WO2022262808A1

WO2022262808A1 - Genetically modified non-human animal with human or chimeric cd20 genes

Info

Publication number: WO2022262808A1
Application number: PCT/CN2022/099152
Authority: WO
Inventors: Lei Zhao; Chang Liu; Chong Li
Original assignee: Biocytogen Pharmaceuticals (Beijing) Co., Ltd.
Priority date: 2021-06-16
Filing date: 2022-06-16
Publication date: 2022-12-22
Also published as: CN114990128A

Abstract

Provided are genetically modified non-human animals that express a human or chimeric (e.g., humanized) CD20, and methods of use thereof.

Description

GENETICALLY MODIFIED NON-HUMAN ANIMAL WITH HUMAN OR CHIMERIC CD20 GENES

CLAIM OF PRIORITY

This application claims the benefit of Chinese Patent Application App. No. CN202110668664.5, filed on June 16, 2021. The entire contents of the foregoing applications are incorporated herein by reference.

TECHNICAL FIELD

This disclosure relates to genetically modified animal expressing human or chimeric (e.g., humanized) CD20, and methods of use thereof.

BACKGROUND

The immune system has developed multiple mechanisms to prevent deleterious activation of immune cells. One such mechanism is the intricate balance between positive and negative co-stimulatory signals delivered to immune cells. Targeting the stimulatory or inhibitory pathways for the immune system is considered to be a potential approach for the treatment of various diseases, e.g., cancers and autoimmune diseases.

The traditional drug research and development for these stimulatory or inhibitory receptors typically use in vitro screening approaches. However, these screening approaches cannot provide the body environment (such as tumor microenvironment, stromal cells, extracellular matrix components and immune cell interaction, etc. ) , resulting in a higher rate of failure in drug development. In addition, in view of the differences between humans and animals, the test results obtained from the use of conventional experimental animals for in vivo pharmacological test may not reflect the real disease state and the interaction at the targeting sites, resulting in that the results in many clinical trials are significantly different from the animal experimental results. Therefore, the development of humanized animal models that are suitable for human antibody screening and evaluation will significantly improve the efficiency of new drug development and reduce the cost for drug research and development.

SUMMAKY

This disclosure relates to transgenic non-human animal with human or chimeric (e.g., humanized) CD20 and methods of use thereof. The animal model can express human CD20 or chimeric CD20 (e.g., humanized CD20) protein in its body. It can be used in the studies on the function of CD20 gene, and can be used in the screening and evaluation of anti-human CD20 antibodies. In addition, the animal models prepared by the methods described herein can be used in drug screening, pharmacodynamics studies; they can also be used to facilitate the development and design of new drugs, and save time and cost. In summary, this disclosure provides a powerful tool for studying the function of CD20 proteins, and a platform for screening hypoglycemic drugs.

In one aspect, the disclosure is related to a genetically-modified, non-human animal whose genome comprises at least one chromosome comprising a sequence encoding a human or chimeric CD20.

In some embodiments, the sequence encoding the human or chimeric CD20 is operably linked to an endogenous regulatory element at the endogenous CD20 gene locus in the at least one chromosome.

In some embodiments, the sequence encoding a human or chimeric CD20 comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to human CD20 (NP_068769.2 (SEQ ID NO: 2) ) .

In some embodiments, the animal is a mammal, e.g., a monkey, a rodent, a mouse, or a rat.

In some embodiments, the animal is a mouse.

In some embodiments, the animal does not express endogenous CD20 or expresses a decreased level of endogenous CD20.

In some embodiments, the animal has one or more cells expressing human or chimeric CD20.

In some embodiments, the expressed human or chimeric CD20 can regulate B cell development.

In some embodiments, the expressed human or chimeric CD20 can maintain normal B cell development.

In one aspect, the disclosure is related to a genetically-modified, non-human animal, wherein the genome of the animal comprises a replacement of a sequence encoding a region of endogenous CD20 with a sequence encoding a corresponding region of human CD20 at an endogenous CD20 gene locus.

In some embodiments, the sequence encoding the corresponding region of human CD20 is operably linked to an endogenous regulatory element at the endogenous CD20 locus, and one or more cells of the animal expresses a human or chimeric CD20.

In some embodiments, the sequence encoding the corresponding region of human CD20 is immediately after endogenous 5’-UTR.

In some embodiments, the sequence that is replaced comprises the full-length coding sequence of endogenous CD20 (e.g., a nucleic acid sequence encoding amino acids 1-291 of SEQ ID NO: 1) .

In some embodiments, the sequence that is replaced comprises exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7, or a part thereof, of the endogenous CD20 gene.

In some embodiments, the sequence that is replaced starts with the start codon and ends with the stop codon of the endogenous mouse CD20 gene.

In some embodiments, the animal is heterozygous with respect to the replacement at the endogenous CD20 gene locus.

In some embodiments, the animal is homozygous with respect to the replacement at the endogenous CD20 gene locus.

In one aspect, the disclosure is related to a method for making a genetically-modified, non-human animal, comprising: replacing in at least one cell of the animal, at an endogenous CD20 gene locus, a sequence encoding a region of endogenous CD20 with a sequence encoding a corresponding region of human CD20.

In some embodiments, the sequence encoding the corresponding region of human CD20 comprises exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7, or a part thereof, of a human CD20 gene.

In some embodiments, the sequence encoding the corresponding region of human CD20 comprises a portion of exon 2, exon 3, exon 4, exon 5, exon 6, and a portion of exon 7 of a human CD20 gene.

In some embodiments, the sequence encoding the corresponding region of human CD20 encodes amino acids 1-297 of SEQ ID NO: 2.

In some embodiments, the sequence encoding a region of endogenous CD20 comprises exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7, or a part thereof, of the endogenous CD20 gene.

In some embodiments, the animal is a mouse, and the sequence encoding a region of endogenous CD20 starts within exon 2 and ends within exon 7 of the endogenous mouse CD20 gene.

In one aspect, the disclosure is related to a method of making a genetically-modified animal cell that expresses a human or chimeric CD20, the method comprising: replacing at an endogenous CD20 gene locus, a nucleotide sequence encoding a region of endogenous CD20 with a nucleotide sequence encoding a corresponding region of human CD20, thereby generating a genetically-modified animal cell that includes a nucleotide sequence that encodes the human or chimeric CD20, wherein the animal cell expresses the human or chimeric CD20.

In some embodiments, the animal is a mouse.

In some embodiments, the nucleotide sequence encoding the human or chimeric CD20 is operably linked to an endogenous CD20 regulatory region, e.g., promoter.

In some embodiments, the animal further comprises a sequence encoding an additional human or chimeric protein.

In some embodiments, the additional human or chimeric protein is programmed cell death protein 1 (PD-1) , programmed cell death ligand 1 (PD-L1) , T-Cell immunoreceptor with Ig and ITIM Domains (TIGIT) , Tumour Necrosis Factor alpha (TNF alpha) , tumor necrosis factor receptor superfamily member 9 (4-1BB) , Tumor necrosis factor ligand superfamily member 9 (4-1BBL) , cytotoxic T-lymphocyte-associated protein 4 (CTLA4) , CD47, Signal regulatory protein α (SIRPα) , OX40, T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) , or CD226.

In some embodiments, the animal or mouse further comprises a sequence encoding an additional human or chimeric protein.

In some embodiments, the additional human or chimeric protein is PD-1, PD-L1, TIGIT, TNFA, 41BB, 41BBL, CTLA4, CD47, SIRPA, OX40, TIM3 or CD226.

In one aspect, the disclosure is related to a method for making a genetically-modified mouse, comprising:

(1) providing a plasmid comprising a human CD20 gene fragment, flanked by a 5' homology arm and a 3' homology arm, wherein the 5' and 3' homology arms target an endogenous CD20 gene;

(2) providing two small guide RNAs (sgRNAs) that target the endogenous CD20 gene;

(3) modifying genome of a fertilized egg or an embryonic stem cell by using the plasmid of step (1) , the sgRNAs of step (2) , and Cas9; and

(4) transplanting the fertilized egg obtained in step (3) into the oviduct of a pseudopregnant female mouse or transplanting the embryonic stem cell obtained in step (3) into a blastocyst which is then transplanted into the oviduct of a pseudopregnant female mouse to produce a child mouse that functionally expresses a humanized CD20 protein,

(5) mating the mouse obtained in step (2) to obtain a homozygote mouse.

In some embodiments, the fertilized egg is modified by CRISPR with sgRNAs that target a 5'-terminal targeting site comprising SEQ ID NO: 24 and a 3'-terminal targeting site comprising SEQ ID NO: 25.

In some embodiments, the mouse has a C57BL/6 background.

In some embodiments, the sequence encoding the humanized CD20 protein is operably linked to an endogenous regulatory element at the endogenous CD20 gene locus.

In some embodiments, the genetically-modified mouse does not express an endogenous CD20 protein.

In some embodiments, the 5' homology arm comprises SEQ ID NO: 22 and the 3' homology arm comprises SEQ ID NO: 23.

In some embodiments, the mouse can be used to test effectiveness of an anti-human CD20 antibody for treating cancer.

