Non-human animals containing modified CACNG1 locus

Abstract

Non-human animal cells and animals comprising a humanized Cacng1 locus, and methods of using the non-human animal cells and animals, are provided. The non-human animal cells or animals comprising a humanized Cacng1 locus express the human CACNG1 protein or a fragment thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 275,582, filed November 4, 2021, the entirety of which is incorporated by reference.

[0002] Sequence Listing The sequence listing set forth in file 11102WO01_ST26.txt is 79 kilobytes, was created on November 4, 2022, and is incorporated by reference herein in its entirety.

[0003] A genetically modified non-human animal (e.g., a rodent such as a mouse or a rat) is described, which comprises a nucleic acid encoding human voltage-dependent calcium channel auxiliary subunit gamma 1 (CACNG1) protein or a part thereof in its genome.Therefore, a genetically modified non-human animal is also described, which expresses human CACNG1 protein or a part thereof, for example, on the surface of skeletal muscle cells.Such a genetically modified non-human animal, which expresses human CACNG1 protein or a part thereof, for example, on the surface of skeletal muscle cells, can be used as a model for preclinical testing of CACNG1-based therapies, for example, CACNG1-based antibodies. [Background technology]

[0004] Skeletal muscles are one of the three significant types of muscle tissue in the human body. Each skeletal muscle contains thousands of muscle fibers bound together by a connective tissue sheath. Skeletal muscles enable humans to move and perform daily activities. Skeletal muscles play an important role in respiratory mechanics and help maintain posture and balance. Skeletal muscles also protect the vital organs in the body.

[0005] Since a myriad of pathologies can result from abnormal skeletal muscle function, suitable animal models in which therapies aimed at treating abnormal muscle function, for example by targeting muscle-specific surface proteins, can be tested, can aid in the study of pathologies associated with skeletal muscle. Summary of the Invention

[0006] Provided herein are genetically modified non-human animals having a recombinant locus encoding the human calcium channel voltage-gated auxiliary subunit gamma 1 (CACNG1) protein. Also provided herein are compositions and methods for producing and using such modified non-human animals.

[0007] Described herein are genetically engineered non-human animal genomes, engineered cells, and non-human animals that contain a heterologous (e.g., human) Cacng1 gene or a portion thereof. In some embodiments, the genetically engineered animals described herein express heterologous (e.g., human) CACNG1 protein from a desired locus (e.g., from a segment of endogenous Cacng1). The non-human animal can be a mammal, such as a rodent (e.g., mouse or rat). The non-human animal cell can be a mammalian cell, such as a rodent cell (e.g., mouse cell or rat cell). The non-human animal genome can be a mammalian nucleic acid, such as a rodent nucleic acid (e.g., mouse nucleic acid or rat nucleic acid).

[0008] In some embodiments, the non-human animal, non-human animal cell, or non-human animal genome comprises a nucleic acid sequence encoding a heterologous (e.g., human) CACNG1 protein or portion thereof.

[0009] In some embodiments, the nucleic acid sequence encoding a heterologous (e.g., human) CACNG1 protein or a portion thereof comprises: (i) a nucleic acid sequence comprising exon 1 or a portion thereof of the human CACNG1 gene; (ii) a nucleic acid sequence comprising exon 2 or a portion thereof of the human CACNG1 gene; (iii) a nucleic acid sequence comprising exon 3 or a portion thereof of the human CACNG1 gene; (iv) a nucleic acid sequence comprising exon 4 or a portion thereof of the human CACNG1 gene; or (v) any combination of (i)-(iv). In some embodiments, the nucleic acid sequence encoding a heterologous (e.g., human) CACNG1 protein or a portion thereof comprises: (i) a nucleic acid sequence comprising exon 1 or a portion thereof of the human CACNG1 gene, (ii) a nucleic acid sequence comprising intron 1 or a portion thereof of the human CACNG1 gene, (iii) a nucleic acid sequence comprising exon 2 or a portion thereof of the human CACNG1 gene, (iv) a nucleic acid sequence comprising intron 2 or a portion thereof of the human CACNG1 gene, (v) a nucleic acid sequence comprising exon 3 or a portion thereof of the human CACNG1 gene, (vi) a nucleic acid sequence comprising intron 3 or a portion thereof of the human CACNG1 gene, (v) a nucleic acid sequence comprising exon 4 or a portion thereof of the human CACNG1 gene, (vii) a nucleic acid sequence of the 3' untranslated region (UTR) of the human CACNG1 gene, or (v) any combination of (i)-(iv). In some embodiments, the nucleic acid sequence encoding the heterologous CACNG1 protein or a portion thereof comprises, consists essentially of, or consists of a nucleic acid sequence selected from the group consisting of the nucleic acid sequence set forth as SEQ ID NO:5, the nucleic acid sequence set forth as SEQ ID NO:27, and the nucleic acid sequence set forth as SEQ ID NO:28.

[0010] In some embodiments, a nucleic acid sequence encoding a heterologous (e.g., human) CACNG1 protein or a portion thereof is integrated into an endogenous Cacng1 locus (of a genome, cell, or non-human animal). In some embodiments, a nucleic acid sequence encoding a heterologous CACNG1 protein or a portion thereof replaces an orthologous endogenous nucleic acid sequence encoding an endogenous CACNG1 protein or a portion thereof (in a non-human animal genome, a non-human animal cell, or an endogenous locus of a non-human animal). In some embodiments, an endogenous Cacng1 locus comprises a heterozygous or homozygous replacement of an endogenous nucleic acid sequence encoding an endogenous CACNG1 protein or a portion thereof with a nucleic acid sequence encoding a heterologous CACNG1 protein or a portion thereof, and the endogenous nucleic acid sequence encoding an endogenous CACNG1 protein or a portion thereof and the nucleic acid sequence encoding a heterologous CACNG1 protein or a portion thereof are orthologous.

[0011] In some embodiments, the heterologous CACNG1 protein or a portion thereof comprises the amino acid sequence of a human CACNG1 protein or a portion thereof. In some embodiments, the heterologous CACNG1 protein or a portion thereof comprises (i) the amino acid sequence set forth as SEQ ID NO:8, (ii) the amino acid sequence set forth as SEQ ID NO:10, (iii) the amino acid sequence set forth as SEQ ID NO:12, (iv) the amino acid sequence set forth as SEQ ID NO:14, (v) the amino acid sequence set forth as SEQ ID NO:16, (vi) the amino acid sequence set forth as SEQ ID NO:18, (vii) the amino acid sequence set forth as SEQ ID NO:20, (viii) the amino acid sequence set forth as SEQ ID NO:22, (ix) the amino acid sequence set forth as SEQ ID NO:24, or (x) any combination of (i)-(ii). In some embodiments, the heterologous CACNG1 protein comprises the amino acid sequence set forth as SEQ ID NO:4.

[0012] In some embodiments, the non-human animal cell described herein expresses heterologous CACNG1 protein or a part thereof on its cell surface, which may be full-length human CACNG1 protein.In some embodiments, the non-human animal cell described herein is a non-human animal skeletal muscle cell that expresses heterologous (e.g., human) CACNG1 protein or a part thereof on its cell surface.

[0013] In some embodiments, the non-human animal cells described herein are non-human animal cells that do not express a heterologous (e.g., human) CACNG1 protein or a portion thereof on their cell surface, e.g., the non-human animal cell is not a skeletal cell, and / or, e.g., the non-human animal cell is a pluripotent cell, an embryonic stem cell, a germ cell, etc.

[0014] In some embodiments, the non-human animal cell is a mouse cell, and the nucleic acid sequence encoding the heterologous CACNG1 protein or a portion thereof comprises, consists essentially of, or consists of the nucleic acid sequence set forth as SEQ ID NO:6.

[0015] In some embodiments, the non-human animal cell described herein comprises a skeletal muscle cell that expresses heterologous CACNG1 protein or a portion thereof on its cell surface. In some embodiments, the non-human animal cell comprises a non-human animal skeletal muscle cell that expresses heterologous CACNG1 protein or a portion thereof (e.g., full-length human CACNG1) protein on its cell surface. In some embodiments, the non-human animal comprises a non-human animal cell that comprises a heterologous (e.g., human) Cacng1 gene or a portion thereof and does not express the heterologous (e.g., human) CACNG1 protein or a portion thereof encoded by the heterologous (e.g., human) Cacng1 gene or a portion thereof on its cell surface, e.g., the non-human animal cell is not a skeletal cell, and / or the non-human animal cell is a pluripotent cell, an embryonic stem cell, a germ cell, etc.

[0016] In some embodiments, the non-human animal described herein is a mouse, the non-human animal cell described herein is a mouse cell, or the non-human animal genome described herein is a mouse nucleic acid, and the nucleic acid sequence encoding the heterologous CACNG1 protein or a portion thereof comprises, consists essentially of, or consists of the nucleic acid sequence set forth as SEQ ID NO:6.

[0017] Also described herein is a chimeric nucleic acid molecule encoding a functional CACNG1 protein comprising a nucleic acid sequence of a modified non-human animal Cacng1 gene encoding a non-human CACNG1 protein or a portion thereof, the modified non-human animal Cacng1 gene comprising a replacement of a nucleic acid sequence encoding a portion of the non-human animal CACNG1 protein with a homologous nucleic acid sequence encoding a heterologous CACNG1 protein or a portion thereof. In some embodiments, the chimeric nucleic acid molecule described herein comprises a nucleic acid sequence of a non-human animal Cacng1 gene that (a) encodes a CACNG1 protein and (b) is modified to comprise a replacement of a sequence encoding a CACNG1 protein or a portion thereof with a homologous sequence encoding a heterologous CACNG1 protein or a portion thereof, the chimeric nucleic acid molecule encoding a functional CACNG1 protein, and optionally the chimeric nucleic acid sequence further comprises a promoter sequence and / or a regulatory sequence of the non-human animal Cacng1 gene. In some embodiments, the homologous nucleic acid sequence comprises: (i) a nucleic acid sequence comprising exon 1 or a portion thereof of the human CACNG1 gene; (ii) a nucleic acid sequence comprising intron 1 or a portion thereof of the human CACNG1 gene; (iii) a nucleic acid sequence comprising exon 2 or a portion thereof of the human CACNG1 gene; (iv) a nucleic acid sequence comprising intron 2 or a portion thereof of the human CACNG1 gene; (v) a nucleic acid sequence comprising exon 3 or a portion thereof of the human CACNG1 gene; (vi) a nucleic acid sequence comprising intron 3 or a portion thereof of the human CACNG1 gene; (v) a nucleic acid sequence comprising exon 4 or a portion thereof of the human CACNG1 gene; (vii) a nucleic acid sequence of the 3' untranslated region (UTR) of the human CACNG1 gene; or (v) any combination of (i)-(iv). In some embodiments, the modified Cacng1 gene further comprises a drug selection cassette. In some embodiments, the chimeric nucleic acid molecule described herein further comprises (i) a 5' homology arm upstream of the modified non-human animal Cacng1 gene, and (ii) a 3' homology arm downstream of the modified non-human animal Cacng1 gene.In some examples, the 5' homology arm and the 3' homology arm can undergo homologous recombination with the non-human animal Cacng1 locus of interest, and after homologous recombination with the non-human animal Cacng1 locus of interest, the modified Cacng1 gene can replace the non-human animal Cacng1 gene at the non-human animal Cacng1 locus of interest, and is operably linked to the endogenous promoter that drives the expression of the non-human animal Cacng1 gene at the non-human animal Cacng1 locus of interest.In certain embodiments, the chimeric nucleic acid described herein has (i) a 5' homology arm that comprises the nucleic acid sequence described as SEQ ID NO:25, and / or (ii) a 3' homology arm that comprises the nucleic acid sequence described as SEQ ID NO:26.In some embodiments, the nucleic acid sequence comprises the nucleic acid sequence described as SEQ ID NO:6.

