (n = 8 per genotype) and stored frozen at -80°C until processing. For each sample, approximately 40 mg of frozen liver fragments were excised from the whole liver. Each fragment was placed in a Dounce homogenizer and homogenized until a homogenous suspension was obtained. The cytoplasmic and membrane fractions of each liver sample were isolated using a detergent-based commercial kit (Thermo, catalog number 89842) according to the manufacturer's "soft tissue" protocol. Once the cytoplasmic and membrane fractions of each sample were isolated, a BCA protein quantification assay (Thermo, catalog number 23225) was performed on two fractions of each sample according to the kit's protocol for microplate analysis. Western blot samples were prepared for each cytoplasmic and membrane fraction of each sample using 5X reducing dye. All samples were prepared at 0.8 μg / μL. Twenty micrograms of total protein from each sample was loaded onto a Western blot gel. hASGR1 was detected using a rabbit anti-hASGR1 polyclonal antibody (Abgent, Catalog No. AP16133a, 1:1,000 dilution in 2.5% blocking milk in TBS-T). Because the antibody cross-reacted with mouse proteins, mouse Asgr1 from littermate mice was also detected using the same antibody. The secondary antibody used for hASGR1 detection was donkey anti-rabbit IgG-HRP (Jackson, Catalog No. 711-035-152, 0.1 μg / mL in 2.5% blocking milk in TBS-T). As a loading control for the cytosolic fraction, GAPDH was detected in all samples using a rabbit anti-GAPDH monoclonal antibody (Cell Signaling, Catalog No. 2118S, 1:10,000 dilution in 2.5% blocking milk in TBS-T). The secondary antibody for GAPDH detection was donkey anti-rabbit IgG-HRP (Jackson, Cat. No. 711-035-152, 0.1 μg / mL in 2.5% blocking milk in TBS-T). As a loading control for the membrane fractions, rabbit anti-transferrin receptor polyclonal antibody was used. Transferrin receptor was detected in all samples using a primary antibody (R&D, Cat. No. AF2472, 0.25 μg / mL in 2.5% blocking milk in TBS-T). The secondary antibody for transferrin receptor detection was donkey anti-goat IgG-HRP (Jackson, Cat. No. 705-035-147, 0.2 μg / mL in 2.5% blocking milk in TBS-T).

[0161] Example 3 Generation of Adeno-Associated Virus Vectors Containing Heterologous Epitopes Next, we conducted an experiment to determine whether the bispecific anti-myc-ASGR1 antibody could retarget scAAV-N587myc viral particles to liver cells expressing humanized ASGR1 in vivo in the mice generated in Example 1. To verify this, viral particles were first generated.

[0162] AAV capsid proteins are modified to contain one of several heterologous epitopes, such as FLAG, c-myc, or hexahistidine, using PCR to generate plasmids encoding the recombinant capsid proteins. Briefly, a sequence encoding FLAG, c-myc, or hexahistidine is inserted in frame after the codon encoding N587 of the AAV2 VP1 capsid protein or Q585 of the AAV6 VP1 capsid protein.

[0163] Adeno-associated virus production was performed using a triple transfection method with HEK293 cells (see, for example, Erik Arden and Joseph M. Metzger, J. Biol Methods. 2016;3(2)). Cells are transfected with the appropriate vector: - Helper plasmid, pHelper (Agilent, Cat#240074); - plasmids encoding wild-type or modified AAV rep / cap genes (e.g., pAAV RC2 (Cell Biolabs, Cat. No. VPK-422), pAAV RC2 / 6 (Cell Biolabs, Cat. No. VPK-426), pAAV RC2-N587myc, pAAV RC2 / 6-Q585myc); and - Seeded one day before PEFpro (Polyplus transfection, New York, NY)-mediated transfection with a plasmid encoding the nucleotide sequence of interest and AAV ITR sequences, such as pscAAV-CMV-eGFP, pAAV-CMVGFP (Agilent, Cat. No. 240074), pAAV-EF1a-eGFP, or pAAV-CAGG-eGFP.