In one aspect, the disclosure is related to a method of determining effectiveness of an anti-CD20 antibody for the treatment of cancer, comprising: administering the anti-CD20 antibody to the animal described herein, wherein the animal has a cancer; and determining the inhibitory effects of the anti-CD20 antibody to the cancer.

In some embodiments, the cancer comprises one or more cells that express CD20.

In some embodiments, the cancer comprises one or more cancer cells that are injected into the animal.

In some embodiments, determining the inhibitory effects of the anti-CD20 antibody to the cancer involves measuring the tumor volume in the animal or measuring fluorescence intensity.

In some embodiments, the cancer is chronic lymphocytic leukemia (CLL) , follicular lymphoma, diffuse large B-cell lymphoma (DLBCL) , Non-Hodgkin lymphoma, Burkitt Lymphoma, mantle cell Lymphoma, marginal zone Lymphoma, Lymphoma lymphoblastic, Hodgkin lymphoma, or Melanoma.

In one aspect, the disclosure is related to a protein comprising an amino acid sequence, wherein the amino acid sequence is one of the following:

an amino acid sequence set forth in SEQ ID NO: 1 or 2;

an amino acid sequence that is at least 90%identical to SEQ ID NO: 1 or 2;

an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 1 or 2;

an amino acid sequence that is different from the amino acid sequence set forth in SEQ ID NO: 1 or 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid; and

an amino acid sequence that comprises a substitution, a deletion and /or insertion of one, two, three, four, five or more amino acids to the amino acid sequence set forth in SEQ ID NO: 1 or 2.

In one aspect, the disclosure is related to a nucleic acid comprising a nucleotide sequence, wherein the nucleotide sequence is one of the following:

a sequence that encodes the protein described herein;

SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 22 or 23;

a sequence that is at least 90%identical to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 22 or 23;

a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 22 or 23; and

a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 22 or 23.

In one aspect, the disclosure is related to a cell comprising the protein described herein and/or the nucleic acid described herein.

In some embodiments, the cell expresses a human or chimeric CD20.

In one aspect, the disclosure is related to an animal comprising the protein described herein and/or the nucleic acid described herein.

In one aspect, the disclosure is related to a method of determining toxicity of an anti-CD20 antibody, the method comprising administering the anti-CD20 antibody to the animal described herein; and determining weight change of the animal.

In some embodiments, the method further comprises performing a blood test (e.g., determining red blood cell count) .

In some embodiments, the expressed human or chimeric CD20 can interact with a human CD20 ligand.

In some embodiments, the expressed human or chimeric CD20 can interact with an endogenous CD20 ligand.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram showing mouse and human CD20 gene loci.

FIG. 2 is a schematic diagram showing humanized CD20 gene locus.

FIG. 3 is a schematic diagram showing a CD20 gene targeting strategy.

FIG. 4 is a schematic diagram showing the FRT recombination process in CD20 gene humanized mice.

FIG. 5A shows PCR identification results ofF 1 generation mice by primers WT-F and WT-R. M is a marker. PC is a positive control. WT is a wild-type control. H ₂O is a water control.

FIG. 5B shows PCR identification results ofF 1 generation mice by primers WT-F and Mut-R. M is a marker. PC is a positive control. WT is a wild-type control. H ₂O is a water control.

FIG. 6 is a schematic diagram showing a CD20 gene targeting strategy.

FIG. 7 shows Southern Blot results of cells after recombination using the 5' Probe and the 3' Probe. WT is a wild-type control. 1EA61-0001, 1EA61-0009 and 1EA61-0010 are mouse numbers.

FIGS. 8A-8C show mRNA transcription results of humanized CD20 gene in CD20 gene humanized mice. “+/+” represents wild-type mouse, and “H/H” represents CD20 gene humanized homozygous mouse.

FIG. 9 shows the alignment between human CD20 amino acid sequence (NP_068769.2; SEQ ID NO: 2) and mouse CD20 amino acid sequence (NP_031667.1; SEQ ID NO: 1) .

FIG. 10 shows the alignment between human CD20 amino acid sequence (NP_068769.2; SEQ ID NO: 2) and rat CD20 amino acid sequence (NP_001386381.1; SEQ ID NO: 43) .

DETAILED DESCRIPTION

This disclosure relates to transgenic non-human animal with human or chimeric (e.g., humanized) CD20, and methods of use thereof.

CD20 is a transmembrane cellular protein that has been validated as a therapeutic target for treatment of B cell malignancies. CD20 is highly expressed by over 95%of B cell lymphocytes throughout their development, from the pre-B cell stage until their final differentiation into plasma cells, but is absent on the hematopoietic stem cell. Moreover, CD20 is believed to exist predominantly as a tetramer on the cell surface. It is also largely believed to be not usually shed or internalized upon antibody binding, meaning that therapeutic antibodies may be expected to recruit immune effectors cells and mediate sustained immunologic activity. The physiological function of CD20 remains unclear, although evidence suggested that it may be involved in calcium signaling downstream of B cell antigen receptor activation.

Experimental animal models are an indispensable research tool for studying the effects of CD20 antibodies. Common experimental animals include mice, rats, guinea pigs, hamsters, rabbits, dogs, monkeys, pigs, fish and so on. However, there are many differences between human and animal genes and protein sequences, and many human proteins cannot bind to the animal's homologous proteins to produce biological activity, leading to that the results of many clinical trials do not match the results obtained from animal experiments. A large number of clinical studies are in urgent need of better animal models. With the continuous development and maturation of genetic engineering technologies, the use of human cells or genes to replace or substitute an animal's endogenous similar cells or genes to establish a biological system or disease model closer to human, and establish the humanized experimental animal models (humanized animal model) has provided an important tool for new clinical approaches or means. In this context, the genetically engineered animal model, that is, the use of genetic manipulation techniques, the use of human normal or mutant genes to replace animal homologous genes, can be used to establish the genetically modified animal models that are closer to human gene systems. The humanized animal models have various important applications. For example, due to the presence of human or humanized genes, the animals can express or express in part of the proteins with human functions, so as to greatly reduce the differences in clinical trials between humans and animals, and provide the possibility of drug screening at animal levels.

CD20

CD20 is a 33-37 kDa non-glycosylated protein expressed on the surface of normal and malignant B lymphocytes, and belongs to the MS4A (membrane-spanning 4-domain family A) protein family. To date, 18 MS4A family members have been identified, besides MS4A1 (encoding CD20) , also the high-affinity immunoglobulin E receptor β subunit (MS4A2/FcεRIβ) or HtM4 gene (MS4A3) . MS4A proteins are transmembrane molecules and they are predicted to share a similar polypeptide sequence and overall topological structure. The majority of MS4A genes, including MS4A1, are localized within a cluster on chromosome 1 lq12 in humans (chromosome 19 in mice) , and two members from a closely related TMEM176 gene family were identified in chromosome region 7q36.1.14.

The MS4A1 gene is 16 kb long, comprises eight exons, and several different CD20 mRNA transcripts have been annotated. The dominant CD20 mRNA variant is 2.8 kb long and uses all eight exons, whereas the second most common form is 263 bases shorter, as it skips exon II. A minor 3.5 kb mRNA results from splicing exons in the upstream region into an internal 3' splice site located in exon I. However, all three transcripts are translated into identical full-length CD20 protein as the translation start codon is localized within exon III. Moreover, other alternative transcripts were identified in malignant B cells, some of them encoding truncated forms of CD20 protein leading to impaired binding of anti-CD20 monoclonal antibodies.

CD20 protein consists of four hydrophobic transmembrane domains, one intracellular and two extracellular domains (large and small loops) with both N-and C-termini residing within the cytosol. Three CD20 isoforms (33, 35 and 37 kDa) resulting from different phosphorylation have been identified, and CD20 phosphorylation was reported to be higher in proliferating malignant B cells than in resting B cells. Normally, CD20 does not form hetero-oligomers, but exists on the cell surface as homodimeric and homo-tetrameric oligomers associated with other cell-surface and cytoplasmic proteins contributing to the signal transduction. Tetraspanin proteins tend to associate with multiple other proteins in membrane microdomains. Energy transfer experiments indicate that CD20 is in close proximity to other tetraspan molecules, such as CD53, CD81, and CD82, forming supramolecular complexes. CD20 is also known to be physically coupled to major histocompatibility complex class II (MHCII) , CD40 molecule, BCR, and the C-terminal src kinase-binding protein (CBP) that interacts with Src kinases such as LYN, FYN, and LCK. Besides the transmembrane form of CD20, circulating CD20 was reported in CLL patients' plasma; however, this is likely to be part of a larger protein complex or a cell membrane fragment originating from cell breakdown.