[0018] Also described are methods of making the non-human animals, non-human animal cells, or non-human animal genomes described herein by inserting a nucleic acid sequence encoding a heterologous CACNG1 protein or a portion thereof into the genome of the non-human animal, the genome of the non-human animal cell, or the genome of the non-human animal. In some embodiments, the non-human animal cell is a non-human animal embryonic stem (ES) cell, and the inserting comprises inserting a nucleic acid sequence encoding a heterologous CACNG1 protein or a portion thereof into the genome of a non-human animal ES cell to form a modified non-human animal ES cell comprising a nucleic acid sequence encoding a heterologous CACNG1 protein or a portion thereof in its genome. In some embodiments, the method comprises introducing the modified non-human animal ES cell into a host embryo cell in vitro. In some embodiments, the method comprises gestating the host embryo cell comprising the modified non-human animal ES cell in a suitable non-human surrogate animal, and allowing the non-human surrogate animal to give birth to a non-human animal progeny comprising a germ cell comprising a nucleic acid sequence encoding a heterologous CACNG1 protein or a portion thereof. In some embodiments, the nucleic acid sequence encoding the heterologous CACNG1 protein or a portion thereof is inserted into the endogenous Cacng1 locus. In such embodiments, the step of inserting comprises replacing the endogenous nucleic acid sequence encoding the endogenous CACNG1 protein or a portion thereof with the nucleic acid sequence encoding the heterologous CACNG1 protein or a portion thereof, and the endogenous nucleic acid sequence encoding the endogenous CACNG1 protein or a portion thereof and the nucleic acid sequence encoding the heterologous CACNG1 protein or a portion thereof are orthologous. In some embodiments, the nucleic acid sequence encoding the heterologous CACNG1 protein or a portion thereof comprises (i) a nucleic acid sequence comprising exon 1 or a portion thereof of the human CACNG1 gene, (ii) a nucleic acid sequence comprising exon 2 or a portion thereof of the human CACNG1 gene, (iii) a nucleic acid sequence comprising exon 3 or a portion thereof of the human CACNG1 gene, (iv) a nucleic acid sequence comprising exon 4 or a portion thereof of the human CACNG1 gene, or (v) any combination of (i)-(iv).In some examples, the nucleic acid sequence encoding a heterologous CACNG1 protein or a portion thereof comprises: (i) a nucleic acid sequence comprising exon 1 or a portion thereof of the human CACNG1 gene, (ii) a nucleic acid sequence comprising intron 1 or a portion thereof of the human CACNG1 gene, (iii) a nucleic acid sequence comprising exon 2 or a portion thereof of the human CACNG1 gene, (iv) a nucleic acid sequence comprising intron 2 or a portion thereof of the human CACNG1 gene, (v) a nucleic acid sequence comprising exon 3 or a portion thereof of the human CACNG1 gene, (vi) a nucleic acid sequence comprising intron 3 or a portion thereof of the human CACNG1 gene, (v) a nucleic acid sequence comprising exon 4 or a portion thereof of the human CACNG1 gene, (vii) a nucleic acid sequence of the 3' untranslated region (UTR) of the human CACNG1 gene, or (v) any combination of (i)-(iv). In some embodiments, the nucleic acid sequence encoding the heterologous CACNG1 protein or portion thereof comprises, consists essentially of, or consists of a nucleic acid sequence selected from the group consisting of the nucleic acid sequence set forth as SEQ ID NO: 5, the nucleic acid sequence set forth as SEQ ID NO: 27, and the nucleic acid sequence set forth as SEQ ID NO: 28. In some embodiments, the heterologous CACNG1 protein or portion thereof comprises the amino acid sequence of a human CACNG1 protein or portion thereof. In some embodiments, the heterologous CACNG1 protein or portion thereof comprises: (i) the amino acid sequence set forth as SEQ ID NO:8 (human cytoplasmic domain 1), (ii) the amino acid sequence set forth as SEQ ID NO:10 (human transmembrane 1), (iii) the amino acid sequence set forth as SEQ ID NO:12 (human extracellular domain 1), (iv) the amino acid sequence set forth as SEQ ID NO:14 (human transmembrane 2), (v) the amino acid sequence set forth as SEQ ID NO:16 (human cytoplasmic domain 2), (vi) the amino acid sequence set forth as SEQ ID NO:18 (human transmembrane 3), (vii) the amino acid sequence set forth as SEQ ID NO:20 (human extracellular domain 3), (viii) the amino acid sequence set forth as SEQ ID NO:22 (human transmembrane 4), (ix) the amino acid sequence set forth as SEQ ID NO:24 (human cytoplasmic domain 4), or (x) any combination of (i)-(ii). In some embodiments, the heterologous CACNG1 protein comprises the amino acid sequence set forth as SEQ ID NO:4.In some embodiments, (i) the non-human animal is a mammal, such as a rodent, (ii) the non-human animal cell is a mammalian cell, such as a rodent cell, or (iii) the non-human animal genome is a mammalian nucleic acid, such as a rodent nucleic acid. In some embodiments, (i) the non-human animal is a rat or a mouse, (ii) the non-human animal cell is a rat cell or a mouse cell, or (iii) the non-human animal genome is a rat nucleic acid or a mouse nucleic acid. In some embodiments, the non-human animal is a mouse, the non-human animal cell is a mouse cell, or the non-human animal genome is a mouse nucleic acid, and the nucleic acid sequence encoding the heterologous CACNG1 protein or a portion thereof comprises, consists essentially of, or consists of the nucleic acid sequence set forth as SEQ ID NO:6.

[0019] In some embodiments, inserting the nucleic acid comprises contacting the genome of the non-human animal, the genome of a non-human animal cell, or the genome of the non-human animal with any of the chimeric nucleic acid molecules of the present disclosure.

[0020] The non-human animal, non-human animal cell, or non-human animal genome can be produced according to any of the methods of the disclosure.

[0021] In some embodiments, the non-human animal described herein comprises an antigen binding protein that binds to heterologous CACNG1 protein, and the non-human animal expresses heterologous CACNG1 protein or its extracellular domain on the surface of skeletal muscle cells.In some embodiments, the heterologous CACNG1 protein is human CACNG1 protein.In some embodiments, the non-human animal is a mouse.

[0022] In some embodiments, the non-human animal, non-human animal cell, or non-human animal genome comprises a knockout mutation of endogenous Cacng1 gene.In some examples, the knockout mutation comprises the deletion of Cacng1 gene or a part thereof.In certain embodiments, the knockout mutation comprises the deletion of the entire coding sequence of Cacng1 gene.

[0023] Also provided herein is a non-human animal, a non-human animal cell, or a non-human animal genome that does not express any CACNG1 protein.

[0024] Also provided herein is a non-human animal, a non-human animal cell, or a non-human animal genome that does not express a skeletal muscle-specific protein, wherein the non-human animal, the non-human animal cell, or the non-human animal genome does not exhibit any grossly mutant phenotype.

[0025] In some embodiments, the present disclosure provides a targeting vector comprising (i) a 5' homology arm and (ii) a 3' homology arm, wherein the 5' homology arm and the 3' homology arm undergo homologous recombination with a non-human animal Cacng1 locus of interest, and following homologous recombination with the non-human animal Cacng1 locus of interest, the targeting vector inserts a knockout mutation within the non-human animal Cacng1 gene at the non-human animal Cacng1 locus of interest.

[0026] In some embodiments, the disclosure provides a method of making a CACNG1 knockout non-human animal, the method comprising modifying the endogenous Cacng1 locus of the non-human animal to contain a knockout mutation. [Brief description of the drawings]

[0027] [Figure 1] FIG. 1 is a graph showing CACNG1 expression (GTEx Portal).

[0028] [Diagram 2] FIG. 2 shows the strategy (not to scale) for the generation of a CACNG1 knockout.

[0029] [Figure 3A] FIG. 3A is a graph showing that CACNG1 gene deletion in mice (CACNG1− / −) does not alter skeletal muscle weight compared to wild-type (WT) control animals.

[0030] [Figure 3B] FIG. 3B is a graph showing that CACNG1 gene deletion in mice (CACNG1− / −) does not alter twitch (1 Hz) or tetanic (125 Hz) contraction force compared to wild-type (WT) control animals.

[0031] [Figure 4A] Figure 4A shows a schematic diagram (not to scale) of humanization of CACNG1 hu / hu mice. Asterisks indicate the location of the upstream primer (7450hTU) and downstream primer (7450hTD) for gain-of-allele assay. The top of the figure shows 12,484bp of sequence derived from human CACNG1 for humanization of non-animal genomes. The bottom of the figure shows 12,795bp of mouse genomic sequence on the Cacng1 locus targeted for deletion.

[0032] [Figure 4B] Figure 4B details the strategy (not to scale) for CACNG1 humanization of the 7450 allele, which includes a Neo (neomycin) self-deletion cassette. The portions of mouse Cacng1, coding exon 1, intron 1, coding exons 2-4 (and intervening introns), and 82 bp of the 3' untranslated region (UTR), are replaced with the corresponding portions of human CACNG1, coding exon 1 sequence, intron 1, coding exons 2-4 (and intervening introns), the complete 3'UTR, and an additional 158 bp after the 3'UTR. The first 15 bp of the coding sequence remain mouse sequence. The loxP-mPrm1-Crei-pA-hUb1-em7-Neo-pA-loxP cassette (4,805 bp) is shown downstream of the human sequence, followed by the remaining mouse 3'UTR.

[0033] [Figure 4C]Figure 4C details the strategy (not to scale) for CACNG1 humanization of the 7451 allele in which the cassette was deleted but the LoxP sites remained. The mouse Cacng1 portion of coding exon 1, intron 1, coding exons 2-4 (and intervening introns), and 82 bp of the 3' untranslated region (UTR) are replaced with the corresponding portions of human CACNG1: coding exon 1 sequence, intron 1, coding exons 2-4 (and intervening introns), the complete 3'UTR, and an additional 158 bp after the 3'UTR. The first 15 bp of the coding sequence remain mouse sequence. The loxP-mPrm1-Crei-pA-hUb1-em7-Neo-pA-loxP cassette (4,805 bp) is shown downstream of the human sequence, followed by the remaining mouse 3'UTR. After cassette deletion, a LoxP and cloning site (77 bp) remains followed by the human 3'UTR.

[0034] [Diagram 5] Figure 5 shows an alignment of human hCACNG1 protein (hCACNG1, SEQ ID NO: 4) and the CACNG1 protein encoded by the 7451 allele (7451, SEQ ID NO: 4) with the mouse CACNG1 protein (mCACNG1, SEQ ID NO: 2). Asterisks indicate residues that remain unchanged. Bold solid lines indicate the transmembrane domain. Underlined residues are those encoded by the introduced human exon. Cytoplasmic and extracellular domains are labeled and illustrated.

[0035] [Figure 6A] FIG. 6A is a graph demonstrating that expression of mouse CACNG1 (mCACNG1) is not detectable by qPCR in CACNG1 hu / hu mouse muscle (left graph), while human CACNG1 (hCACNG1) is expressed in CACNG1 hu / hu but not wild-type (WT) mouse muscle (right graph).

[0036] [Figure 6B]FIG. 6B is an image showing live staining of a single skeletal muscle myofiber with 100 nM Alexa 647-conjugated human-specific α-CACNG1 antibody, showing binding to myofibers isolated from CACNG1 hu / hu mice but not to myofibers isolated from wild-type (WT) mice.

[0037] [Figure 6C] Figure 6C shows cryofluorescence tomography (CryoFT) images of CACNG1 hu / hu mice injected with 10 mg / kg Alexa 647-conjugated human-specific α-CACNG1 antibody, 6 days after injection, demonstrating high specificity for skeletal muscle compared to isotype control antibody.

[0038] [Figure 7] 7 is an image showing tissue sections from CACNG1hu / hu mice administered 10 mg / kg Alexa 647-conjugated human-specific α-CACNG1 antibody, showing binding to skeletal muscle 6 days after injection. The top image shows endogenous Alexa 647 signal from the antibody injected in vivo, and the bottom image shows an overlay of Alexa 647 antibody binding with co-stained laminin and DAPI to visualize muscle morphology.

[0039] [Figure 8A] Figure 8A provides annotation of the cytoplasmic domain (amino acids 1–10, 131–135, and 206–223), transmembrane domain (amino acids 11–29, 110–130, 136–156, and 181–205), and extracellular domain (amino acids 30–109, and 157–180) of the mouse Cacng1 protein, designated as NP_031608.1.

[0040] [Figure 8B]Figure 8B provides annotation of the mouse coding DNA sequence (CDS) for nucleic acid sequences encoding the cytoplasmic domain (nucleic acids 1-30, 391-405, and 616-669), transmembrane domain (nucleic acids 31-87, 328-390, 406-468, and 541-615), and extracellular domain (nucleic acids 88-327, and 469-540).

[0041] [Figure 9A] Figure 9A provides annotation of the cytoplasmic domain (amino acids 1–10, 130–134, and 205–222), transmembrane domain (amino acids 11–29, 109–129, 135–155, and 180–204), and extracellular domain (amino acids 30–108, and 156–179) of the human CACNG1 protein, designated as NP_000718.1.

[0042] [Figure 9B] FIG. 9B provides annotation of the nucleic acid sequences encoding the cytoplasmic, transmembrane, and extracellular domains of the human coding DNA sequence (CDS).

[0043] [Figure 10A] FIG. 10A provides an exemplary sequence of the CACNG1 protein encoded by the 7451 allele.

[0044] [Figure 10B] FIG. 10B provides an exemplary sequence of the mouse / human CACNG1 nucleic acid coding sequence (CDS), including the 3′ untranslated sequence.