[0164] Seventy-two hours after transfection, the medium was collected and the cells were lysed in a buffer solution containing 50 mM Tris-HCl, 150 mM NaCl, and 0.5% sodium deoxycholate (Sigma, Cat. #D6750-100G). Benzonase (Sigma, St. Louis, MO) was then added to both the medium and the cell lysate at a final concentration of 0.5 U / μL, followed by incubation at 37°C for 60 minutes. The cell lysate was spun down at 4000 rpm for 30 minutes. The cell lysate and medium were mixed together and precipitated with PEG8000 (Teknova, Cat. #P4340) at a final concentration of 8%. The precipitate was resuspended in 400 nM NaCl and centrifuged at 10,000 g for 10 minutes. Virus in the supernatant was precipitated by ultracentrifugation at 149,000 g for 3 hours and titered by qPCR.

[0165] For qPCR to titer AAV genomes, AAV samples were treated with DNase I (Thermofisher Scientific, Cat. No. EN0525) for 1 hour at 37°C and then diluted with DNA extract All Reagents (Th The PCR product was then lysed using a Thermofisher Scientific product (catalog number 4403319). Encapsidated viral genomes were quantified using a QuantStudio 3 Real-Time PCR System (Thermofisher Scientific) using primers directed against the AAV2 ITR. The sequences of the AAV2 ITR primers were 5'-GGAACCCCTAGTGATGGAGTT-3' (forward ITR, SEQ ID NO: 36) and 5'-CGGCCTCAGTGAGCGA-3' (reverse ITR, SEQ ID NO: 37) (Aurnhammer et al., 2012), derived from the left and right AAV internal inverted repeat (ITR) sequences, respectively. The sequence of the AAV2 ITR probe was 5'-6-FAM-CACTCCCTCTCTGCGCGCTCG-TAMRA-3' (SEQ ID NO: 38) (Aurnhammer). (2012) Hum. Gene Ther. Methods 23:18-28). After a 10-minute activation step at 95°C, 40 PCR cycles were performed, each consisting of 15 seconds at 95°C and 30 seconds at 60°C. TaqMan Universal PCR Master Mix (Thermofisher Scientific, Cat. No. 4304437) was used for qPCR. DNA plasmid (Agilent, Cat. No. 240074) was used as a standard to determine absolute titers.

[0166] An adeno-associated viral vector containing a capsid containing a c-myc epitope was constructed. In this example, the c-myc epitope (EQKLISEEDL, SEQ ID NO: 25) was inserted between amino acids N587 and R588 of the AAV2 VP1 capsid protein. Specifically, the nucleotide sequence encoding the c-myc epitope (GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG, SEQ ID NO: 26) was inserted into the plasmid pAAV RC2 (Cell Biolabs, Inc., San Diego, CA), and the modified pAAV RC2-N587Myc plasmid was used to encode a modified capsid protein for an AAV viral vector with neutral tropism.

[0167] Specifically, a first polymerase chain reaction (PCR) product containing (in the 5' to 3' direction) a BsiW1 restriction enzyme site, the nucleotide sequence between positions 3050 and 3773 of pAAV RC2, and a c-myc epitope overhang nucleotide sequence, and a second PCR product containing (in the 5' to 3' direction) a c-myc epitope overhang nucleotide sequence, the nucleotide sequence between positions 3774 and 4370 of pAAV RC2, and a Pme1 restriction enzyme site were generated using the primers listed in Table 4. Li et al. The pAAV RC2-N587Myc plasmid (i.e., a pAAV RC2 plasmid modified to encode a c-myc epitope between amino acids N587 and R588 of the VP1 capsid protein) was generated by digesting pAAV RC2 with BsiW1 (New England Biolabs, R0553L) and Pme1 (New England Biolabs, R0560L) and inserting the two PCR products via ligation-independent cloning, as described in [Crossref], [Web of Science ® ...

[0168] Table 4. [Table 4]

[0169] Specifically, a gblock DNA fragment containing positions 3700 and 3940 of pAAV RC2 / 6, which has a c-myc epitope sequence inserted between positions 3757 and 3758, was ordered from Integrated DNA Technologies (Coralville, IA). The pAAV RC2 / 6-Q585Myc plasmid was generated by inserting the gblock fragment into pAAV RC2 / 6 digested with MscI (New England Biolabs, catalog no. R0534L) and AflII (New England Biolabs, catalog no. R0520L) via ligation-independent cloning, as described in Li et al. (2012) Methods Mol. Biol. 852:51-59.