CD20 is a general B-cell marker expressed by the majority of B cells starting from late pre-B lymphocytes (it is not expressed by pro-B lymphocytes) , and its expression is lost in terminally differentiated plasmablasts and plasma cells. Recently, a subset of CD20+ T cells with immune-regulatory and pro-inflammatory activity has been described; however, the clinical relevance of this remains to be determined. In B-cell malignancies, the level of CD20 expression is extremely variable depending on the specific neoplasm, with the lowest CD20 expression usually being observed in patients with CLL and the highest CD20 cell-surface expression on DLBCL and hairy cell leukemia cells. Within CLL, it was noted that CD20 expression was also relatively higher in a disease subtype with a mutated variable region of immunoglobulin gene (IGHV) than in the subtype with unmutated IGHV. Some studies described that higher CD20 expression levels correlate with longer overall survival in patients with B-cell lymphomas treated with rituximab, although this remains controversial. Notably, CD20 levels are heterogeneous not only among patients with the same malignancy, but also within the intraclonal cell subpopulations in an individual patient.

The biological function of CD20 in B cells and its physiological ligand, if any, remain unclear. Some light on CD20 function has been shed by a case report of a patient with a common variable immunodeficiency and CD20 loss caused by a homozygous mutation in an exon 5 splicing site ofMS4A1. The mutation led to alternative splicing with complete deletion of exon 5 and insertion of intron sequences and thus a truncated form of MS4A1 mRNA. Due to this homozygous mutation, the patient completely lacked cell-surface CD20. This did not disturb precursor B-cell differentiation in the bone marrow, as the patient had normal serum IgM levels and normal B-cell numbers. However, CD20 deficiency resulted in a reduced number of circulating memory B cells, reduced isotype switching of Ig, and decreased IgG antibody levels. In agreement with this observation, challenging the patient's primary B cells in vitro using T-dependent and T-independent antigens led to the normal proliferation and secretion of IgM but reduced production of IgG. Given these data it is surprising that after repeated vaccinations the patient displayed a reduced ability to respond to T-independent antigens (pneumococcal polysaccharide vaccine) , but a normal reaction to T-dependent antigens (anti-tetanus toxoid IgG) .

A detailed description of CD20 and its function can be found in, e.g., Pavlasova, Gabriela, and Marek Mraz. "The regulation and function of CD20: an “enigma” of B-cell biology and targeted therapy. " Haematologica 105.6 (2020) : 1494, which is incorporated by reference in its entirety.

In mice, CD20 gene locus has seven exons, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and exon 7 (FIG. 1) . The mouse Ms4al gene (Gene ID: 12482) is located in Chromosome 19 of the mouse genome, which is located from 11227043 to 11243513 ofNC_000085.7 (GRCm39 (GCF_000001635.27) ) . The 5’-UTR is from 11,243,605 to 11,243,412, and 11236186 to 11236200, exon 1 is from 11,243,605 to 11,243,412, the first intron is from 11,243,411 to 11,236,201, exon 2 is from 11,236,200 to 11,236,048, the second intron is from 11,236,047 to 11,235,617, exon 3 is from 11,235,616 to 11,235,497, the third intron is from 11,235,496 to 11,233,997, exon 4 is from 11,233,996 to 11,233,937, the fourth intron is from 11,233,936 to 11,232,056, exon 5 is from 11,232,055 to 11,231,819, the fifth intron is from 11,231,818 to 11,230,645, exon 6 is from 11,230,644 to 11,230,543, the sixth intron is from 11,230,542 to 11,229,248, exon 7 is from 11,229,247 to 11,227,039, the 3'-UTR is from 11227043 to 11229028, base on transcript NM_007641.6. All relevant information for mouse Ms4al locus can be found in the NCBI website with Gene ID: 12482, which is incorporated by reference herein in its entirety. The location for each exon and each region in mouse CD20 nucleotide sequence and amino acid sequence is listed below:

Table 1

In human, CD20 gene locus has seven exons, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and exon 7 (FIG. 1) . The human MS4A1 gene (Gene ID: 931) is located in Chromosome 11 of the mouse genome, which is located from 60455847 to 60470752 of NC_000011.10 (GRCh38. p14 (GCF_000001405.40) ) . The 5’-UTR is from 60,455,905 to 60,455,945, and 60,462,359 to 60,462,374, exon 1 is from 60,455,905 to 60,455,945, the first intron is from 60,455,946 to 60,462,358, exon 2 is from 60,462,359 to 60,462,533, the second intron is from 60,462,534 to 60,463,001, exon 3 is from 60,463,002 to 60,463,121, the third intron is from 60,463,122 to 60,464,287, exon 4 is from 60,464,288 to 60,464,344, the fourth intron is from 60,464,345 to 60,465,920, exon 5 is from 60,465,921 to 60,466,157, the fifth intron is from 60,466,158 to 60,466,958, exon 6 is from 60,466,959 to 60,467,060, the sixth intron is from 60,467,061 to 60,468,249, exon 7 is from 60,468,250 to 60,470,016, the 3’-UTR is from 60,468,469 to 60,470,016, base on transcript NM_021950.4. All relevant information for human MS4A1 locus can be found in the NCBI website with Gene ID: 7315, which is incorporated by reference herein in its entirety. The location for each exon and each region in the human CD20 nucleotide sequence and amino acid sequence is listed below:

Table 2

FIG. 9 shows the alignment between human CD20 amino acid sequence (NP_068769.2; SEQ ID NO: 2) and mouse CD20 amino acid sequence (NP_031667.1; SEQ ID NO: 1) . Thus, the corresponding amino acid residue or region between human and mouse CD20 can be found in FIG. 9.

CD20 genes, proteins, and locus of the other species are also known in the art. For example, the gene ID for CD20 in Rattus norvegicus (rat) is 309217, the gene ID for CD20 in Macaca mulatta (Rhesus monkey) is 696843, and the gene ID for CD20 in Pan troglodytes (chimpanzee) is 112204125. The relevant information for these genes (e.g., intron sequences, exon sequences, amino acid residues of these proteins) can be found, e.g., in NCBI database, which is incorporated by reference herein in its entirety. FIG. 10 shows the alignment between human CD20 amino acid sequence (NP_068769.2; SEQ ID NO: 2) and rat CD20 amino acid sequence (NP_001386381.1; SEQ ID NO: 43) . Thus, the corresponding amino acid residue or region between human and rodent CD20 can be found in FIG. 10.

The present disclosure provides human or chimeric (e.g., humanized) CD20 nucleotide sequence and/or amino acid sequences. In some embodiments, the entire sequence of mouse exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, extracellular regions, transmembrane regions, and/or cytoplasmic regions are replaced by the corresponding human sequence. In some embodiments, a “region” or “portion” of mouse exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, extracellular regions, transmembrane regions, and/or cytoplasmic regions are replaced by the corresponding human sequence. The term “region” or “portion” can refer to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 2000, 2500 or 2900 nucleotides, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, or 290 amino acid residues. In some embodiments, the “region” or “portion” can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%identical to exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, extracellular regions, transmembrane regions, or cytoplasmic regions. In some embodiments, a region, a portion, or the entire sequence of mouse exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7 (e.g., a portion of exon 2, exon 3, exon 4, exon 5, exon 6, and a portion of exon 7) are replaced by a region, a portion, or the entire sequence of the human exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7 (e.g., a portion of exon 2, exon 3, exon 4, exon 5, exon 6, and a portion of exon 7) .

In some embodiments, the present disclosure is related to a genetically-modified, non-human animal whose genome comprises a chimeric (e.g., humanized) CD20 nucleotide sequence. In some embodiments, the chimeric (e.g., humanized) CD20 nucleotide sequence encodes a CD20 protein comprising one or more extracellular regions, one or more transmembrane regions, and/or one or more cytoplasmic regions. In some embodiments, the genome of the animal comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 22 or 23.

In some embodiments, the genetically-modified non-human animal described herein comprises a sequence encoding a human or humanized CD20 protein. In some embodiments, the humanized CD20 protein comprises one or more human cytoplasmic regions (e.g., the first cytoplasmic region, the second cytoplasmic region, or the third cytoplasmic region) . In some embodiments, the humanized CD20 protein comprises one or more endogenous cytoplasmic regions (e.g., the first cytoplasmic region, the second cytoplasmic region, or the third cytoplasmic region) . In some embodiments, the humanized CD20 protein comprises one or more human transmembrane regions (e.g., the first transmembrane region, the second transmembrane region, the third transmembrane region, or the fourth transmembrane region) . In some embodiments, the humanized CD20 protein comprises one or more endogenous transmembrane regions (e.g., the first transmembrane region, the second transmembrane region, the third transmembrane region, or the fourth transmembrane region) . In some embodiments, the humanized CD20 protein comprises one or more human extracellular regions (e.g., the first extracellular region or the second extracellular region) . In some embodiments, the humanized CD20 protein comprises one or more endogenous extracellular regions (e.g., the first extracellular region or the second extracellular region) .

In some embodiment, the first cytoplasmic region is human or humanized. In some embodiment, the second cytoplasmic region is human or humanized. In some embodiment, the third cytoplasmic region is human or humanized. In some embodiment, the first transmembrane region is human or humanized. In some embodiment, the second transmembrane region is human or humanized. In some embodiment, the third transmembrane region is human or humanized. In some embodiment, the fourth transmembrane region is human or humanized. In some embodiment, the first extracellular region is human or humanized. In some embodiment, the second extracellular region is human or humanized.