[0045] [Figure 11-1] Figure 11 provides the nucleic acid sequence of the 7450 allele. CACNG1 humanized region with Neo self-deletion cassette = mouse (lowercase)_human_XhoI_LoxP_Prm_Crei_sv40 polya (lowercase)-hUbi-em7 (lowercase)-NEO-PGK polyA_LoxP_ICeUI_mouse (lowercase). [Figure 11-2] Same as above. [Figure 11-3] Same as above.

[0046] [Figure 12-1] Figure 12 provides the nucleic acid sequence of the 7450 allele. CACNG1 humanized region with Neo self-deletion cassette = [ka] [Figure 12-2] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] definition The terms "protein," "polypeptide," and "peptide" are used interchangeably herein and include any length of polymeric form of amino acids, including coded and non-coded amino acids, and chemically or biochemically modified or derivatized amino acids. The term also includes modified multimers, such as, for example, polypeptides with modified peptide backbones. The term domain may refer to any portion of a protein or polypeptide having a particular function or structure.

[0048] Proteins are considered 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).

[0049] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein and include polymeric forms of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, or analogs or modifications thereof. Nucleic acids and polynucleotides can include single-stranded, double-stranded, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers that contain purine, pyrimidine, or other naturally occurring, chemically modified, biochemically modified, non-natural, or derivatized nucleotide bases.

[0050] Nucleic acids are considered to have a "5' end" and a "3' end" because mononucleotides are reacted such that the 5' phosphate of the pentose ring of one mononucleotide is linked in one orientation to its adjacent 3' oxygen via a phosphodiester bond to produce an oligonucleotide. 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 a 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 may be considered to have a 5' end and a 3' end, even if the inner oligonucleotide is a larger one. In either a linear or circular DNA molecule, the dispersing elements are referred to as being "upstream" or 5' elements of the "downstream" or 3' elements.

[0051] 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 to achieve stable integration of the nucleic acid into the genome of the cell.

[0052] The term "targeting vector" refers to a recombinant nucleic acid that can be introduced by homologous recombination, ligation by non-homologous end joining, or any other recombinant means into a target location within a cell genome.

[0053] The term "viral vector" refers to a recombinant nucleic acid that contains at least one element of viral origin and that contains elements sufficient or permissive for packaging into a viral vector particle. The vectors and / or particles can be used to transfer DNA, RNA, or other nucleic acids into cells, either ex vivo or in vivo. Many forms of viral vectors are known.

[0054] The term "wild-type" includes entities that have a structure and / or activity that is present under a normal state or background (as compared to a mutant, disease state, altered state, etc.). Wild-type genes and polypeptides often exist in multiple alternative forms (e.g., alleles).

[0055] The phrase "gross mutant phenotype" refers to a significant difference or change in phenotype between the engineered non-human mice of the present disclosure and the "wild type."

[0056] The term "endogenous" refers to a nucleic acid sequence that occurs naturally in a cell or a non-human animal. For example, an endogenous Cacng1 sequence of a non-human animal refers to a natural Cacng1 sequence that occurs naturally in the Cacng1 locus of the non-human animal.

[0057] An "exogenous" molecule or sequence includes a molecule or sequence that is not normally present in a cell in that form. Normal presence includes presence in relation to a particular developmental stage and environmental conditions of the cell. An exogenous molecule or sequence may include, for example, a mutated form of a corresponding endogenous sequence in the cell, such as a humanized form of an endogenous sequence, or may include a sequence that corresponds to an endogenous sequence not within the cell but in a different form (i.e., not within a chromosome). In contrast, an endogenous molecule or sequence includes a molecule or sequence that is normally present in that form in a particular cell at a particular developmental stage under particular environmental conditions.

[0058] The term "heterologous" when used in reference to a nucleic acid or protein indicates that the nucleic acid or protein comprises at least two moieties that do not naturally occur together in the same molecule. For example, the term "heterologous" when used in reference to a nucleic acid moiety or protein moiety indicates that the nucleic acid or protein comprises two or more subsequences that do not naturally occur in the same relationship to each other (e.g., are not linked together). As an example, a "heterologous" region of a nucleic acid vector is a segment of nucleic acid within or attached to another nucleic acid molecule that is not naturally found associated with the other molecule. For example, a heterologous region of a nucleic acid vector can include a coding sequence flanked by sequences that are not naturally found associated with the coding sequence. Similarly, a "heterologous" region of a protein is a segment of amino acids within or attached to another peptide molecule that is not naturally found associated with the other peptide molecule (e.g., a fusion protein or tagged protein). Similarly, a nucleic acid or protein can include a heterologous label, or a heterologous secretion or localization sequence.

[0059] "Codon optimization" refers to the use of codon degeneracy, as seen by the multiplicity of combinations of three base pair codons that define amino acids, and generally involves the process of modifying a nucleic acid sequence to enhance expression in a particular host cell 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. For example, a nucleic acid encoding a Cas9 protein can be modified to replace a codon with a more frequently used codon compared to the native nucleic acid sequence in a given prokaryotic or eukaryotic cell, including, for example, a bacterial cell, a yeast cell, a human cell, a non-human cell, a mammalian cell, a rodent cell, a mouse cell, a rat cell, a hamster cell, or any other host cell. Codon usage tables are readily available, for example, in 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 by reference in its entirety for all purposes. Computer algorithms are also available that are directed to codon optimization of a particular sequence for expression in a particular host (see, eg, Gene Forge).

[0060] The term "locus" refers to a specific location of a gene (or sequence of interest), DNA sequence, polypeptide-encoding sequence or position on a chromosome of the genome of an organism. For example, "Cacng1 locus" can refer to a specific location of the Cacng1 gene, Cacng1 DNA sequence, Cacng1 coding sequence or Cacng1 position on a chromosome of the genome of an organism that is specified for where such sequence is located. "Cacng1 locus" can include regulators of the Cacng1 gene, including, for example, enhancers, promoters, 5' and / or 3' untranslated regions (UTRs), or combinations thereof.

[0061] The term "gene" refers to a DNA sequence in a chromosome that codes for a product (e.g., an RNA product and / or a polypeptide product) and includes the coding region interspersed with non-coding introns, as well as sequences adjacent to the coding region on 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 proximal (e.g., within 10 kb) or distal to the coding region of the gene, and they affect the level or rate of transcription and translation of the gene.

[0062] The term "allele" refers to a variant form of a gene. Some genes have multiple 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, and heterozygous when the two alleles are different.

[0063] A "promoter" is a regulatory region of DNA, typically including a TATA box, which can provide instructions to RNA polymerase II to begin RNA synthesis at the appropriate transcription initiation site for a particular polynucleotide sequence. A promoter can further include other regions that affect the rate of transcription initiation. The promoter sequences disclosed herein regulate the transcription of an operably linked polynucleotide. The promoter can be active in one or more of the cell types disclosed herein (e.g., eukaryotic cells, non-human mammalian cells, human cells, rodent cells, pluripotent cells, one-cell stage embryos, differentiated cells, or combinations thereof). The promoter can be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a temporally restricted promoter (e.g., a developmentally regulated promoter), or a spatially restricted promoter (e.g., a cell-specific or tissue-specific promoter). Examples of promoters can be found, for example, in WO 2013 / 176772, which is incorporated by reference in its entirety for all purposes.

[0064] "Operable linkage" or "operably linked" includes the juxtaposition of two or more components (e.g., a promoter and another sequence element) in such a manner that both components function normally such that at least one of the components can mediate the function and affect at least one of the other components. For example, a promoter can be operably linked to a coding sequence if the promoter controls the transcription level of the coding sequence depending on 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., a regulatory sequence can act at a distance to control the transcription of the coding sequence), and the like.

[0065] The term "variant" refers to a nucleotide sequence that differs from the most prevalent sequence in a population (eg, differs by a single nucleotide) or a protein sequence that differs from the most prevalent sequence in a population (eg, differs by a single amino acid).

[0066] 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 may 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.

[0067] "Sequence identity" or "identity" in the context of two polynucleotide or polypeptide sequences refers to the residues in the two sequences that are the same when aligned for maximum correspondence over a defined comparison window. When using percentage sequence identity with respect to proteins, residue positions that are not identical often differ by conservative amino acid substitutions, where an amino acid residue is replaced with another amino acid residue that has similar chemical properties (such as 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 upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity". Means for making this adjustment are well known. Typically, such means include ranking conservative substitutions as partial mismatches rather than full-length mismatches, thereby increasing the percentage of sequence identity. Thus, for example, conservative substitutions are given a score between zero and one, where identical amino acids are given a score of 1 and non-conservative substitutions are given a score of zero. The conservative substitution score is calculated, for example, as implemented in the PC / GENE program (Intelligenetics, Mountain View, Calif.).

[0068] "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 within the comparison window may contain additions or deletions (i.e., gaps) compared 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 in both sequences where identical nucleic acid bases or amino acid residues exist to obtain the number of matching positions, dividing the number of matching positions by the total number of positions within the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Unless otherwise specified (e.g., the shorter sequence includes a heterologous sequence linked to it), the comparison window is the full length of the shorter sequence of the two sequences to be compared.

[0069] Unless otherwise specified, sequence identity / similarity values ​​include values ​​obtained using GAP Version 10 or any equivalent program using the following parameters: % identity and % similarity for nucleotide sequences using a GAP Weight of 50 and a Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for amino acid sequences using a GAP Weight of 8 and a Length Weight of 2, and the BLOSUM62 scoring matrix. "Equivalent programs" include any sequence comparison program that generates alignments and percent sequence identity matches for any two sequences of interest that have identical nucleotide or amino acid residue matches when compared to the corresponding alignments generated by GAP Version 10.

[0070] The term "conservative amino acid substitution" refers to the substitution of an amino acid that is normally present in a sequence with a different amino acid that has a similar size, charge, or polarity. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue, such as isoleucine, valine, or leucine, with another non-polar residue. Similarly, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue with another polar residue, such as between arginine and lysine, between glutamine and asparagine, or between glycine and serine. Furthermore, the substitution of a basic residue, such as lysine, arginine, or histidine, with another basic residue, or the substitution of one acidic residue, such as aspartic acid or glutamic acid, with another acidic residue, are further examples of conservative substitutions. Examples of non-conservative substitutions include substitutions of a non-polar (hydrophobic) amino acid residue, e.g., isoleucine, valine, leucine, alanine, or methionine, for a polar (hydrophilic) residue, e.g., cysteine, glutamine, glutamic acid, or lysine, and / or substitutions of a polar residue for a non-polar residue. Exemplary amino acid classes are summarized below. [Table 2]

[0071] A "homologous" sequence (e.g., a nucleic acid sequence) includes a sequence that is either identical or substantially similar to a known reference sequence, such as 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 can include, for example, orthologous and paralogous sequences. Homologous genes are those that are derived from a common ancestral DNA sequence, for example, typically through a speciation event (orthologous genes) or through a gene duplication event (paralogous genes). "Orthologous" genes include genes from different species that have evolved from a common ancestral gene by speciation. Orthologs typically retain the same function during evolution. "Paralogues" genes include genes that are related by intragenomic duplication. Paralogs can develop new functions during evolution.

[0072] 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.

[0073] The term "reporter gene" refers to a nucleic acid having a sequence encoding a gene product (typically an enzyme) that is easily and quantitatively evaluated when a construct containing a reporter gene sequence operably linked to a heterologous promoter and / or enhancer element is introduced into a cell that contains (or can be made to contain) the factors necessary for activation of the promoter and / or enhancer element. Examples of reporter genes include, but are not limited to, the gene encoding β-galactosidase (lacZ), the bacterial chloramphenicol acetyltransferase (cat) gene, the firefly luciferase gene, the gene encoding β-glucuronidase (GUS), and genes encoding fluorescent proteins. "Reporter protein" refers to the protein encoded by the reporter gene.

[0074] The term "fluorescent reporter protein" as used herein means a reporter protein that is detectable based on fluorescence, which may be either directly from the reporter protein, from the activity of the reporter protein on a fluorogenic substrate, or from the protein having affinity for binding to a fluorescently tagged compound. Examples of fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, eGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, and ZsGreenl), yellow fluorescent proteins (e.g., YFP, eYFP, Citrine, Venus, YPet, PhiYFP, and ZsYellowl), blue fluorescent proteins (e.g., BFP, eBFP, eBFP2, Azurite, mKalamal, GFPuv, Sapphire, and T-sapphire), cyan fluorescent proteins (e.g., CFP, eCFP, Cerulean, CyPet, AmCyanl, and Midoriishi-C). yan), red fluorescent proteins (e.g., RFP, mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611, mRaspberry, mStrawberry and Jred), orange fluorescent proteins (e.g., mOrange, mKO, Kusabira-Orange, monomeric Kusabira-Orange, mTangerine and tdTomato), and any other suitable fluorescent protein whose presence in a cell is detectable by flow cytometry.