[0170] Specifically, pscAAV-CMV-eGFP was generated by introducing the GFP fragment into the pscAAV MCS vector (Cell Biolabs, catalog number VPK-430) using the BamHI and NotI restriction enzyme sites. The pAAV-EF1a-eGFP and pAAV-CAGG-eGFP plasmids were generated by de novo synthesis at Thermofisher Scientific (Waltham, MA).

[0171] Example 4. Bispecific antibody-mediated internalization of scAAV-N587Myc particles in vivo in mice with a humanized ASGR1 locus To determine whether the bispecific anti-myc-ASGR1 antibody could retarget the scAAV2-N587myc-CMV-eGFP viral vector to hepatocytes expressing hASGR1 in vivo, 1 × 10 cells were injected into mice genetically modified to express hASGR1 in their hepatocytes on a C57BL / 6 background, and into control wild-type C57BL / 6 mice. 11Mice were intravascularly injected with wild-type scAAV2-CMV-eGFP alone or with the scAAV2-N587myc-CMV-eGFP viral vector combined with a bispecific anti-myc-ASGR1 antibody at a viral genome-to-antibody molecule ratio of 1:8 (titered by qPCR). Controls included mice injected with saline [250 mM NaCl] or the scAAV2-N587myc-CMV-eGFP viral vector alone. Ten days after injection, mice were sacrificed and transcardially perfused with 4% PFA. Liver, kidney, and heart organs were collected and dehydrated in 15% sucrose followed by 30% sucrose. Organs were then cryosectioned onto slides and stained with chicken anti-EGFP antibody (Jackson ImmunoResearch Labs, Inc., West Grove, PA) and Alexa-488-conjugated anti-chicken secondary antibody (Jackson ImmunoResearch Labs, Inc., West Grove, PA) (Figures 8A-8C). Livers from transgenic animals engineered to express ASGR1 in the liver and injected with wild-type scAAV2-CMV-eGFP or scAAV2-N587myc-CMV-eGFP combined with a bispecific anti-myc-ASGR1 antibody (Figures 8A(i) and 8A(iv)), and from wild-type C57BL / 6 mice injected with wild-type scAAV2-CMV-eGFP (Figures 8A(i) and 8A(iv)). GFP-positive cells were detected in the livers of wild-type C57BL / 6 animals (Fig. 8A(v)). GFP was not detected in any spleen or kidney samples (Fig. 8B and 8C), nor in any liver, spleen, or kidney samples from animals injected with saline or the scAAV2-N587myc-CMV-eGFP viral vector alone (Fig. 8A(ii, iii, vi, vii)), nor in any liver samples collected from wild-type C57BL / 6 animals injected with scAAV2-N587myc-CMV-eGFP in combination with the bispecific anti-myc-ASGR1 antibody (Fig. 8A(viii)). In summary, the combination of the scAAV2-N587myc-CMV-eGFP viral vector and the bispecific anti-myc-ASGR1 antibody infected only hASGR1-expressing hepatocytes. This strongly suggests that the scAAV2-CMV-eGFP viral vector was inactivated by modification of the capsid protein, that the intrinsic tropism of the scAAV viral vector could be neutralized, for example, by the c-myc epitope, and that such a viral vector could be specifically reactivated, for example, by being specifically retargeted to liver cells in vivo, by a bispecific anti-myc-ASGR1 antibody.