In some embodiments, the genetically-modified non-human animal described herein comprises a human or humanized CD20 gene. In some embodiments, the humanized CD20 gene comprises 7 exons. In some embodiments, the humanized CD20 gene comprises humanized exon 1, humanized exon 2, humanized exon 3, humanized exon 4, humanized exon 5, humanized exon 6, and/or humanized exon 7. In some embodiments, the humanized CD20 gene comprises humanized intron 1, humanized intron 2, humanized intron 3, humanized intron 4, humanized intron 5, and/or humanized intron 6. In some embodiments, the humanized CD20 gene comprises human or humanized 5' UTR. In some embodiments, the humanized CD20 gene comprises human or humanized 3' UTR. In some embodiments, the humanized CD20 gene comprises endogenous 5' UTR. In some embodiments, the humanized CD20 gene comprises endogenous 3' UTR.

In some embodiments, the present disclosure also provides a chimeric (e.g., humanized) CD20 nucleotide sequence and/or amino acid sequences, wherein in some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%of the sequence are identical to or derived from mouse CD20 mRNA sequence (e.g., NM_007641.6) , mouse CD20 amino acid sequence (e.g., SEQ ID NO: 1) , or a portion thereof (e.g., exon 1, a portion of exon 2, and a portion of exon 7) ; and in some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%of the sequence are identical to or derived from human CD20 mRNA sequence (e.g., NM_021950.4) , human CD20 amino acid sequence (e.g., SEQ ID NO: 2) , or a portion thereof (e.g., a portion of exon 2, exon 3, exon 4, exon 5, exon 6, and a portion of exon 7) .

In some embodiments, the sequence encoding amino acids 1-291 of mouse CD20 (SEQ ID NO: 1) is replaced. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human CD20 (e.g., amino acids 1-297 of human CD20 (SEQ ID NO: 2) ) .

In some embodiments, the nucleic acids as described herein are operably linked to a promotor or regulatory element, e.g., an endogenous mouse CD20 promotor, an inducible promoter, an enhancer, and/or mouse or human regulatory elements.

In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that are different from part of or the entire mouse CD20 nucleotide sequence (e.g., a portion of exon 2, exon 3, exon 4, exon 5, exon 6, and a portion of exon 7 of NM_007641.6) .

In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is the same as part of or the entire mouse CD20 nucleotide sequence (e.g., exon 1, a portion of exon 2, and a portion of exon 7 of NM_007641.6) .

In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is different from part of or the entire human CD20 nucleotide sequence (e.g., exon 1, a portion of exon 2, and a portion of exon 7 of NM_021950.4) .

In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is the same as part of or the entire human CD20 nucleotide sequence (e.g., a portion of exon 2, exon 3, exon 4, exon 5, exon 6, and a portion of exon 7 of NM_021950.4) .

In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is different from part of or the entire mouse CD20 amino acid sequence (e.g., NP_031667.1 (SEQ ID NO: 1) ) .

In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is the same as part of or the entire mouse CD20 amino acid sequence (e.g., NP_031667.1 (SEQ ID NO: 1) ) .

In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is different from part of or the entire human CD20 amino acid sequence (e.g., NP_068769.2 (SEQ ID NO: 2) ) .

In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is the same as part of or the entire human CD20 amino acid sequence (e.g., NP_068769.2 (SEQ ID NO: 2) ) .

The present disclosure also provides a humanized CD20 mouse amino acid sequence, wherein the amino acid sequence is selected from the group consisting of:

a) an amino acid sequence shown in SEQ ID NO: 1 or 2;

b) an amino acid sequence having a homology of at least 90%with or at least 90%identical to the amino acid sequence shown in SEQ ID NO: 1 or 2;

c) an amino acid sequence encoded by a nucleic acid sequence, wherein the nucleic acid sequence is able to hybridize to a nucleotide sequence encoding the amino acid shown in SEQ ID NO: 1 or 2 under a low stringency condition or a strict stringency condition;

d) an amino acid sequence having a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence shown in SEQ ID NO: 1 or 2;

e) an amino acid sequence that is different from the amino acid sequence shown in SEQ ID NO: 1 or 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or

f) an amino acid sequence that comprises a substitution, a deletion and /or insertion of one or more amino acids to the amino acid sequence shown in SEQ ID NO: 1 or 2.

The present disclosure also relates to a CD20 nucleic acid (e.g., DNA or RNA) sequence, wherein the nucleic acid sequence can be selected from the group consisting of:

a) a nucleic acid sequence as shown in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 22 or 23, or a nucleic acid sequence encoding a homologous CD20 amino acid sequence of a humanized mouse CD20;

b) a nucleic acid sequence that is able to hybridize to the nucleotide sequence as shown in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 22 or 23 under a low stringency condition or a strict stringency condition;

c) a nucleic acid sequence that has a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the nucleotide sequence as shown in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 22 or 23;

d) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence has a homology of at least 90%with or at least 90%identical to the amino acid sequence shown in SEQ ID NO: 1 or 2;

e) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence has a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%with, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence shown in SEQ ID NO: 1 or 2;

f) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence is different from the amino acid sequence shown in SEQ ID NO: 1 or 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; and/or

g) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence comprises a substitution, a deletion and /or insertion of one or more amino acids to the amino acid sequence shown in SEQ ID NO: 1 or 2.

The present disclosure further relates to a CD20 genomic DNA sequence of a humanized mouse. The DNA sequence is obtained by reverse transcription of the mRNA obtained by transcription thereof is consistent with or complementary to the DNA sequence homologous to the sequence shown in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 22 or 23.

The disclosure also provides an amino acid sequence that has a homology of at least 90%with, or at least 90%identical to the sequence shown in SEQ ID NO: 1 or 2, and has protein activity. In some embodiments, the homology with the sequence shown in SEQ ID NO: 1 or 2 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. In some embodiments, the foregoing homology is at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, or 85%.

In some embodiments, the percentage identity with the sequence shown in SEQ ID NO: 1 or 2 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. In some embodiments, the foregoing percentage identity is at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, or 85%.

The disclosure also provides a nucleotide sequence that has a homology of at least 90%, or at least 90%identical to the sequence shown in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 22 or 23, and encodes a polypeptide that has protein activity. In some embodiments, the homology with the sequence shown in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 22 or 23 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. In some embodiments, the foregoing homology is at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, or 85%.

In some embodiments, the percentage identity with the sequence shown in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 22 or 23 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. In some embodiments, the foregoing percentage identity is at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, or 85%.

The disclosure also provides a nucleic acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to any nucleotide sequence as described herein, and an amino acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to any amino acid sequence as described herein. In some embodiments, the disclosure relates to nucleotide sequences encoding any peptides that are described herein, or any amino acid sequences that are encoded by any nucleotide sequences as described herein. In some embodiments, the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides. In some embodiments, the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acid residues.

In some embodiments, the amino acid sequence (i) comprises an amino acid sequence; or (ii) consists of an amino acid sequence, wherein the amino acid sequence is any one of the sequences as described herein.

In some embodiments, the nucleic acid sequence (i) comprises a nucleic acid sequence; or (ii) consists of a nucleic acid sequence, wherein the nucleic acid sequence is any one of the sequences as described herein.

To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes) . The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. For example, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

The percentage of residues conserved with similar physicochemical properties (percent homology) , e.g. leucine and isoleucine, can also be used to measure sequence similarity. Families of amino acid residues having similar physicochemical properties have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine) , acidic side chains (e.g., aspartic acid, glutamic acid) , uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine) , nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) , beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine) . The homology percentage, in many cases, is higher than the identity percentage.

Cells, tissues, and animals (e.g., mouse) are also provided that comprise the nucleotide sequences as described herein, as well as cells, tissues, and animals (e.g., mouse) that express human or chimeric (e.g., humanized) CD20 from an endogenous non-human CD20 locus.

Genetically modified animals

As used herein, the term “genetically-modified non-human animal” refers to a non-human animal having exogenous DNA in at least one chromosome of the animal's genome. In some embodiments, at least one or more cells, e.g., at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%of cells of the genetically-modified non-human animal have the exogenous DNA in its genome. The cell having exogenous DNA can be various kinds of cells, e.g., hepatocytes, lymphocytes, monocytes, macrophages, endothelial cells, epithelial cells, CD34+thymocytes, neurons or tumor cells. In some embodiments, the cell is an islet alpha cell or an islet beta cell of pancreas. In some embodiments, genetically-modified non-human animals are provided that comprise a modified endogenous CD20 locus that comprises an exogenous sequence (e.g., a human sequence) , e.g., a replacement of one or more non-human sequences with one or more human sequences. The animals are generally able to pass the modification to progeny, i.e., through germline transmission.

As used herein, the term “chimeric gene” or “chimeric nucleic acid” refers to a gene or a nucleic acid, wherein two or more portions of the gene or the nucleic acid are from different species, or at least one of the sequences of the gene or the nucleic acid does not correspond to the wild-type nucleic acid in the animal. In some embodiments, the chimeric gene or chimeric nucleic acid has at least one portion of the sequence that is derived from two or more different sources, e.g., sequences encoding different proteins or sequences encoding the same (or homologous) protein of two or more different species. In some embodiments, the chimeric gene or the chimeric nucleic acid is a humanized gene or humanized nucleic acid.