[0075] 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) occurs primarily through two conservative DNA repair pathways, 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.

[0076] NHEJ involves the repair of double-strand breaks in a nucleic acid by direct ligation of the cut ends to each other or to a foreign sequence without the need for a homologous template. Ligation of non-contiguous sequences by NHEJ can often result in deletions, insertions, or translocations near the double-strand break site. For example, NHEJ can also result in targeted integration of foreign donor nucleic acid through direct ligation of the cut ends to the ends of the foreign donor nucleic acid (i.e., NHEJ capture). Such targeted integration by NHEJ can be preferred for the insertion of foreign donor nucleic acid when the homology directed repair (HDR) pathway is not readily available (e.g., non-dividing cells, primary cells, and cells in which homology-based DNA repair is rarely performed). In addition, in contrast to homology directed repair, knowledge of large regions of sequence identity adjacent to the cleavage site is not required, which can be beneficial when attempting targeted insertion into organisms with genomes with limited knowledge of the genome sequence. This integration can proceed via ligation of blunt ends between the exogenous donor nucleic acid and the cleaved genomic sequence, or via ligation of sticky ends (i.e. ends with 5' or 3' overhangs) using an exogenous donor nucleic acid flanked by overhangs that are compatible with the ends generated by the nuclease agent in the cleaved genomic sequence.See, for example, US Patent Publication No. 2011 / 020722, WO 2014 / 033644, WO 2014 / 089290, and Maresca et al. (2013) Genome Res. 23(3):539-546, each of which is incorporated herein by reference in its entirety and for all purposes.If blunt ends are ligated, resection 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.

[0077] Recombination can also occur via homology directed repair (HDR) or homologous recombination (HR). HDR or HR includes forms 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., the 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 include mismatch repair of heteroduplex DNA that forms between the broken target and the donor, and / or synthesis-dependent strand annealing, in which the donor is used to resynthesize the genetic information, which becomes part of the target, and / or related processes. 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.

[0078] The term "antigen-binding protein" includes any protein that binds to an antigen. Examples of antigen-binding proteins include antibodies, antigen-binding fragments of antibodies, multispecific antibodies (e.g., bispecific antibodies), scFVs, bis-scFVs, diabodies, triabodies, tetrabodies, V-NARs, VHHs, VLs, F(abs), F(abs)2s, DVDs (dual variable domain antigen-binding proteins), SVDs (single variable domain antigen-binding proteins), bispecific T-cell engagers (BiTEs), or Davisbodies (U.S. Patent No. 8,586,713, incorporated herein by reference in its entirety for all purposes).

[0079] 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 non-covalently bound to each other. For example, an antibody or a fragment thereof can be operatively linked (e.g., by chemical conjugation, genetic fusion, non-covalent binding, 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.

[0080] The term "antigen" refers to a substance, either an entire 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 that have been appropriately genetically engineered to break immune tolerance.

[0081] 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 non-contiguous amino acids arranged in close proximity by tertiary folding of one or more proteins. Epitopes formed from contiguous amino acids (also known as linear epitopes) typically are retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding (also known as structural epitopes) are typically lost on treatment with denaturing solvents. Typically, an epitope contains at least 3, more commonly at least 5, 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 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996), which is incorporated by reference in its entirety and for all purposes.

[0082] The antibody paratopes described herein generally comprise at least the complementarity determining regions (CDRs) that specifically recognize a heterologous epitope (eg, the CDR3 regions of the heavy and / or light chain variable domains).

[0083] The term "antibody" includes immunoglobulin molecules that contain 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 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) 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 be abbreviated as HCDR1, HCDR2, and HCDR3, and light chain CDRs may be abbreviated as LCDR1, LCDR2, and LCDR3). The term "high affinity" antibody refers to an antibody that has a high affinity for its target epitope of about 10 -9 M or less (for example, about 1×10 -9 M, 1×10 -10 M, 1×10 -11 M, or approximately 1 x 10 -12 M)K D In one embodiment, the antibody has the formula: D is measured by surface plasmon resonance, e.g., BIACORE™, and in another embodiment, K D is measured by ELISA.

[0084] The term "bispecific antibody" includes antibodies that can selectively bind two or more epitopes. Bispecific antibodies generally include two different heavy chains, each of which specifically binds to a different epitope, i.e., 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 orders of magnitude lower than the affinity of the first heavy chain for the second epitope, or vice versa. The epitopes recognized by bispecific antibodies 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-terminus 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 can bind each heavy chain, or can bind each heavy chain and bind one or more of the epitopes bound by the heavy chain antigen binding region, or can bind each heavy chain and bind one or both of the heavy chains to one or both of the epitopes.

[0085] 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 includes (from N-terminus to C-terminus) the variable domain followed by a CDR, a CDR and a FR, and a combination thereof.H 1 domain, hinge, C H 2 domain, and C H The functional fragment of the heavy chain has three domains. D The heavy chain variable domain is encoded by a variable region nucleotide sequence and generally corresponds to the V domain 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 for 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".

[0086] The term "light chain" includes immunoglobulin light chain sequences from any organism, including 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. A 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 LThe light chain V gene segment and the light chain J gene segment of various organisms include gene segments. The sequences, locations, and nomenclature of the light chain V gene segment and the light chain J gene segment of 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". Light chains include, for example, light chains that do not selectively bind to 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.

[0087] As used herein, the term "complementarity determining region" or "CDR" includes an amino acid sequence encoded by a nucleic acid sequence of an immunoglobulin gene of an organism, which normally (i.e., in a wild-type animal) resides between two framework regions in the variable region of a light or heavy chain of an immunoglobulin molecule (e.g., an antibody or a T cell receptor). CDRs can be encoded, for example, by germline or rearranged sequences, and by, for example, naive or mature B 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, for example, as a result of splicing or joining of these sequences (e.g., VDJ rearrangement to form heavy chain CDR3).

[0088] Specific binding of an antigen-binding protein to a target antigen has an affinity of at least 10 6 , 10 7 , 10 8 , 10 9 , or 10 10 M -1Specific binding includes binding that is detectably higher in magnitude than and distinguishable from non-specific 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 type), while non-specific 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.

[0089] "Optional" or "optionally" means that the event or circumstance described thereafter may or may not occur, and is meant to include examples where the said event or circumstance occurs and examples where it does not occur.

[0090] The designation of a range of values ​​includes every integer within that range or all integers defining the range, and all subranges defined by the integers in the range.

[0091] Unless otherwise clear from the context, the term "about" encompasses values ​​within the standard error (eg, SEM) of measurement of the specified value.

[0092] The term "and / or" means and encompasses any and all possible combinations of one or more of the associated listed items, as well as the absence of combinations when interpreted in the alternative ("or").

[0093] The term "or" refers to any one member of a particular list and also includes any combination of members of that list.

[0094] The singular articles "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a protein" or "at least one protein" can include a plurality of proteins, including combinations thereof.

[0095] Statistically significant means p≦0.05. (Mode for carrying out the invention)

[0096] I. Overview Disclosed herein are non-human animal cells, animals, and genomes that contain an exogenous sequence that is found to be specifically expressed in skeletal muscle, and reagents for producing the same. In some embodiments, the exogenous sequence is integrated into the endogenous locus of a gene.

[0097] Skeletal muscle is one of the three significant types of muscle tissue in the human body. Each skeletal muscle is made up of thousands of muscle fibers bound together by connective tissue sheaths. The individual bundles of muscle fibers in a skeletal muscle are known as fasciculi. The outermost connective tissue sheath that surrounds the entire muscle is known as the epimysium. The connective tissue sheath that covers each fasciculi is known as the epimysium, and the innermost sheath that surrounds the individual muscle fibers is known as the endomysium. Each muscle fiber contains myofibrils, which contain multiple myofilaments.

[0098] When bundled together, all myofibrils are arranged in a unique striated pattern that forms the sarcomere, the basic contractile unit of skeletal muscle. The two most significant myofilaments are actin and myosin filaments that are characteristically arranged to form various bands on skeletal muscle. Stem cells that differentiate into mature muscle fibers are known as muscle satellite cells that can reside between the basement membrane and the sarcolemma (the cell membrane that surrounds the striated muscle fiber cells). When stimulated by growth factors, stem cells differentiate and proliferate to form new muscle fiber cells.

[0099] The main function of skeletal muscle is carried out through the intrinsic excitation-contraction coupling process of skeletal muscle. As muscles are connected to bone-tendons, muscle contraction leads to the movement of that bone that allows the execution of a specific movement. Skeletal muscles also provide structural support and help maintain body posture. Skeletal muscles also act as a storage source of amino acids that can be used by different organs of the body to synthesize organ-specific proteins. Skeletal muscles also play a central role in maintaining homeostasis (thermostasis) and act as a source of energy during starvation. CACNG1 protein was found to be specifically expressed in skeletal muscle.

[0100] In some embodiments, the present disclosure provides non-human animal cells and non-human animals having heterologous Cacng1 sequences in the genome of the non-human animal cells or non-human animals provided herein.The heterologous Cacng1 sequences can be inserted into endogenous Cacng1 locus, thus providing non-human animal cells and non-human animals having genetically modified endogenous Cacng1 locus.

[0101] In some embodiments, provided herein is a nucleic acid encoding a heterologous sequence encoding at least a portion of the Cacng1 sequence, and a method for producing a non-human animal cell and a non-human animal having such a nucleic acid. In some embodiments, such a nucleic acid has a sequence that facilitates editing of the non-human animal (e.g., a loxP site) adjacent to the sequence encoding the Cacng1 gene.

[0102] In some embodiments, provided herein are antibodies against chimeric CACNG1 proteins produced by the non-human animal cells and / or non-human animals of the present disclosure.

[0103] In some embodiments, the disclosure provides methods that can be used to generate such non-human animals (e.g., rodents such as rats or mice), cells and / or tissues derived from such non-human animals, and nucleotides (e.g., targeting vectors, genomes, etc.).

[0104] In some embodiments, the present disclosure also provides a non-human animal genome comprising the endogenous CACNG1 locus genetically modified with heterologous Cacng1 sequence.In some embodiments, the heterologous Cacng1 sequence encodes human CACNG1 protein sequence.In some examples, all or part of the CACNG1 domain is encoded by the segment of endogenous Cacng1 locus that is deleted and replaced with heterologous Cacng1 sequence.

[0105] In some embodiments, non-human animals are provided that comprise a humanized Cacng1 locus and express a humanized or chimeric CACNG1 protein from the humanized Cacng1 locus, as well as methods of using the non-human animals (e.g., rodents such as rats or mice), cells and tissues derived from the non-human animals, and nucleotides useful in producing the animals (e.g., targeting vectors, genomes, etc.).

[0106] In some embodiments, described herein are non-human animals comprising a genetically modified Cacng1 locus encoding a modified CACNG1 protein, wherein the modified CACNG1 protein comprises a domain of a human CACNG1 sequence, and wherein all or a portion of the domain is encoded by a segment of the endogenous Cacng1 locus that has been deleted and replaced with an orthologous human CACNG1 sequence, and the non-human animal expresses the modified Cacng1 protein.

[0107] In some embodiments, a domain of the human CACNG1 sequence is encoded by a segment of the endogenous Cacng1 locus that has been deleted and replaced with a heterologous sequence. Such a domain can be a human Cacng1 extracellular domain. Suitable sequences encoding the extracellular domain contemplated by the present disclosure include a human extracellular domain corresponding to amino acids 30-108 (SEQ ID NO: 12), amino acids 156-179 (SEQ ID NO: 20), or both, of the CACNG1 protein upon translation in a given cell.

[0108] In some embodiments, at least two domains of the human CACNG1 sequence are encoded by segments of the endogenous Cacng1 locus in the humanized mouse model. Non-limiting illustrative examples of domains of the human CACNG1 sequence include the cytoplasmic domain, the transmembrane domain, and the extracellular domain. In some examples, all or part of each domain can be encoded by a segment of the endogenous Cacng1 locus that has been deleted and replaced with an orthologous human CACNG1 sequence. In other examples, the cytoplasmic domain and the extracellular domain can be optionally encoded by the endogenous genome. In some embodiments, all or part of both the cytoplasmic domain and the transmembrane domain are encoded by a segment of the endogenous Cacng1 locus that has been deleted and replaced with an orthologous human CACNG1 sequence. In some embodiments, all of the cytoplasmic domain, the transmembrane domain, and the extracellular domain are encoded by a segment of the endogenous Cacng1 locus that has been deleted and replaced with an orthologous human CACNG1 sequence. The latter incorporates multiple humanized domains of the human CACNG1 gene into a non-human genome, while the former allows for humanization of the extracellular membrane while preserving endogenous domains of domains understood to be located within the membrane and intracellularly. A suitable sequence encoding a cytoplasmic domain of the present disclosure upon translation in a cell produces a human cytoplasmic domain corresponding to amino acids 1-10 (SEQ ID NO:8), amino acids 130-134 (SEQ ID NO:16), amino acids 205-222 (SEQ ID NO:24), or any combination thereof, of the CACNG1 protein. A suitable sequence encoding a transmembrane domain of the present disclosure upon translation in a cell produces a human transmembrane domain corresponding to amino acids 11-29 (SEQ ID NO:10), amino acids 109-129 (SEQ ID NO:14), amino acids 135-155 (SEQ ID NO:18), amino acids 180-204 (SEQ ID NO:22), or any combination thereof, of the CACNG1 protein. Consequently, in some alternative embodiments, all or part of the cytoplasmic or transmembrane domain is encoded by an endogenous Cacng1 sequence of the non-human animal.