[0172] Similarly, to determine whether the bispecific anti-myc-ASGR1 antibody could retarget the ssAAV2-N587myc-CAGG-eGFP viral vector to hepatocytes expressing hASGR1 in vivo, 2.18 × 10 cells were injected into mice genetically modified to express hASGR1 in their hepatocytes on a C57BL / 6 background and into control wild-type C57BL / 6 mice. 11Mice were intravascularly injected with wild-type ssAAV2-CAGG-eGFP viral vector (titered by qPCR) alone or with the ssAAV2-N587myc-CAGG-eGFP viral vector and bispecific anti-myc-ASGR1 antibody at a viral genome to antibody molecule ratio of 1:4. Controls included mice injected with PBS or the ssAAV2-N587myc-CAGG-eGFP viral vector alone. Four weeks after injection, mice were sacrificed and transcardially perfused with 4% PFA. Liver, kidney, and heart organs were collected and dehydrated in 15% sucrose followed by 30% sucrose. Organs were then cryosectioned onto slides and stained with a chicken anti-EGFP antibody (Jackson ImmunoResearch Labs, Inc., West Grove, PA) and an Alexa-488-conjugated anti-chicken secondary antibody (Jackson ImmunoResearch Labs, Inc., West Grove, PA) (Figures 9A-9O). GFP-positive cells were detected in livers from transgenic animals engineered to express ASGR1 in the liver and injected with wild-type ssAAV2-CAGG-eGFP or ssAAV2-N587myc-CAGG-eGFP combined with a bispecific anti-myc-ASGR1 antibody (Figures 9E-9F, 9P-9R), and in livers from wild-type C57BL / 6 mice injected with wild-type ssAAV2-CAGG-eGFP (Figures 9B-9C). Surprisingly, the infection efficiency of ssAAV2-N587myc-CAGG-eGFP combined with bispecific anti-myc-ASGR1 antibody was much higher than that of WT ssAAV2-CAGG-GFP (Figures 9E–9F, 9P–9R). GFP was not or barely detectable in liver samples from all animals injected with saline or the ssAAV2-N587myc-CAGG-eGFP viral vector alone (Figures 9A, 9D, 9G–9L), as well as in liver samples collected from wild-type C57BL / 6 animals injected with ssAAV2-N587myc-CAGG-eGFP combined with bispecific anti-myc-ASGR1 antibody (Figures 9M–9O).In summary, the combination of ssAAV2-N587myc-CAGG-eGFP viral vector and bispecific anti-myc-ASGR1 antibody infected only hASGR1-expressing (hepatic) cells. This indicates that the ssAAV2-N587myc-CAGG-eGFP viral vector was inactivated by capsid protein modification, that the inherent tropism of the scAAV viral vector could be neutralized by, for example, the c-myc epitope, and that such viral vectors could be targeted to liver cells in vivo, for example, by bispecific anti-myc-ASGR1 antibody. This strongly suggests that the IL-1 receptor could be specifically reactivated, i.e., specifically retargeted to the cytoplasm.

[0173] This example demonstrates that humanized ASGR1 mice can be used to validate therapeutic candidates that specifically bind to human ASGR1 for targeted delivery to liver cells.

Claims

1. A nucleic acid comprising a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO: 3, wherein the nucleic acid further comprises a nucleic acid sequence selected from the group consisting of the nucleic acid sequence set forth in SEQ ID NO: 18, the nucleic acid sequence set forth in SEQ ID NO: 19, the nucleic acid sequence set forth in SEQ ID NO: 20, and the nucleic acid sequence set forth in SEQ ID NO:

23.

2. The nucleic acid of claim 1, comprising the nucleic acid sequence set forth in SEQ ID NO:

24.

3. The nucleic acid of claim 1, comprising the nucleic acid sequence set forth in SEQ ID NO:

21.

4. A polypeptide encoded by the nucleic acid of any one of claims 1 to 3, wherein the polypeptide binds to desialylated galactose-terminated and N-acetylgalactosamine-terminated glycoproteins.

5. A targeting vector comprising the nucleic acid of any one of claims 1 to 3 flanked by 5' and 3' homology arms corresponding to 5' and 3' target sites within the endogenous mouse Asgr1 locus.

6. 1. A method for producing a mouse comprising a genetically modified endogenous Asgr1 locus encoding a chimeric human / non-human Asgr1 protein, comprising: (a) contacting a mouse ES cell with the targeting vector of claim 5, wherein the targeting vector recombines with the endogenous mouse Asgr1 locus, thereby generating a genetically modified mouse ES cell comprising a genetically modified endogenous Asgr1 locus encoding a chimeric human / non-human Asgr1 protein; (b) introducing the genetically modified mouse ES cells into a mouse host embryo; (c) implanting the mouse host embryo into a surrogate mouse mother and gestation to produce the mouse comprising the genetically modified endogenous Asgr1 locus encoding the chimeric human / non-human Asgr1 protein; Including, a method.

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