As used herein, the term “chimeric protein” or “chimeric polypeptide” refers to a protein or a polypeptide, wherein two or more portions of the protein or the polypeptide are from different species, or at least one of the sequences of the protein or the polypeptide does not correspond to wild-type amino acid sequence in the animal. In some embodiments, the chimeric protein or the chimeric polypeptide has at least one portion of the sequence that is derived from two or more different sources, e.g., same (or homologous) proteins of different species. In some embodiments, the chimeric protein or the chimeric polypeptide is a humanized protein or a humanized polypeptide.

As used herein, the term “humanized protein” or “humanized polypeptide” refers to a protein or a polypeptide, wherein at least a portion of the protein or the polypeptide is from the human protein or human polypeptide. In some embodiments, the humanized protein or polypeptide is a human protein or polypeptide.

As used herein, the term “humanized nucleic acid” refers to a nucleic acid, wherein at least a portion of the nucleic acid is from the human. In some embodiments, the entire nucleic acid of the humanized nucleic acid is from human. In some embodiments, the humanized nucleic acid is a humanized exon. A humanized exon can be e.g., a human exon or a chimeric exon.

In some embodiments, the chimeric gene or the chimeric nucleic acid is a humanized CD20 gene or a humanized CD20 nucleic acid. In some embodiments, at least one or more portions of the gene or the nucleic acid is from the human CD20 gene, at least one or more portions of the gene or the nucleic acid is from a non-human CD20 gene. In some embodiments, the gene or the nucleic acid comprises a sequence that encodes an CD20 protein. The encoded CD20 protein is functional or has at least one activity of the human CD20 protein or the non-human CD20 protein, e.g., transporting calcium.

In some embodiments, the chimeric protein or the chimeric polypeptide is a humanized CD20 protein or a humanized CD20 polypeptide. In some embodiments, at least one or more portions of the amino acid sequence of the protein or the polypeptide is from a human CD20 protein, and at least one or more portions of the amino acid sequence of the protein or the polypeptide is from a non-human CD20 protein. The humanized CD20 protein or the humanized CD20 polypeptide is functional or has at least one activity of the human CD20 protein or the non-human CD20 protein.

The genetically modified non-human animal can be various animals, e.g., a mouse, rat, rabbit, pig, bovine (e.g., cow, bull, buffalo) , deer, sheep, goat, chicken, cat, dog, ferret, primate (e.g., marmoset, rhesus monkey) . For the non-human animals where suitable genetically modifiable embryonic stem (ES) cells are not readily available, other methods are employed to make a non-human animal comprising the genetic modification. Such methods include, e.g., modifying a non-ES cell genome (e.g., a fibroblast or an induced pluripotent cell) and employing nuclear transfer to transfer the modified genome to a suitable cell, e.g., an oocyte, and gestating the modified cell (e.g., the modified oocyte) in a non-human animal under suitable conditions to form an embryo. These methods are known in the art, and are described, e.g., in A. Nagy, et al., “Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition) , ” Cold Spring Harbor Laboratory Press, 2003, which is incorporated by reference herein in its entirety.

In one aspect, the animal is a mammal, e.g., of the superfamily Dipodoidea or Muroidea. In some embodiments, the genetically modified animal is a rodent. The rodent can be selected from a mouse, a rat, and a hamster. In some embodiments, the genetically modified animal is from a family selected from Calomyscidae (e.g., mouse-like hamsters) , Cricetidae (e.g., hamster, New World rats and mice, voles) , Muridae (true mice and rats, gerbils, spiny mice, crested rats) , Nesomyidae (climbing mice, rock mice, with-tailed rats, Malagasy rats and mice) , Platacanthomyidae (e.g., spiny dormice) , and Spalacidae (e.g., mole rates, bamboo rats, and zokors) . In some embodiments, the genetically modified rodent is selected from a true mouse or rat (family Muridae) , a gerbil, a spiny mouse, and a crested rat. In some embodiments, the non-human animal is a mouse.

In some embodiments, the animal is a mouse of a C57BL strain selected from C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola. In some embodiments, the mouse is a 129 strain selected from the group consisting of a strain that is 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV, 129S1/SvIm) , 129S2, 129S4, 129S5, 129S9/SvEvH, 129S6 (129/SvEvTac) , 129S7, 129S8, 129T1, 129T2. These mice are described, e.g., in Festing et al., Revised nomenclature for strain 129 mice, Mammalian Genome 10: 836 (1999) ; Auerbach et al., Establishment and Chimera Analysis of 129/SvEv-and C57BL/6-Derived Mouse Embryonic Stem Cell Lines (2000) , both of which are incorporated herein by reference in the entirety. In some embodiments, the genetically modified mouse is a mix of the 129 strain and the C57BL/6 strain. In some embodiments, the mouse is a mix of the 129 strains, or a mix of the BL/6 strains. In some embodiments, the mouse is a BALB strain, e.g., BALB/c strain. In some embodiments, the mouse is a mix of a BALB strain and another strain. In some embodiments, the mouse is from a hybrid line (e.g., 50%BALB/c-50%12954/Sv; or 50%C57BL/6-50%129) . In some embodiments, the non-human animal is a rodent. In some embodiments, the non-human animal is a mouse having a BALB/c, A, A/He, A/J, A/WySN, AKR, AKR/A, AKR/J, AKR/N, TA1, TA2, RF, SWR, C3H, C57BR, SJL, C57L, DBA/2, KM, NIH, ICR, CFW, FACA, C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL (C57BL/10Cr and C57BL/Ola) , C58, CBA/Br, CBA/Ca, CBA/J, CBA/st, or CBA/H background.

In some embodiments, the animal is a rat. The rat can be selected from a Wistar rat, an LEA strain, a Sprague Dawley strain, a Fischer strain, F344, F6, and Dark Agouti. In some embodiments, the rat strain is a mix of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.

The animal can have one or more other genetic modifications, and/or other modifications, that are suitable for the particular purpose for which the humanized CD20 animal is made. For example, suitable mice for maintaining a xenograft (e.g., a human cancer or tumor) , can have one or more modifications that compromise, inactivate, or destroy the immune system of the non-human animal in whole or in part. Compromise, inactivation, or destruction of the immune system of the non-human animal can include, for example, destruction of hematopoietic cells and/or immune cells by chemical means (e.g., administering a toxin) , physical means (e.g., irradiating the animal) , and/or genetic modification (e.g., knocking out one or more genes) . Non-limiting examples of such mice include, e.g., NOD mice, SCID mice, NOD/SCID mice, IL2Rγknockout mice, NOD/SCID/γc ^null mice (Ito, M. et al., NOD/SCID/γc ^null mouse: an excellent recipient mouse model for engraftment of human cells, Blood 100 (9) : 3175-3182, 2002) , nude mice, and Rag 1 and/or Rag2 knockout mice. These mice can optionally be irradiated, or otherwise treated to destroy one or more immune cell type. Thus, in various embodiments, a genetically modified mouse is provided that can include a humanization of at least a portion of an endogenous non-human CD20 locus, and further comprises a modification that compromises, inactivates, or destroys the immune system (or one or more cell types of the immune system) of the non-human animal in whole or in part. In some embodiments, modification is, e.g., selected from the group consisting of a modification that results in NOD mice, SCID mice, NOD/SCID mice, IL-2Rγ knockout mice, NOD/SCID/γc ^null mice, nude mice, Ragl and/or Rag2 knockout mice, NOD-Prkdc ^scid IL-2rγ ^null mice, NOD-Rag 1 ^-/--IL2rg ^-/- (NRG) mice, Rag 2 ^-/--IL2rg ^-/- (RG) mice, and a combination thereof. These genetically modified animals are described, e.g., in US20150106961, which is incorporated herein by reference in its entirety. In some embodiments, the mouse can include a replacement of all or part of CD20 coding sequence with human CD20 coding sequence.

Genetically modified non-human animals that comprise a modification of an endogenous non-human CD20 locus. In some embodiments, the modification can comprise a human nucleic acid sequence encoding at least a portion of a CD20 protein (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%identical to the CD20 protein sequence) . Although genetically modified cells are also provided that can comprise the modifications described herein (e.g., ES cells, somatic cells) , in many embodiments, the genetically modified non-human animals comprise the modification of the endogenous CD20 locus in the germline of the animal.

Genetically modified animals can express a human CD20 and/or a chimeric (e.g., humanized) CD20 from endogenous mouse loci, wherein the endogenous mouse CD20 gene has been replaced with a human CD20 gene and/or a nucleotide sequence that encodes a region of human CD20 sequence or an amino acid sequence that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70&, 80%, 90%, 95%, 96%, 97%, 98%, or 99%identical to the human CD20 sequence. In various embodiments, an endogenous non-human CD20 locus is modified in whole or in part to comprise human nucleic acid sequence encoding at least one protein-coding sequence of a CD20 protein.