[0109] In some embodiments, the non-human animal or non-human animal genome described herein encodes an orthologous human CACNG1 sequence in place of the endogenous Cacng1 sequence of mouse. In some embodiments, the non-human animal or non-human animal genome comprises a sequence selected from the group consisting of the nucleic acid sequence set forth as SEQ ID NO:5, the nucleic acid sequence set forth as SEQ ID NO:27, and the nucleic acid sequence set forth as SEQ ID NO:28.

[0110] In some embodiments, the human CACNG1 sequence is a human CACNG1 sequence encoded by a segment of the endogenous Cacng1 locus that has been deleted and replaced with a human Cacng1 sequence encoding the entire γ1 domain of a voltage-gated calcium channel.

[0111] In some embodiments, the non-human animal or non-human animal genome described herein is heterozygous for the endogenous Cacng1 locus genetically modified.In some embodiments, the non-human animal or non-human animal genome is homozygous for the endogenous Cacng1 locus genetically modified.

[0112] In some embodiments, a segment of the endogenous Cacng1 locus is deleted and replaced with an exogenous Cacng1 sequence. In some of these examples, the deleted endogenous Cacng1 locus may include a segment of the 3' untranslated region, a segment of coding exon 1, a segment of intron 1, a segment of coding exon 2, a segment of intron 2, a segment of coding exon 3, a segment of intron 3, a segment of coding exon 4, or a combination of the aforementioned segments of the endogenous Cacng1 locus.

[0113] In some embodiments, the human CACNG1 sequence may be used to replace a locus in a non-human animal or cell. In such embodiments, the orthologous human CACNG1 sequence replacing a segment of the endogenous locus may include any one of the segments of the 3' untranslated region of the human CACNG1 sequence, exon 1 of the human CACNG1 sequence, intron 1 of the human CACNG1 sequence, exon 2 of the human CACNG1 sequence, intron 2 of the human CACNG1 sequence, exon 2 of the human CACNG1 sequence, intron 3 of the human CACNG1 sequence, exon 3 of the human CACNG1 sequence, intron 4 of the human CACNG1 sequence, exon 4 of the human CACNG1 sequence, or any combination thereof.

[0114] In some embodiments, the non-human animal is a mammal, or the non-human animal genome is a mammalian genome. In some embodiments, the non-human animal can be a rodent, or the non-human animal genome can be a rodent genome. In some embodiments, the non-human animal can be a rat or a mouse, or the non-human animal genome can be a rat genome or a mouse genome.

[0115] In some embodiments, the heterologous sequence integrated into the genome of the non-human animal or non-human animal genome encodes a human Cacng1 extracellular domain, a human Cacng1 transmembrane domain, and a human Cacng1 domain.

[0116] In some embodiments, the heterologous sequence integrated into the genome of the non-human animal or non-human animal genome encodes at least two domains of the human CACNG1 sequence, non-limiting examples of the two or more domains include a first cytoplasmic domain, a first transmembrane domain, a first extracellular domain, a second transmembrane domain, a second cytoplasmic domain, a third transmembrane domain, a second extracellular domain, a fourth transmembrane domain, and a third cytoplasmic domain.

[0117] In some embodiments, the heterologous sequence to be integrated into the genome of the non-human animal or non-human animal genome comprises a suitable sequence to encode amino acids 1-10 (cytoplasmic domain), amino acids 11-29 (transmembrane domain), amino acids 30-108 (extracellular domain), amino acids 109-129 (transmembrane domain), amino acids 130-134 (cytoplasmic domain), amino acids 135-155 (transmembrane domain), amino acids 156-179 (extracellular domain), amino acids 180-204 (transmembrane domain), amino acids 205-222 cytoplasmic domain, or any suitable combination thereof.

[0118] In some embodiments, a non-human animal cell is provided herein that comprises a genetically modified endogenous Cacng1 locus that encodes a modified CACNG1 protein, the modified Cacng1 protein comprising a domain of a human CACNG1 sequence, and all or part of the domain is encoded by a segment of the endogenous Cacng1 locus that is deleted and replaced with an orthologous human CACNG1 sequence. The non-human animal cell can be a skeletal muscle cell, a pluripotent cell, an ES cell, or a germ cell.

[0119] In some embodiments, the disclosure further provides methods of making the non-human animals or any of the reagents necessary to make the non-human animals described herein.

[0120] A. Voltage-gated calcium channel auxiliary subunit gamma 1 (CACNG1) The cells and non-human animals described herein generally contain exogenous sequences encoding segments of the CACNG1 protein (e.g., human CACNG1 protein domains). Voltage-gated calcium channels are generally composed of five subunits. The protein encoded by the CACNG1 gene represents one of these subunits. Furthermore, the protein encoded by the CACNG1 gene gamma is one of two known gamma subunit proteins. This particular gamma subunit is part of the skeletal muscle 1,4-dihydropyridine-sensitive calcium channel, an integral membrane protein that plays a role in excitation-contraction coupling. This gene is part of the functionally diverse eight-member protein subfamily of the PMP-22 / EMP / MP20 family, located within a cluster with two family members that function as transmembrane AMPA receptor regulatory proteins (TARPs).

[0121] The gene encoding human CACNG1 (CACNG1) is located on the long arm of chromosome 17. CACNG1 contains four exons and is approximately 12,244 bases in length.

[0122] An exemplary sequence of human CACNG1 is assigned NCBI accession number NM_0007582.2 (see FIG. 4A). An exemplary sequence of mouse Cacng1 is assigned NCBI accession number NM_000727.4 (see FIG. 4A). An exemplary human CACNG1 protein is assigned UniProt accession number O70578 (see FIG. 4A and FIG. 5). An exemplary mouse CACNG1 protein is assigned UniProt accession number Q06432 (see FIG. 4A and FIG. 5). An example of a human or humanized CACNG1 protein encoded by a modified non-human Cacng1 locus (e.g., 7451) is described in FIG. 5. An exemplary rat CACNG1 protein is assigned NCBI reference sequence: NP_062128.1. An exemplary orangutan Cacng1 protein is assigned NCBI reference sequence: XP_002827789.2.

[0123] In some embodiments, the present disclosure provides a non-human animal, a non-human animal cell, or a non-human animal genome comprising a nucleic acid sequence encoding a heterologous CACNG1 protein or a portion thereof. Such a nucleic acid sequence encoding a heterologous CACNG1 protein or a portion thereof may comprise (i) a nucleic acid sequence comprising exon 1 or a portion thereof of the human CACNG1 gene, (ii) a nucleic acid sequence comprising exon 2 or a portion thereof of the human CACNG1 gene, (iii) a nucleic acid sequence comprising exon 3 or a portion thereof of the human CACNG1 gene, (iv) a nucleic acid sequence comprising exon 4 or a portion thereof of the human CACNG1 gene, or (v) any combination of (i)-(iv).

[0124] Furthermore, the nucleic acid sequence integrated into the genome of the non-human animal, non-human animal cell, or non-human animal genome described herein may include an intron. In some embodiments, the nucleic acid sequence encoding the heterologous CACNG1 protein or a portion thereof includes (i) a nucleic acid sequence including exon 1 or a portion thereof of the human CACNG1 gene, (ii) a nucleic acid sequence including intron 1 or a portion thereof of the human CACNG1 gene, (iii) a nucleic acid sequence including exon 2 or a portion thereof of the human CACNG1 gene, (iv) a nucleic acid sequence including intron 2 or a portion thereof of the human CACNG1 gene, (v) a nucleic acid sequence including exon 3 or a portion thereof of the human CACNG1 gene, (vi) a nucleic acid sequence including intron 3 or a portion thereof of the human CACNG1 gene, (v) a nucleic acid sequence including exon 4 or a portion thereof of the human CACNG1 gene, (vii) a nucleic acid sequence of the 3' untranslated region (UTR) of the human CACNG1 gene, or (v) any combination of (i)-(iv).

[0125] In some embodiments, the non-human animal, non-human animal cell, or non-human animal genome described herein encodes a humanized coding region of CACNG1 protein (i.e., some mouse regulatory regions and selected human non-coding / coding regions). In some embodiments, the nucleic acid sequence encoding a heterologous CACNG1 protein or a portion thereof may comprise, consist essentially of, or consist of a nucleic acid sequence encoding a humanized mouse / human CACNG1 protein, such as a nucleic acid sequence selected from the group consisting of the nucleic acid sequence described as SEQ ID NO:5, the nucleic acid sequence described as SEQ ID NO:27, and the nucleic acid sequence described as SEQ ID NO:28. Any such nucleic acid may be integrated in the endogenous Cacng1 locus. In some embodiments, the nucleic acid sequence encoding a heterologous CACNG1 protein or a portion thereof may replace an orthologous endogenous nucleic acid sequence encoding an endogenous CACNG1 protein or a portion thereof.

[0126] In some embodiments, the disclosure provides a non-human animal, non-human animal cell, or non-human animal genome, wherein a heterologous CACNG1 protein, or a portion thereof, comprises: (i) the amino acid sequence set forth as SEQ ID NO:8, (ii) the amino acid sequence set forth as SEQ ID NO:10, (iii) the amino acid sequence set forth as SEQ ID NO:12, (iv) the amino acid sequence set forth as SEQ ID NO:14, (v) the amino acid sequence set forth as SEQ ID NO:16, (vi) the amino acid sequence set forth as SEQ ID NO:18, (vii) the amino acid sequence set forth as SEQ ID NO:20, (viii) the amino acid sequence set forth as SEQ ID NO:22, (ix) the amino acid sequence set forth as SEQ ID NO:24, or (x) any combination of (i)-(ii).

[0127] B. Engineered Cacng1 Non-Human Animals The present disclosure provides non-human animals with loss of function of CACNG1 protein, and chimeric animals (e.g., transgenic rodents expressing humanized CACNG1 protein). Humanized Cacng1 locus can be a Cacng1 locus in which the entire Cacng1 gene is replaced with a corresponding orthologous human CACNG1 sequence, or a Cacng1 locus in which only a part of the Cacng1 gene is replaced with a corresponding orthologous human CACNG1 sequence (i.e., humanized). Optionally, the corresponding orthologous human CACNG1 sequence is modified to be codon-optimized based on codon usage in the non-human animal. The replaced (i.e., humanized) region can include coding regions, such as exons, non-coding regions, such as introns, non-translated regions, or regulatory regions (e.g., promoters, enhancers, or transcriptional repressor binding elements), or any combination thereof. As an example, exons corresponding to one, two, three, four, or all four exons of the human CACNG1 gene can be humanized. For example, exons corresponding to exons 1-4 of the human CACNG1 gene can be humanized. Alternatively, a region of Cacng1 that encodes an epitope recognized by an anti-human CACNG1 antigen-binding protein can be humanized. As another example, one or more or all of the N-terminal cytoplasmic domain, the transmembrane domain, or the intracellular domain can be humanized. For example, all or a portion of the region of the Cacng1 locus that encodes the extracellular domain can be humanized, all or a portion of the region of the Cacng1 locus that encodes the cytoplasmic domain can be humanized, and / or all or a portion of the region of the Cacng1 locus that encodes the transmembrane domain can be humanized. In one example, all or only a portion of the region of the Cacng1 locus that encodes the transmembrane domain is humanized, all or only a portion of the region of the Cacng1 locus that encodes the cytoplasmic domain is humanized, or all or only a portion of the region of the Cacng1 locus that encodes the extracellular domain (i.e., the region that can serve as an epitope) is humanized.For example, the region of the Cacng1 locus that codes for the extracellular domain can be humanized so that a chimeric Cacng1 protein is produced with an endogenous N-terminal cytoplasmic domain, an endogenous transmembrane domain, and a humanized transmembrane domain (epitope). Similarly, introns corresponding to one, two, three, or all four introns of the human CACNG1 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 particular example, the 3'UTR is humanized, but 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 with replaced human orthologous sequences. For example, a humanized Cacng1 locus can include an endogenous non-human animal Cacng1 promoter.

[0128] The Cacng1 protein encoded by the humanized Cacng1 locus may include one or more domains derived from a mammalian CACNG1 protein (e.g., human). For example, the Cacng1 protein may include one or more or all of the human extracellular domain, the human CACNG1 transmembrane domain, and the human CACNG1 cytoplasmic domain. As an example, the Cacng1 protein may include only the human CACNG1 extracellular domain. Optionally, the Cacng1 protein encoded by the humanized Cacng1 locus may also further include one or more domains derived from an endogenous (i.e., natural) non-human animal Cacng1 protein.

[0129] A domain derived from the human CACNG1 protein can be encoded by a fully humanized sequence (i.e., the entire sequence encoding the domain is replaced with the orthologous human CACNG1 sequence) or can be encoded by a partially humanized sequence (i.e., a portion of the sequence encoding the domain is replaced with the orthologous human CACNG1 sequence, and the remaining endogenous (i.e., native) sequence encoding the domain encodes the same amino acids as the orthologous human CACNG1 sequence, such that the encoded domain is identical to that domain in the human CACNG1 protein).

[0130] As an example, the Cacng1 protein encoded by the humanized Cacng1 locus can include a human CACNG1 extracellular domain (e.g., a human epitope). Optionally, the human CACNG1 transmembrane domain comprises, consists essentially of, or consists of a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in Figure 9A, and the CACNG1 protein retains the activity of native CACNG1 (i.e., retains its function in skeletal muscle).

[0131] As another example, the CACNG1 protein encoded by the humanized Cacng1 locus can include a human transmembrane or cytoplasmic CACNG1 domain. Optionally, the human CACNG1 extracellular domain comprises, consists essentially of, or consists of a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to FIG. 9A, and the Cacng1 protein retains the activity of native CACNG1.

[0132] Optionally, the humanized Cacng1 locus may include other elements. Examples of such elements may include a selection cassette, a reporter gene, a recombinase recognition site, or other elements. Alternatively, the humanized Cacng1 locus may lack other elements (e.g., 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, and the two exons encoding the Cre recombinase are separated by an intron, preventing its expression in prokaryotic cells. Such recombinases may further include a nuclear localization signal to promote localization to the nucleus (e.g., NLS-Crei). Recombinase recognition sites include nucleotide sequences that are recognized by site-specific recombinases and can function as substrates for recombination events. Examples of recombinase recognition sites include FRT, FRT11, FRT71, attp, att, rox, and lox sites, such as loXp, lox511, lox2272, lox66, lox71, loxM2, and lox5171.

[0133] Other elements, such as a reporter gene or a selection cassette, may be a self-deletion cassette flanked by recombinase recognition sites. See, for example, U.S. Pat. No. 8,697,851 and U.S. Patent Application Publication No. 2013 / 0312129, each of which is incorporated herein by reference in its entirety for all purposes. As an example, the self-deletion cassette may include a Crei gene (comprising two exons encoding Cre recombinase, separated by an intron) operably linked to a mouse Prm1 promoter, and a neomycin resistance gene operably linked to a human ubiquitin promoter. By employing the Prm1 promoter, the self-deletion cassette may be specifically deleted in male germ cells of the F0 animal. A polynucleotide encoding a selection marker may be operably linked to a promoter active in the cells being targeted. Examples of promoters are described elsewhere herein. As another specific example, a self-deletion selection cassette can 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.

[0134] An example of a humanized Cacng1 locus (e.g., a humanized mouse Cacng1 locus) is a locus in which coding exons 1-4 have been replaced with corresponding human sequences flanked by a Neo self-deletion cassette. These exons encode the coding domain of Cacng1. The coding exon 1, intron 1, coding exons 2-4 (and intervening introns), and a portion of the 82 bp 3' untranslated region (UTR) of mouse Cacng1 are replaced with the corresponding portions of human CACNG1 coding exon 1 sequences, intron 1, coding exons 2-4 (and intervening introns), the complete 3'UTR, and an additional 158 bp after the 3'UTR, with the first 15 bp of the coding sequence remaining as mouse sequences, providing such a non-human animal. See Figures 4B and 4C.

[0135] Cacng1 hu / hu mouse

[0136] The present disclosure contemplates cells and non-human animals that comprise an exogenous Cacng1 locus. In some embodiments, a cell or non-human animal that comprises a heterologous Cacng1 locus can express a heterologous CACNG1 protein, or a chimeric CACNG1 protein in which one or more fragments of a native Cacng1 protein are replaced with a corresponding fragment from a heterologous CACNG1 sequence (e.g., all or part of the extracellular domain, all of the CACNG1 coding region).

[0137] In some embodiments, the cells and non-human animals disclosed herein comprise an exogenous nucleic acid sequence encoding amino acids 1-10, amino acids 11-29, amino acids 30-108, amino acids 109-129, amino acids 130-134, amino acids 135-155, amino acids 156-179, amino acids 180-204, amino acids 205-222, and / or combinations thereof, of a human CACNG1 protein.

[0138] Loss of function Cacng1 - / - mouse

[0139] Using gene editing technology, CACNG1- / - Mice were generated to determine whether the deletion of CACNG1 affects skeletal muscle mass or function. Some of the non-human animals described herein lack the Cacng1 locus, so these non-human animals can provide a holistic understanding of the effects of loss of function on Cacng1 protein.

[0140] In some embodiments, a non-human animal, a non-human animal cell, or a non-human animal genome is provided that comprises a knockout mutation of an endogenous Cacng1 gene. In some embodiments, such a knockout mutation may comprise a deletion of the Cacng1 gene or a portion thereof. In some examples, the knockout mutation may comprise a deletion of the entire coding sequence of the Cacng1 gene. In some embodiments, the Cacng1 - / - The human animal genome does not express any CACNG1 protein.

[0141] In some embodiments, the non-human animal, non-human animal cell, or non-human animal genome does not exhibit any gross mutant phenotype (i.e., does not exhibit any measurable characteristics, particularly muscle strength, structure, or functional traits, that are statistically significant from their wild-type counterparts).

[0142] C. Non-human cells and animals containing a heterologous Cacng1 locus Provided herein are non-human animal cells and non-human animals that comprise the humanized Cacng1 locus described herein.The cells or non-human animals can be heterozygous or homozygous for the humanized Cacng1 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.

[0143] The non-human animal cell provided herein can be, for example, any non-human cell that contains a Cacng1 locus or genomic locus that is homologous or orthologous to the human CACNG1 locus. The cell can be a eukaryotic cell, including, for example, a fungal cell (e.g., yeast), a plant cell, an animal cell, a mammalian cell, a non-human mammalian cell, and a human cell. The animal can be, for example, a mammal, a fish, or a bird. The mammalian cell can be, for example, a non-human mammalian cell, a rodent cell, a rat cell, a mouse cell, or a hamster cell. Other non-human mammals include, for example, non-human primates monkeys, apes, orangutans, cats, dogs, rabbits, horses, bulls, deer, bison, livestock (e.g., bovine species such as cows, steers, etc.; ovine species such as sheep, goats, etc.; and porcine species such as pigs and hogs, etc.). Birds include, for example, chickens, turkeys, ostriches, geese, ducks, etc. Domesticated and agricultural animals are also included. The term "non-human" excludes humans.

[0144] The cells can also be in any kind of undifferentiated or differentiated state. For example, the cells can be totipotent, pluripotent (e.g., human pluripotent or non-human pluripotent such as mouse embryonic stem (ES) or rat ES cells), or non-pluripotent. Totipotent cells include undifferentiated cells that can give rise to any cell type, and pluripotent cells include undifferentiated cells that retain the ability to develop into multiple differentiated cell types. Such pluripotent and / or totipotent cells can be, for example, ES or ES-like cells, such as induced pluripotent stem (iPS) cells. ES cells include embryo-derived totipotent or pluripotent cells that can be introduced into an embryo to 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 any cell of the three vertebrate germ layers (endoderm, ectoderm, and mesoderm).

[0145] The cell provided herein can be a germ cell (e.g., sperm or oocyte). The cell can be a mitotically competent or mitotically inactive cell, a meiotically competent or meiotically inactive cell. Similarly, the cell disclosed herein can also be a primary somatic cell or a cell that is not a primary somatic cell. Somatic cells include any cell that is not a gamete, germ cell, gametocyte, or undifferentiated stem cell. For example, the cell disclosed herein can be a muscle cell, such as a skeletal muscle cell.

[0146] Suitable cells provided herein also include primary cells. Primary cells include cells or cell cultures directly isolated from organisms, organs or tissues. Primary cells include cells that are not transformed or immortalized. Primary cells include any cells obtained from organisms, organs or tissues that have not previously been passaged in tissue culture, or have previously been passaged in tissue culture but cannot be passaged indefinitely in tissue culture. Such cells can be isolated by conventional techniques and include, for example, muscle cells (e.g., skeletal muscle cells).

[0147] Other suitable cells provided herein include immortalized cells. Immortalized cells include cells from multicellular organisms that do not normally proliferate indefinitely, but can avoid normal cell senescence by mutation or modification and continue to divide instead. Such mutation or modification can occur naturally or be induced intentionally. An example of an immortalized cell line is a myofiber cell line. Immortalized or primary cells include cells that can be used for cell culture or for expressing recombinant genes or recombinant proteins.

[0148] 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.

[0149] The cells provided herein can be normal, healthy cells, or can be diseased or mutated cells.

[0150] Non-human animals comprising the humanized Cacng1 locus described herein can be produced by methods described elsewhere herein. Animals can be, for example, mammals, fish, or birds. Non-human mammals include, for example, non-human primates monkeys, apes, orangutans, cats, dogs, horses, bulls, deer, bison, sheep, rabbits, rodents (e.g., mice, rats, hamsters, and guinea pigs) and livestock (e.g., bovine species, such as cows and steers; ovine species, such as sheep and goats; and porcine species, such as pigs and hogs). Birds include, for example, chickens, turkeys, ostriches, geese, and ducks. Domesticated animals and agricultural animals are also included. The term "non-human animals" excludes humans. Preferred non-human animals include, for example, rodents, such as mice and rats.

[0151] Non-human animals can be from any genetic background. For example, suitable mice can be from 129, C57BL / 6, 129 and C57BL / 6 hybrids, BALB / c, or Swiss Webster. Examples of 129 strains include 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / Svlm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, and 129T2. See, for example, Festing et al. (1999) Mammalian Genome 10:836, which is incorporated herein by reference in its entirety for all purposes. Examples of C57BL strains include C57BL / A, C57BL / An, C57BL / GrFa, C57BL / Kal_wN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr and C57BL / Ola. Suitable mice 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).

[0152] 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 the Fischer rat strain, e.g., Fisher F344 or Fisher F6. Rats may be obtained from strains derived from hybrids of two or more of the above strains. For example, suitable rats may be from the DA or ACI strain. The ACI rat strain is dark agouti in color with white abdomen and feet and RT1. av1The Dark Agouti (DA) rat strain has agouti-colored fur and is characterized by the RT1 haplotype. The strain is available from a variety of sources, including Harlan Laboratories. av1 The rat is characterized by being a haplotype.The rat is available from various sources, including Charles River and Harlan Laboratories.Some suitable rats can be from inbred rat lines.See, for example, US Patent Application Publication No. 2014 / 0235933, which is incorporated herein by reference in its entirety for all purposes.

[0153] III. Methods for generating non-human animals containing a heterologous Cacng1 locus As disclosed elsewhere herein, various methods are provided for producing non-human animals that contain heterologous Cacng1 locus.Any convenient method or protocol for producing genetically modified organisms is suitable for producing such genetically modified non-human animals.See, for example, Cho et al.(2009)Current Protocols in Cell Biology 42:19.11:19.11.1-19.11.22 and Gama Sosa et al.(2010)Brain Struct.Funct.214(2-3):91-109, each of which is incorporated herein by reference in its entirety for all purposes.Such genetically modified non-human animals can be produced, for example, through targeted gene knock-in at Cacng1 locus.

[0154] For example, a method for producing a non-human animal comprising a humanized Cacng1 locus may include (1) modifying the genome of a pluripotent cell to comprise a humanized Cacng1 locus, (2) identifying or selecting a genetically modified pluripotent cell comprising a humanized Cacng1 locus, (3) introducing the genetically modified pluripotent cell into an embryonic cell of a non-human animal host in vitro, and (4) implanting the host embryonic cell into a surrogate mother for gestation. Optionally, the host embryo (e.g., non-human ES cell) comprising the modified pluripotent cell may be incubated to the blastocyst stage and then implanted into a surrogate mother for gestation to produce an F0 non-human animal. The surrogate mother may then produce an F0 generation non-human animal comprising a humanized Cacng1 locus.

[0155] 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.

[0156] The screening step may include, for example, a quantitative assay to evaluate the allelic alteration (MOA) of parental chromosomes. For example, the quantitative assay may be carried out 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.