In some embodiments, the genetically modified mice express the human CD20 and/or chimeric CD20 (e.g., humanized CD20) from endogenous loci that are under control of mouse promoters and/or mouse regulatory elements. The replacement (s) at the endogenous mouse loci provide non-human animals that express human CD20 or chimeric CD20 (e.g., humanized CD20) in appropriate cell types and in a manner that does not result in the potential pathologies observed in some other transgenic mice known in the art. The human CD20 or the chimeric CD20 (e.g., humanized CD20) expressed in animal can maintain one or more functions of the wild-type mouse or human CD20 in the animal. For example, human or non-human CD20 ligands can bind to the expressed CD20. Furthermore, in some embodiments, the animal does not express endogenous CD20. In some embodiments, the animal expresses a decreased level of endogenous CD20 as compared to a wild-type animal. As used herein, the term “endogenous CD20” refers to CD20 protein that is expressed from an endogenous CD20 nucleotide sequence of the non-human animal (e.g., mouse) before any genetic modification.

The genome of the animal can comprise a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to human CD20 (NP_068769.2) (SEQ ID NO: 2) .

The genome of the genetically modified animal can comprise a replacement at an endogenous CD20 gene locus of a sequence encoding a region of endogenous CD20 with a sequence encoding a corresponding region of human CD20. In some embodiments, the sequence that is replaced is any sequence within the endogenous CD20 gene locus, e.g., exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, 5'-UTR, 3'-UTR, intron 1, intron 2, intron 3, intron 4, intron 5, and/or intron 6, etc. In some embodiments, the sequence that is replaced is within the regulatory region of the endogenous CD20 gene. In some embodiments, the sequence that is replaced is exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, or a portion thereof, of an endogenous mouse CD20 gene locus.

In some embodiments, the non-human animal can have, at an endogenous CD20 gene locus, a nucleotide sequence encoding a chimeric human/non-human CD20 polypeptide, wherein a human portion of the chimeric human/non-human CD20 polypeptide comprises a portion of human CD20, and wherein the animal expresses a functional CD20 on a surface of a cell of the animal. The human portion of the chimeric human/non-human CD20 polypeptide can comprise an amino acid sequence encoded by exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, or a portion thereof, of human CD20. In some embodiments, the human portion of the chimeric human/non-human CD20 polypeptide can comprise a sequence that is at least 80%, 85%, 90%, 95%, or 99%identical to amino acids 1-297 of SEQ ID NO: 2. In some embodiments, the human portion of the chimeric human/non-human CD20 polypeptide comprises one or more (e.g., 1, 2, 3, or 4) human CD20 transmembrane regions. In some embodiments, the human portion of the chimeric human/non-human CD20 polypeptide comprises one or more (e.g., 1, 2, or 3) human CD20 cytoplasmic regions. In some embodiments, the human portion of the chimeric human/non-human CD20 polypeptide comprises one or more (e.g., 1 or 2) human CD20 extracellular regions. In some embodiments, the first extracellular region is humanized. In some embodiments, the second extracellular region is humanized.

In some embodiments, the non-human portion of the chimeric human/non-human CD20 polypeptide comprises one or more (e.g., 1, 2, 3, or 4) transmembrane regions, one or more (e.g., 1, 2, or 3) cytoplasmic regions, and/or one or more (e.g., 1 or 2) extracellular regions of an endogenous non-human CD20 polypeptide.

Furthermore, the genetically modified animal can be heterozygous with respect to the replacement at the endogenous CD20 locus, or homozygous with respect to the replacement at the endogenous CD20 locus.

In some embodiments, the humanized CD20 locus lacks a human CD20 5'-UTR. In some embodiment, the humanized CD20 locus comprises an endogenous (e.g., mouse) 5'-UTR. In some embodiments, the humanization comprises an endogenous (e.g., mouse) 3'-UTR. In appropriate cases, it may be reasonable to presume that the mouse and human CD20 genes appear to be similarly regulated based on the similarity of their 5'-flanking sequence. As shown in the present disclosure, humanized CD20 mice that comprise a replacement at an endogenous mouse CD20 locus, which retain mouse regulatory elements but comprise a humanization of CD20 encoding sequence, do not exhibit pathologies. Both genetically modified mice that are heterozygous or homozygous for humanized CD20 are grossly normal.

The present disclosure further relates to a non-human mammal generated through the method mentioned above. In some embodiments, the genome thereof contains human gene (s) .

In some embodiments, the non-human mammal is a rodent, and preferably, the non-human mammal is a mouse.

In some embodiments, the non-human mammal expresses a protein encoded by a humanized CD20 gene.

In addition, the present disclosure also relates to a non-human mammal model for cancer, characterized in that the non-human mammal model is obtained through the methods as described herein. In some embodiments, the non-human mammal is a rodent (e.g., a mouse) .

The present disclosure further relates to a cell or cell line, or a primary cell culture thereof derived from the non-human mammal or an offspring thereof, or an animal model (e.g., cancer model) induced from the non-human mammal or an offspring thereof; the tissue (e.g., pancreas or kidney) , organ or a culture thereof derived from the non-human mammal or an offspring thereof, or the animal model.

The present disclosure also provides non-human mammals produced by any of the methods described herein. In some embodiments, a non-human mammal is provided; and the genetically modified animal contains the DNA encoding human or humanized CD20 in the genome of the animal.

In some embodiments, the non-human mammal comprises the genetic construct as described herein (e.g., gene construct as shown in FIGS. 2, 3, and 6) . In some embodiments, a non-human mammal expressing human or humanized CD20 is provided. In some embodiments, the tissue-specific expression of human or humanized CD20 protein is provided.

In some embodiments, the expression of human or humanized CD20 in a genetically modified animal is controllable, as by the addition of a specific inducer or repressor substance. In some embodiments, the specific inducer is selected from Tet-Off System/Tet-On System, or Tamoxifen System.

Non-human mammals can be any non-human animal known in the art and which can be used in the methods as described herein. Preferred non-human mammals are mammals, (e.g., rodents) . In some embodiments, the non-human mammal is a mouse.

Genetic, molecular and behavioral analyses for the non-human mammals described above can performed. The present disclosure also relates to the progeny produced by the non-human mammal provided by the present disclosure mated with the same or other genotypes.

The present disclosure also provides a cell line or primary cell culture derived from the non-human mammal or a progeny thereof. A model based on cell culture can be prepared, for example, by the following methods. Cell cultures can be obtained by way of isolation from a non-human mammal, alternatively cells can be obtained from the cell culture established using the same constructs and the standard cell transfection techniques. The integration of genetic constructs containing DNA sequences encoding human CD20 protein can be detected by a variety of methods.

There are many analytical methods that can be used to detect exogenous DNA, including methods at the level of nucleic acid (including the mRNA quantification approaches using reverse transcriptase polymerase chain reaction (RT-PCR) or Southern blotting, and in situ hybridization) and methods at the protein level (including histochemistry, immunoblot analysis and in vitro binding studies) . In addition, the expression level of the gene of interest can be quantified by ELISA techniques well known to those skilled in the art. Many standard analysis methods can be used to complete quantitative measurements. For example, transcription levels can be measured using RT-PCR and hybridization methods including RNase protection, Southern blot analysis, RNA dot analysis (RNAdot) analysis. Immunohistochemical staining, flow cytometry, Western blot analysis can also be used to assess the presence of human or humanized CD20 protein.

Vectors

The present disclosure relates to a targeting vector, comprising: a) a DNA fragment homologous to the 5' end of a region to be altered (5' homology arm) , which is selected from the CD20 gene genomic DNAs in the length of 100 to 10,000 nucleotides; b) a desired/donor DNA sequence encoding a donor region; and c) a second DNA fragment homologous to the 3' end of the region to be altered (3' homology arm) , which is selected from the CD20 gene genomic DNAs in the length of 100 to 10,000 nucleotides.

In some embodiments, a) the DNA fragment homologous to the 5' end of a conversion region to be altered (5' arm) is selected from the nucleotide sequences that have at least 90%homology to the NCBI accession number NC_000085.7; c) the DNA fragment homologous to the 3' end of the region to be altered (3' arm) is selected from the nucleotide sequences that have at least 90%homology to the NCBI accession number NC_000085.7.

In some embodiments, the length of the selected genomic nucleotide sequence in the targeting vector can be more than about 3 kb, about 3.5 kb, about 4 kb, about 4.5 kb, about 5 kb, about 5.5 kb, or about 6 kb.

In some embodiments, the region to be altered is exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7 of CD20 gene (e.g., exon 2, exon 3, exon 4, exon 5, exon 6, and exon 7 of mouse CD20 gene) .

The targeting vector can further include one or more selectable markers, e.g., positive and/or negative selectable markers. In some embodiments, the positive selectable marker is a Neo gene or Neo cassette. In some embodiments, the negative selectable marker is a DTA gene.

In some embodiments, the sequence of the 5' homology arm is shown in SEQ ID NO: 3; and the sequence of the 3' homology arm is shown in SEQ ID NO: 4.