[0157] Other examples of suitable quantitative assays include fluorescent in situ hybridization (FISH), comparative genomic hybridization, isothermal DNA amplification, quantitative hybridization to immobilized probes, INVADER® probes, TAQMAN® molecular beacon probes, or ECLIPSE™ probe technology (see, e.g., U.S. Patent Application Publication No. 2005 / 0144655, which is incorporated by reference in its entirety and for all purposes).

[0158] An example of a suitable pluripotent cell is an embryonic stem (ES) cell (e.g., a mouse ES cell or a rat ES cell). Modified pluripotent cells can be generated through recombination, for example, by (a) introducing into a cell one or more targeting vectors comprising an insert nucleic acid flanked by 5' and 3' homology arms corresponding to 5' and 3' target sites, the insert nucleic acid comprising a heterologous Cacng1 locus, and (b) identifying at least one cell comprising the insert nucleic acid integrated into its genome at the target genomic locus. Alternatively, modified pluripotent cells can be generated by (a) introducing into a cell (i) a nuclease agent, where the nuclease agent induces a nick or double-stranded break at a recognition site in a target genomic locus, and (ii) one or more targeting vectors comprising an insert nucleic acid flanked by 5' and 3' homology arms corresponding to the 5' and 3' target sites located sufficiently close to the recognition site, where the insert nucleic acid comprises a heterologous Cacng1 locus, and (c) identifying at least one cell comprising a modification (e.g., integration of the insert nucleic acid) at the target genomic locus. Any nuclease agent that induces a nick or double-stranded break at a desired recognition site can be used. Examples of suitable nucleases include TAL effector nucleases (TALENs), zinc finger nucleases (ZFNs), meganucleases, and CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats) / CRISPR-associated (Cas) systems, or components of such systems (e.g., CRISPR / Cas9).See, for example, US Patent Application Publication No. 2013 / 0309670 and US Patent Application Publication No. 2015 / 0159175, each of which is incorporated herein by reference in its entirety for all purposes.

[0159] Donor cells can be introduced into host embryo at any cell stage, such as blastocyst stage or before morula stage (i.e. 4-cell stage or 8-cell stage). Progeny that can transmit genetic modification through germline are generated.See, for example, U.S. Patent No. 7,294,754, which is incorporated herein by reference in its entirety for all purposes.

[0160] Alternatively, the method of producing a non-human animal as described elsewhere herein may include (1) modifying the genome of a one-cell stage embryo to contain a heterologous Cacng1 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 for gestation. Offspring capable of transmitting the genetic modification through the germ line are generated.

[0161] Nuclear transfer techniques can also be used to generate non-human mammals. Briefly, methods for nuclear transfer can 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 the enucleated oocyte to form a reconstituted cell, (4) implanting the reconstituted cell into the uterus of an animal to form an embryo, and (5) allowing the embryo to develop. In the methods, oocytes are generally taken from dead animals, but can also be isolated from either the oviduct and / or ovary of a live animal. Injecting the donor cell or donor nucleus into the enucleated oocyte to form a reconstituted cell can be done by microinjection of the donor cell under the zona pellucida prior to fusion. Fusion can be induced by 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 can be activated by electrical and / or non-electrical means before, during, and / or after fusion of the nuclear donor with the recipient oocyte. Activation methods include electrical pulses, chemically induced bombardment, sperm penetration, increasing divalent cation levels in the oocyte, and reducing phosphorylation of cellular proteins in the oocyte (as by kinase inhibitors). The activated reconstituted cells or embryos can be cultured in culture medium and then implanted into the uterus of an animal. See, for example, U.S. Patent Application Publication No. 2008 / 0092249, WO 1999 / 005266, U.S. Patent Application Publication No. 2004 / 0177390, WO 2008 / 017234, and U.S. Patent No. 7,612,250, each of which is incorporated herein by reference in its entirety and for all purposes.

[0162] Various methods presented herein allow the generation of genetically modified non-human F0 animals, in which the cells of the genetically modified F0 animals contain humanized Cacng1 locus. It is recognized that the number of cells in the F0 animals that have heterologous Cacng1 locus will vary depending on the method used to generate the F0 animals. By introducing donor ES cells from the corresponding organism (e.g., 8-cell stage mouse embryo) into pre-morula stage embryos, for example, via VELOCIMOUSE® method, the proportion of the cell population of the F0 animal that contains cells that have the nucleotide sequence of interest, including targeted genetic modification, will be increased. For example, at least 50%, 60%, 65%, 70%, 75%, 85%, 86%, 87%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cell contributions of the non-human F0 animal can comprise a cell population having the targeted modification.

[0163] The cells of the genetically modified F0 animal can be heterozygous for the heterologous Cacng1 locus or can be homozygous for the heterologous Cacng1 locus.

[0164] All patent applications, websites, other publications, accession numbers, etc. cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item was specifically and individually indicated to be incorporated by reference. Where various 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 to which the accession number refers, as appropriate. Similarly, where different versions of a publication, website, etc. have been published at different times, the version most recently published as of the effective filing date of this application is intended, unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the present invention may be used in combination with any other, unless specifically indicated otherwise. The present invention has been described in some detail by way of illustration and example, for purposes of clarity and understanding, but it will be apparent that certain changes and modifications may be made within the scope of the appended claims.

[0165] In some embodiments, the disclosure provides a method of making a non-human animal, non-human animal cell, or non-human animal genome as described herein, comprising inserting a nucleic acid sequence encoding a heterologous CACNG1 protein or a portion thereof into the genome of the non-human animal, the genome of the non-human animal cell, or the non-human animal genome.

[0166] Various chimeric nucleic acids can be specifically used for such purposes. In some embodiments, chimeric nucleic acid molecules encoding functional CACNG1 protein comprising the nucleic acid sequence of modified non-human animal Cacng1 gene, wherein modified non-human animal Cacng1 gene comprises the replacement of the nucleic acid sequence encoding a part of non-human animal CACNG1 protein with a homologous nucleic acid sequence encoding a heterologous CACNG1 protein or a part thereof, can be used in gene editing of cells or genomes as described herein. In some examples, the chimeric nucleic acid molecule comprises (i) a nucleic acid sequence comprising exon 1 or a portion thereof of the human CACNG1 gene, (ii) a nucleic acid sequence comprising intron 1 or a portion thereof of the human CACNG1 gene, (iii) a nucleic acid sequence comprising exon 2 or a portion thereof of the human CACNG1 gene, (iv) a nucleic acid sequence comprising intron 2 or a portion thereof of the human CACNG1 gene, (v) a nucleic acid sequence comprising exon 3 or a portion thereof of the human CACNG1 gene, (vi) a nucleic acid sequence comprising intron 3 or a portion thereof of the human CACNG1 gene, (v) a nucleic acid sequence comprising exon 4 or a portion thereof of the human CACNG1 gene, (vii) a nucleic acid sequence of the 3' untranslated region (UTR) of the human CACNG1 gene, or (v) any combination of (i)-(iv). In some examples, the chimeric molecule provides a drug selection cassette.

[0167] In some embodiments, the chimeric nucleic acid molecule described herein comprises (i) a 5' homology arm upstream of modified non-human animal Cacng1 gene, and (ii) a 3' homology arm downstream of modified non-human animal Cacng1 gene.In some embodiments, the 5' homology arm and the 3' homology arm are configured to undergo homologous recombination with the non-human animal Cacng1 locus of interest, and after homologous recombination with the non-human animal Cacng1 locus of interest, the modified Cacng1 gene replaces the non-human animal Cacng1 gene at the non-human animal Cacng1 locus of interest, and is operably linked to the endogenous promoter that drives the expression of the non-human animal Cacng1 gene at the non-human animal Cacng1 locus of interest.In some embodiments, the chimeric nucleic acid molecule has (i) a 5' homology arm that comprises the nucleic acid sequence described as SEQ ID NO:25, and / or (ii) a 3' homology arm that comprises the nucleic acid sequence described as SEQ ID NO:26. In some embodiments, the chimeric nucleic acid molecule comprises the nucleic acid sequence set forth as SEQ ID NO:6.

[0168] A brief description of the sequence The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases and three-letter code for amino acids. The nucleotide sequences follow the standard convention of beginning at the 5'-terminus of the sequence and proceeding to the 3'-terminus (i.e., left to right on each line). Only one strand of each nucleotide sequence is shown, but the complementary strand is understood to be included by any reference to the presented strand. The amino acid sequences follow the standard convention of beginning at the amino-terminus of the sequence and proceeding to the carboxy-terminus (i.e., left to right on each line).

[0169] [Table 1] EXAMPLES

[0170] Example 1. CACNG1 is specifically expressed in skeletal muscle Skeletal muscle dihydropyridine receptors (DHPRs) are L-type calcium channels involved in excitation-contraction coupling. Skeletal muscle DHPRs consist of five subunits, with the α1s subunit playing a key role in muscle contraction through physical interaction with ryanodine receptors that regulate calcium release from the sarcoplasmic reticulum. The γ1 subunit (CACNG1) was found to be highly and specifically expressed in skeletal muscle (Figure 1A). Thus, a number of different methods were utilized to generate mice with humanized Cacng1 for use in validating liver-specific delivery of various therapeutic agents.

[0171] Example 2. CACNG1 knockout mice (CACNG1 - / - ) generation and analysis The inventors CACNG1 - / - Mice were generated and bred to determine whether deletion of CACNG1 affected skeletal muscle mass or function. A Cacng1 ablation construct was designed as follows: A bacterial artificial chromosome containing the Cacng1 genomic sequence was modified such that a loxP-transfected lacZ reporter cassette containing a neomycin resistance gene under the control of the human UBC (ubiquitin) promoter replaced 224 bp of Cacng1 coding exon 1 starting immediately after the initiation ATG. The cassette was cloned such that the lacZ coding sequence was in frame with the initiation ATG, leaving 5 bp of Cacng1 coding exon 1 3' following the cassette. (See FIG. 2) This construct was electroporated into 100% C57Bl / 6NTac embryonic stem cells. Successful targeted clones were identified by TaqMan analysis. Cacng1- / + mice were generated using the VelociGene method (Valenzuela 2003 Nat Biotech PMID:12730667, Poueymirou 2007 Nat Biotech PMID:17187059) and bred to homozygosity (CACNG1- / -) as needed. In the F0 germline, the resistance cassette was removed using the self-deletion technique.

[0172] Adult (5-7 months old) male wild-type (WT) mice and CACNG1 - / - Muscle tissues from mice were carefully dissected and weighed to assess muscle mass, and isolated extensor digitorum longus (EDL) muscles were measured for contractility ex vivo to assess muscle function. Muscle contractility measurements were performed using an Aurora Scientific 1300A instrument. EDL muscles were carefully excised and attached to the muscle physiological function apparatus via sutures. The muscles were then equilibrated at optimal length in Krebs-Henseleit buffer acidified with 95% O2 / 5% CO2, and then stimulated with supramaximal biphasic current to elicit twitch responses. After twitch stimulation, force-contraction frequency tetanic curves were generated via stimulation at 40 Hz, 60 Hz, 80 Hz, 100 Hz, and 125 Hz with a 2-minute pause between stimuli. Maximum force production was recorded at 100 Hz. CACNG1 was significantly associated with a 10-fold increase in the tibial and viscoelastic responses in wild-type (WT) and CACNG1 mice. - / - Twitch and tetanus force were similar between the mice and rats and therefore do not appear to play a major role in regulating muscle function, see Figures 3A and 3B.

[0173] Example 3. CACNG1 humanized mice (CACNG1 hu / hu ) generation and analysis The Cacng1 targeting construct was designed as follows: A bacterial artificial chromosome containing the complete mouse Cacng1 genomic sequence was modified to humanize the Cacng1 locus. The mouse Cacng1 portion of coding exon 1, intron 1, coding exons 2-4 (and intervening introns), and 82 bp of the 3' untranslated region (UTR) are replaced with human CACNG1 sequence consisting of coding exon 1 sequence except for 15 bp at the start of human CACNG1 (this start sequence remains mouse), intron 1, coding exons 2-4 (and intervening introns), the complete 3'UTR, and an additional 158 bp after the 3'UTR. See Figures 2A-2C. A self-deleted neomycin resistance cassette was inserted downstream of the human sequence, followed by the remainder of the mouse 3'UTR. See Figures 2A-2C showing the target site after deletion of the self-deleted neomycin resistance cassette. This targeting vector was then electroporated into 50% C57Bl / 6NTac / 50% 129SvEvTac embryonic stem cell line. Successfully targeted clones were identified by TaqMan analysis. Cacng1+ / + mice were generated using the VelociGene (copyright) method (Valenzuela 2003 Nat Biotech PMID:12730667, Poueymirou 2007 Nat Biotech PMID:17187059) and backcrossed to C57Bl / 6NTac as required. The antibiotic resistance cassette was removed in the F0 male germline using the self-deletion technique.

[0174] Gene expression analysis

[0175] Total RNA was isolated from tissues via TRIzol homogenization and chloroform phase separation followed by purification with MagMAX-96 for Microarrays Total RNA Isolation Kit. Genomic DNA was removed using RNAse-free DNAse set and mRNA was reverse transcribed to cDNA using SuperScript VILO Master Mix. cDNA was amplified with SensiFAST Probe Lo-Rox using the 12K Flex System. Taqman gene expression assays were used to determine human (h) and mouse (m) CACNG1 expression relative to m18S (endogenous control) and data were analyzed using the comparative Ct method (ΔΔCt). The Taqman primer / probe sequences were fwd-GGCGAGAGCTCGGAGATC (SEQ ID NO: 30), rev-GGCTGCCCAGGATGATGAAG (SEQ ID NO: 31), and probe-TCGAATTCACCACTCAGAAGGAGTACA (SEQ ID NO: 32) for hCACNG1, and fwd-CCGTGCACAACAAAGACAAGAG (SEQ ID NO: 33), rev-GCTCTCCCCTGGGTTGAAG (SEQ ID NO: 34), and probe-TGTGAGCACGTCACACCATCAGG (SEQ ID NO: 35) for mCACNG1. hu / hu It was not detectable by qPCR in mouse muscle (left graph), whereas human CACNG1 (hCACNG1) was hu / hu Graphs demonstrating that the gene is expressed in mouse muscle but not in wild-type (WT) mouse muscle (right graph).

[0176] Isolation of single muscle fibers and live cell staining

[0177] Single muscle fibers were cultured from adult male CACNG1 hu / huCACNG1 was isolated from mouse gastrocnemius muscle. The muscle was carefully dissected and digested with 700U / mL collagenase in DMEM for 60 minutes. Single myofibers were isolated with a blunt glass Pasteur pipette, and after several rounds of digestion and washing, the myofibers were seeded overnight on low-attachment tissue culture plates. The following morning, human-specific Alexa 647-conjugated CACNG1 antibody was added to the live single myofibers at 100nM concentration for either 30 minutes or 4 hours. The myofibers were then washed with DMEM, fixed in 4% PFA, and stained with DAPI. The single myofibers were then transferred to microscope slides, mounted with Fluoromount, and imaged with an LSM880 confocal microscope. See Figure 3B. The experiment shows live staining of single skeletal muscle myofibers with 100nM Alexa 647-conjugated human-specific α-CACNG1 antibody, which inhibits CACNG1. hu / hu The results show binding to muscle fibers isolated from mice, but not to muscle fibers isolated from wild type (WT) mice.

[0178] Cryofluorescence tomography (CryoFT) imaging of antibody distribution

[0179] Adult male CACNG1 hu / hu Mice were tail vein injected with 10 mg / kg of human-specific Alexa 647-conjugated CACNG1 antibody, Alexa 647-conjugated isotype control antibody, or saline. Six days after injection, mice were euthanized with CO2, whole body frozen, and evaluated using CryoFT processing and imaging. See Figure 3C. CACNG1 injected with 10 mg / kg of Alexa 647-conjugated human-specific α-CACNG1 antibody hu / hu Images of mice show high specificity for skeletal muscle compared to isotype control antibody 6 days after injection.

[0180] Immunofluorescence imaging of antibody distribution

[0181] Adult male CACNG1 hu / huMice were subcutaneously injected with 10 mg / kg human-specific Alexa 647-conjugated CACNG1 antibody, Alexa 647-conjugated isotype control antibody, or saline. Six days after injection, mice were perfused transcardially with PBS, and gastrocnemius / plantaris / soleus muscle complexes were immersed in OCT embedding medium and frozen in liquid nitrogen-cooled isopentane. Tissues were cryosectioned at 12 μm thickness, then fixed in 4% PFA and stained with laminin and DAPI. Slides were mounted with Fluoromount and imaged on an Axioscan slide scanner. See Figure 3D. The top panel shows endogenous Alexa 647 signal from the antibody injected in vivo, and the bottom panel shows an overlay of Alexa 647 antibody binding with co-stained laminin and DAPI to visualize muscle morphology.

Claims

1. A non-human animal cell, wherein the non-human animal cell comprises a nucleic acid sequence encoding a heterologous calcium voltage-gated channel accessory subunit gamma 1 (CACNG1) protein or a portion thereof.

2. the nucleic acid sequence encoding the heterologous CACNG1 protein or a portion thereof (a) Below (i) exon 1 of the human CACNG1 gene or a part thereof; (ii) intron 1 of the human CACNG1 gene or a part thereof; (iii) exon 2 of the human CACNG1 gene or a part thereof; (iv) intron 2 of the human CACNG1 gene or a part thereof; (v) exon 3 of the human CACNG1 gene or a part thereof; (vi) intron 3 of the human CACNG1 gene or a part thereof; (vii) exon 4 of the human CACNG1 gene or a part thereof; (viii) the 3' untranslated region (UTR) of the human CACNG1 gene, or (ix) Any combination of (i) to (viii) and / or (b) a nucleic acid sequence selected from the group consisting of the nucleic acid sequence set forth as SEQ ID NO:5, the nucleic acid sequence set forth as SEQ ID NO:27, and the nucleic acid sequence set forth as SEQ ID NO:

28. The non-human animal cell of claim 1 , comprising:

3. the nucleic acid sequence encoding the heterologous CACNG1 protein or the portion thereof is (a) at the endogenous Cacng1 locus, or (b) at the endogenous Cacng1 locus and replaces an orthologous endogenous nucleic acid sequence encoding the endogenous CACNG1 protein or a portion thereof; The non-human animal cell according to claim 1.

4. the non-human animal cell comprises an endogenous Cacng1 locus, and the endogenous Cacng1 locus comprises a heterozygous or homozygous replacement of an endogenous nucleic acid sequence encoding an endogenous CACNG1 protein or a portion thereof with the nucleic acid sequence encoding the heterologous CACNG1 protein or a portion thereof; The non-human animal cell according to claim 1 , wherein the endogenous nucleic acid sequence encoding the endogenous CACNG1 protein or a portion thereof and the nucleic acid sequence encoding the heterologous CACNG1 protein or a portion thereof are orthologous.

5. the heterologous CACNG1 protein or portion thereof (a) comprising the amino acid sequence of human CACNG1 protein or a portion thereof; (b) Below (i) SEQ ID NO: 8; (ii) SEQ ID NO: 10, (iii) SEQ ID NO: 12, (iv) SEQ ID NO: 14, (v) SEQ ID NO: 16, (vi) SEQ ID NO: 18, (vii) SEQ ID NO: 20, (viii) SEQ ID NO: 22, (ix) SEQ ID NO: 24, or (x) Any combination of (i) to (ix) or comprising the amino acid sequence described as (c) comprising the amino acid sequence set forth as SEQ ID NO:4; or (d) comprising a full-length human CACNG1 protein, and the non-human animal cell expresses the full-length human CACNG1 protein on its cell surface; The non-human animal cell according to claim 1.

6. The non-human animal cell according to claim 1 , wherein the non-human animal cell is a mammalian cell, a rodent cell, a rat cell or a mouse cell.

7. The non-human animal cell according to claim 1, wherein the non-human animal cell is a non-human animal skeletal muscle cell that expresses the heterologous CACNG1 protein or the portion thereof on its cell surface.

8. the non-human animal cell is a non-human animal cell that does not express the heterologous CACNG1 protein or the part thereof on its cell surface, (a) the non-human animal cell is not a skeletal cell; (b) the non-human animal cell is a pluripotent cell; (c) the non-human animal cell is an embryonic stem cell, or (d) the non-human animal cell is a germ cell; The non-human animal cell according to claim 1.

9. The non-human animal cell of claim 1, wherein the non-human animal cell is a mouse cell, and the nucleic acid sequence encoding the heterologous CACNG1 protein or the portion thereof comprises the nucleic acid sequence set forth as SEQ ID NO:

6.

10. A non-human animal, comprising a nucleic acid sequence encoding a heterologous calcium voltage-gated channel accessory subunit gamma 1 (CACNG1) protein or a portion thereof.

11. A chimeric nucleic acid molecule, comprising: (a) a nucleic acid sequence of a non-human animal Cacng1 gene that encodes a CACNG1 protein; and (b) a nucleic acid sequence of a non-human animal Cacng1 gene that is modified to include a replacement of the sequence encoding the CACNG1 protein or a portion thereof with a homologous sequence encoding a heterologous CACNG1 protein or a portion thereof; A chimeric nucleic acid molecule, wherein the chimeric nucleic acid molecule encodes a functional CACNG1 protein.

12. The chimeric nucleic acid molecule of claim 11 , wherein the sequence of the nucleic acid further comprises a promoter sequence and / or a regulatory sequence of the non-human animal Cacng1 gene.

13. (a) the homologous sequence is (i) a nucleic acid sequence comprising exon 1 of the human CACNG1 gene or a part thereof; (ii) the nucleic acid sequence of intron 1 of the human CACNG1 gene or a part thereof; (iii) a nucleic acid sequence comprising exon 2 of the human CACNG1 gene or a part thereof; (iv) the nucleic acid sequence of intron 2 of the human CACNG1 gene or a part thereof; (v) a nucleic acid sequence comprising exon 3 of the human CACNG1 gene or a part thereof; (vi) the nucleic acid sequence of intron 3 of the human CACNG1 gene or a part thereof; (vii) a nucleic acid sequence comprising exon 4 of the human CACNG1 gene or a part thereof; (viii) the nucleic acid sequence of the 3' untranslated region (UTR) of the human CACNG1 gene; or (ix) any combination of (i) to (viii), including: (b) the modified non-human animal Cacng1 gene further comprises a drug selection cassette; (c) the chimeric nucleic acid molecule is (i) a 5' homologous arm upstream of the modified non-human animal Cacng1 gene, and / or a 5' homologous arm comprising the nucleic acid sequence set forth as SEQ ID NO: 25; and (ii) a 3' homologous arm downstream of the modified non-human animal Cacng1 gene, and / or a 3' homologous arm comprising the nucleic acid sequence set forth as SEQ ID NO: 26; further comprising the 5' and 3' homologous arms undergo homologous recombination with the Cacng1 locus of the non-human animal of interest; and after homologous recombination with the non-human animal Cacng1 locus of interest, the modified non-human animal Cacng1 gene replaces the non-human animal Cacng1 gene at the non-human animal Cacng1 locus of interest and is operably linked to an endogenous promoter driving expression of the non-human animal Cacng1 gene at the non-human animal Cacng1 locus of interest; and / or (d) the nucleic acid sequence of the chimeric nucleic acid molecule comprises the nucleic acid sequence set forth as SEQ ID NO:6; The chimeric nucleic acid molecule of claim 12.

14. A method for producing the non-human animal cell of claim 1, comprising inserting the nucleic acid sequence encoding the heterologous CACNG1 protein or a portion thereof into the genome of the non-human animal cell.

15. the non-human animal cells are non-human animal embryonic stem (ES) cells; and The method of claim 14, wherein the inserting comprises inserting the nucleic acid sequence encoding the heterologous CACNG1 protein or a portion thereof into the genome of the non-human animal ES cell to form a modified non-human animal ES cell comprising the nucleic acid sequence encoding the heterologous CACNG1 protein or a portion thereof in the genome of the non-human animal ES cell.

16. 15. The method of claim 14, wherein inserting comprises contacting the genome of the non-human animal cell with the chimeric nucleic acid molecule of claim 13.

17. 15. The method of claim 14, wherein inserting comprises contacting the genome of the non-human animal cell with the chimeric nucleic acid molecule of claim 11.

18. 11. A method for producing a non-human animal according to claim 10, comprising inserting a nucleic acid sequence encoding a heterologous CACNG1 protein or a portion thereof into the genome of a non-human animal embryonic stem (ES) cell to form a modified non-human animal ES cell comprising a nucleic acid sequence encoding a heterologous CACNG1 protein or a portion thereof in the genome of the non-human animal ES cell; introducing the non-human animal ES cells into host embryonic cells in vitro; gestation of a host embryonic cell comprising a modified non-human animal ES cell in a non-human surrogate mother animal, wherein after said gestation, a non-human animal offspring comprising germ cells comprising a nucleic acid sequence encoding the heterologous CACNG1 protein or a portion thereof is born by the non-human surrogate mother animal.

19. The non-human animal of claim 10, comprising an antigen-binding protein that binds to the heterologous CACNG1 protein, wherein the non-human animal expresses the heterologous CACNG1 protein or its extracellular domain on the surface of skeletal muscle cells.

20. The non-human animal of claim 19 , wherein the heterologous CACNG1 protein is a human CACNG1 protein, and / or the non-human animal is a mouse.

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