In some embodiments, the sequence of the 5' homology arm is shown in SEQ ID NO: 22; and the sequence of the 3' homology arm is shown in SEQ ID NO: 23.

In some embodiments, the sequence is derived from human (e.g., 60462375-60468468 of NC_000011.10) . For example, the target region in the targeting vector is a part or entirety of the nucleotide sequence of a human CD20 gene, preferably exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7 of the human CD20 gene. In some embodiments, the nucleotide sequence of the humanized CD20 encodes the entire or the part of human CD20 protein with the NCBI accession number NP_068769.2 (SEQ ID NO: 2) .

The disclosure also relates to a cell comprising the targeting vectors as described above.

In addition, the present disclosure further relates to a non-human mammalian cell, having any one of the foregoing targeting vectors, and one or more in vitro transcripts of the construct as described herein. In some embodiments, the cell includes Cas9 mRNA or an in vitro transcript thereof.

In some embodiments, the genes in the cell are heterozygous. In some embodiments, the genes in the cell are homozygous.

In some embodiments, the non-human mammalian cell is a mouse cell. In some embodiments, the cell is a fertilized egg cell. In some embodiments, the cell is an embryonic stem cell.

Methods of making genetically modified animals

Genetically modified animals can be made by several techniques that are known in the art, including, e.g., nonhomologous end-joining (NHEJ) , homologous recombination (HR) , zinc finger nucleases (ZFNs) , transcription activator-like effector-based nucleases (TALEN) , and the clustered regularly interspaced short palindromic repeats (CRISPR) -Cas system. In some embodiments, homologous recombination is used. In some embodiments, CRISPR-Cas9 genome editing is used to generate genetically modified animals. Many of these genome editing techniques are known in the art, and is described, e.g., in Yin et al., "Delivery technologies for genome editing, " Nature Reviews Drug Discovery 16.6 (2017) : 387-399, which is incorporated by reference in its entirety. Many other methods are also provided and can be used in genome editing, e.g., micro-injecting a genetically modified nucleus into an enucleated oocyte, and fusing an enucleated oocyte with another genetically modified cell.

Thus, in some embodiments, the disclosure provides replacing in at least one cell of the animal, at an endogenous CD20 gene locus, a sequence encoding a region of an endogenous CD20 with a sequence encoding a corresponding region of human or chimeric CD20. In some embodiments, the replacement occurs in a germ cell, a somatic cell, a blastocyst, or a fibroblast, etc. The nucleus of a somatic cell or the fibroblast can be inserted into an enucleated oocyte.

FIG. 3 shows a humanization strategy for a mouse CD20 locus. In FIG. 3, the targeting strategy involves a vector comprising the 5' homologous arm, human CD20 gene fragment, 3' homologous arm. The process can involve replacing endogenous CD20 sequence with human sequence by homologous recombination. In some embodiments, the cleavage at the upstream and the downstream of the target site (e.g., by zinc finger nucleases, TALEN or CRISPR) can result in DNA double strands break, and the homologous recombination is used to replace endogenous CD20 sequence with human CD20 sequence.

FIG. 6 shows a humanization strategy for a mouse CD20 locus. In FIG. 6, the targeting strategy involves a vector comprising the 5' homologous arm, human CD20 gene fragment, 3' homologous arm. The process can involve replacing endogenous CD20 sequence with human sequence by homologous recombination. In some embodiments, the cleavage at the upstream and the downstream of the target site (e.g., by zinc finger nucleases, TALEN or CRISPR) can result in DNA double strand breaks, and the homologous recombination is used to replace endogenous CD20 sequence with human CD20 sequence.

In some embodiments, sgRNAs targeting SEQ ID NO: 24 and SEQ ID NO: 25 are used to make the cleavage at the upstream and the downstream of the target site, to create DNA double strand breaks.

Thus, in some embodiments, the methods for making a genetically modified, humanized animal, can include the step of replacing at an endogenous CD20 locus (or site) , a nucleic acid encoding a sequence encoding a region of endogenous CD20 with a sequence encoding a corresponding region of human CD20. The sequence can include a region (e.g., a part or the entire region) of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7 of a human CD20 gene. In some embodiments, the sequence includes a region of exon 2, exon 3, exon 4, exon 5, exon 6, and a region of exon 7 of a human CD20 gene (e.g., nucleic acids 116-1009 of NM_021950.4) . In some embodiments, the endogenous CD20 locus is exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7 of mouse CD20. In some embodiments, the sequence includes a region of exon 2, exon 3, exon 4, exon 5, exon 6, and a region of exon 7 of mouse CD20 gene (e.g., nucleic acids 118-993 of NM_007641.6) .

In some embodiments, the methods of modifying a CD20 locus of a mouse to express a chimeric human/mouse CD20 peptide can include the steps of replacing at the endogenous mouse CD20 locus a nucleotide sequence encoding a mouse CD20 with a nucleotide sequence encoding a human CD20, thereby generating a sequence encoding a chimeric human/mouse CD20.

In some embodiments, the nucleotide sequences as described herein do not overlap with each other (e.g., the first nucleotide sequence, the second nucleotide sequence, and/or the third nucleotide sequence do not overlap) . In some embodiments, the amino acid sequences as described herein do not overlap with each other.

The present disclosure further provides a method for establishing a CD20 gene humanized animal model, involving the following steps:

(a) providing the cell (e.g. a fertilized egg cell) based on the methods described herein;

(b) culturing the cell in a liquid culture medium;

(c) transplanting the cultured cell to the fallopian tube or uterus of the recipient female non-human mammal, allowing the cell to develop in the uterus of the female non-human mammal;

(d) identifying the germline transmission in the offspring genetically modified humanized non-human mammal of the pregnant female in step (c) .

(1) providing a plasmid comprising a human CD20 gene fragment, flanked by a 5' homology arm (e.g., SEQ ID NO: 22) and a 3' homology arm (e.g., SEQ ID NO: 23) , wherein the 5' and 3' homology arms target an endogenous CD20 gene;

(5) mating the child mouse obtained in step (2) to obtain a homozygote mouse,

wherein the fertilized egg is modified by CRISPR with sgRNAs that target a 5'-terminal targeting site comprising SEQ ID NO: 24 and a 3'-terminal targeting site comprising SEQ ID NO: 25.

In some embodiments, the non-human mammal in the foregoing method is a mouse (e.g., a C57BL/6 mouse) .

In some embodiments, the non-human mammal in step (c) is a female with pseudopregnancy (or false pregnancy) .

In some embodiments, the fertilized eggs for the methods described above are C57BL/6 fertilized eggs. Other fertilized eggs that can also be used in the methods as described herein include, but are not limited to, FVB/N fertilized eggs, BALB/c fertilized eggs, DBA/1 fertilized eggs and DBA/2 fertilized eggs.

Fertilized eggs can come from any non-human animal, e.g., any non-human animal as described herein. In some embodiments, the fertilized egg cells are derived from rodents. The genetic construct can be introduced into a fertilized egg by microinjection of DNA. For example, by way of culturing a fertilized egg after microinjection, a cultured fertilized egg can be transferred to a false pregnant non-human animal, which then gives birth of a non-human mammal, so as to generate the non-human mammal mentioned in the methods described above.

Methods of using genetically modified animals

Replacement of non-human genes in a non-human animal with homologous or orthologous human genes or human sequences, at the endogenous non-human locus and under control of endogenous promoters and/or regulatory elements, can result in a non-human animal with qualities and characteristics that may be substantially different from a typical knockout- plus-transgene animal. In the typical knockout-plus-transgene animal, an endogenous locus is removed or damaged and a fully human transgene is inserted into the animal's genome and presumably integrates at random into the genome. Typically, the location of the integrated transgene is unknown; expression of the human protein is measured by transcription of the human gene and/or protein assay and/or functional assay. Inclusion in the human transgene of upstream and/or downstream human sequences are apparently presumed to be sufficient to provide suitable support for expression and/or regulation of the transgene.

In some cases, the transgene with human regulatory elements expresses in a manner that is unphysiological or otherwise unsatisfactory, and can be actually detrimental to the animal. The disclosure demonstrates that a replacement with human sequence at an endogenous locus under control of endogenous regulatory elements provides a physiologically appropriate expression pattern and level that results in a useful humanized animal whose physiology with respect to the replaced gene are meaningful and appropriate in the context of the humanized animal's physiology.

Genetically modified animals that express human or humanized CD20 protein, e.g., in a physiologically appropriate manner, provide a variety of uses that include, but are not limited to, developing therapeutics for human diseases and disorders, and assessing the toxicity and/or the efficacy of these human therapeutics in the animal models.

In various aspects, genetically modified animals are provided that express human or humanized CD20, which are useful for testing agents that can decrease or block the interaction between CD20 and CD20 ligands or the interaction between CD20 and anti-human CD20 antibodies, testing whether an agent can increase or decrease calcium transportation, and/or determining whether an agent is an CD20 agonist or antagonist. The genetically modified animals can be, e.g., an animal model of a human disease, e.g., the disease is induced genetically (aknock-in or knockout) . In some embodiments, the anti-CD20 antibody blocks or inhibits the CD20-related signaling pathway.

In some embodiments, the genetically-modified animals can be used for determining effectiveness of a CD20 modulator (e.g., an anti-CD20 antibody) for treating cancer. The methods involve administering the anti-CD20 antibody (e.g., an anti-human CD20 antibody) to the animal as described herein; and determining the tumor volume of the animal. The method can further include comparing the tumor volume of the animal with tumor volume of a reference animal. In some embodiments, the reference animal is not administered with the anti-CD20 antibody, or administered with an isotype control (e.g., human IgG4) . In some embodiments, the reference animal is not genetically modified (e.g., a wild-type animal) . In some embodiments, the reference animal includes an endogenous CD20 gene locus. In some embodiments, the reference animal has the same background as the animal used for determining effectiveness of an anti-CD20 antibody for the treatment of cancer.

In some embodiments, the genetically-modified animals can be used for determining effectiveness of a CD20 modulator (e.g., an anti-CD20 antibody) , optionally in combination with one or more additional therapeutic agents (e.g. a second therapeutic agent) , for treating cancer.

The present disclosure also provides methods of determining toxicity of a CD20 modulator (e.g., anti-CD20 antibodies) . The methods involve administering the antibody to the animal as described herein. The animal is then evaluated for its weight change, red blood cell count, hematocrit, and/or hemoglobin. In some embodiments, the antibody can decrease the red blood cells (RBC) , hematocrit, or hemoglobin by more than 20%, 30%, 40%, or 50%. In some embodiments, the animals can have a weight that is at least 5%, 10%, 20%, 30%, or 40%smaller than the weight of the control group (e.g., average weight of the animals that are not treated with the antibody) .

The present disclosure also relates to the use of the animal model generated through the methods as described herein in the development of a product related to an immunization processes of human cells, the manufacturing of a human antibody, or the model system for a research in pharmacology, immunology, microbiology and medicine.

In some embodiments, the disclosure provides the use of the animal model generated through the methods as described herein in the production and utilization of an animal experimental disease model of an immunization processes involving human cells, the study on a pathogen, or the development of a new diagnostic strategy and/or a therapeutic strategy.

The disclosure also relates to the use of the animal model generated through the methods as described herein in the screening, verifying, evaluating or studying the CD20 gene function, human CD20 antibodies, drugs or efficacies for human CD20 targeting sites.

In some embodiments, the genetically-modified non-human animals described herein can be used to generated animal models for preparation and screening of drugs for the treatment of cancer.

In some embodiments, the CD20 modulator is selected from CAR-T and small-molecular drugs. In some embodiments, the CD20 modulator is an anti-CD20 antibody or antigen-binding fragment thereof.

In some embodiments, the genetically modified animals can be used for determining effectiveness of an anti-CD20 antibody for the treatment of cancer. The methods involve administering the anti-CD20 antibody (e.g., anti-human CD20 antibody) to the animal as described herein, wherein the animal has a tumor; and determining the inhibitory effects of the anti-CD20 antibody to the tumor. The inhibitory effects that can be determined include, e.g., a decrease of tumor size or tumor volume, a decrease of tumor growth, a reduction of the increase rate of tumor volume in a subject (e.g., as compared to the rate of increase in tumor volume in the same subject prior to treatment or in another subject without such treatment) , a decrease in the risk of developing a metastasis or the risk of developing one or more additional metastasis, an increase of survival rate, and an increase of life expectancy, etc. The tumor volume in a subject can be determined by various methods, e.g., as determined by direct measurement, MRI or CT.

In some embodiments, the tumor comprises one or more cancer cells (e.g., human or mouse cancer cells) that are injected into the animal. In some embodiments, the anti-CD20 antibody prevents CD20 ligands from binding to CD20. In some embodiments, the anti-CD20 antibody does not prevent CD20 ligands from binding to CD20.

In some embodiments, the genetically modified animals can be used for determining whether an anti-CD20antibody is a CD20agonist or antagonist. In some embodiments, the methods as described herein are also designed to determine the effects of the agent (e.g., anti-CD20antibodies) on CD20, e.g., whether the agent can stimulate immune cells or inhibit immune cells, whether the agent can increase or decrease the production of cytokines, whether the agent can activate or deactivate immune cells, whether the agent can upregulate the immune response or downregulate immune response, and/or whether the agent can induce complement mediated cytotoxicity (CMC) or antibody dependent cellular cytoxicity (ADCC) . In some embodiments, the genetically modified animals can be used for determining the effective dosage of a therapeutic agent for treating a disease in the subject, e.g., cancer, or autoimmune diseases.

The inhibitory effects on tumors can also be determined by methods known in the art, e.g., measuring the tumor volume in the animal, and/or determining tumor (volume) inhibition rate (TGI _TV) . The tumor growth inhibition rate can be calculated using the formula TGI _TV (%) = (1 -TVt/TVc) x 100, where TVt and TVc are the mean tumor volume (or weight) of treated and control groups.

In some embodiments, the anti-CD20 antibody is designed for treating various cancers. As used herein, the term “cancer” refers to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. The term “tumor” as used herein refers to cancerous cells, e.g., a mass of cancerous cells. Cancers that can be treated or diagnosed using the methods described herein include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. In some embodiments, the agents described herein are designed for treating or diagnosing a carcinoma in a subject. The term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. In some embodiments, the cancer is renal carcinoma or melanoma. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.

In some embodiments, the cancer types as described herein include, but not limited to, lymphoma, non-small cell lung cancer (NSCLC) , leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, stomach cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma. In some embodiments, the leukemia is selected from acute lymphocytic (lymphoblastic) leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myelogenous leukemia. In some embodiments, the lymphoma is selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T cell lymphoma, and Waldenstrom macroglobulinemia. In some embodiments, the sarcoma is selected from osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma. In some embodiments, the cancer types include B-cell leukemias and lymphomas such as chronic lymphocytic leukemia (CLL) , follicular lymphoma, and diffuse large B-cell lymphoma (DLBCL) .

In some embodiments, the antibody is designed for treating various immune disorder or immune-related diseases (e.g., psoriasis, allergic rhinitis, sinusitis, asthma, rheumatoid arthritis, atopic dermatitis, chronic obstructive pulmonary disease (COPD) , chronic bronchitis, emphysema, eczema, osteoarthritis, rheumatoid arthritis, systemic lupus erythematosus, polymyalgia rheumatica, autoimmune hemolytic anemia, systemic vasculitis, pernicious anemia, inflammatory bowel disease, ulcerative colitis, Crohn's disease, or multiple sclerosis) . Thus, the methods as described herein can be used to determine the effectiveness of an anti-CD20 antibody in inhibiting immune response.

In some embodiments, the immune disorder or immune-related diseases described here include allergy, asthma, myocarditis, nephritis, hepatitis, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, primary thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, self-immune liver disease, diabetes, pain, or neurological disorders.

In some embodiments, the antibody is designed for reducing inflammation (e.g., inflammatory bowel disease, chronic inflammation, asthmatic inflammation, periodontitis, or wound healing) . Thus, the methods as described herein can be used to determine the effectiveness of an antibody for reducing inflammation. In some embodiments, the inflammation described herein includes degenerative inflammation, exudative inflammation, serous inflammation, fibrinitis, suppurative inflammation, hemorrhagic inflammation, necrotitis, catarrhal inflammation, proliferative inflammation, specific inflammation, tuberculosis, syphilis, leprosy, or lymphogranuloma.

In some embodiments, the antibody is designed for treating disorders of bone mineralization, e.g., rickets, renal diseases (renal osteodystrophy, Fanconi syndrome) , tumor-induced osteomalacia, hypophosphatasia, McCune-Albright syndrome, or osteogenesis imperfecta with mineralization defect (syndrome resembling osteogenesis imperfecta (SROI) . In some embodiments, the disorder of bone mineralization is osteoporosis.

The present disclosure also provides methods of determining toxicity of an antibody (e.g., anti-CD20 antibody) . The methods involve administering the antibody to the animal as described herein. The animal is then evaluated for its weight change, red blood cell count, hematocrit, and/or hemoglobin. In some embodiments, the antibody can decrease the red blood cells (RBC) , hematocrit, or hemoglobin by more than 20%, 30%, 40%, or 50%. In some embodiments, the animals can have a weight that is at least 5%, 10%, 20%, 30%, or 40%smaller than the weight of the control group (e.g., average weight of the animals that are not treated with the antibody) .

The disclosure also relates to the use of the animal model generated through the methods as described herein in the screening, verifying, evaluating or studying the CD20 gene function, human CD20 antibodies, drugs for human CD20 targeting sites, the drugs or efficacies for human CD20 targeting sites, the drugs for immune-related diseases and antitumor drugs.

In some embodiments, the disclosure provides a method to verify in vivo efficacy of TCR-T, CAR-T, and/or other immunotherapies (e.g., T-cell adoptive transfer therapies) . For example, the methods include transplanting human tumor cells into the animal described herein, and applying human CAR-T to the animal with human tumor cells. Effectiveness of the CAR-T therapy can be determined and evaluated. In some embodiments, the animal is selected from the CD20 gene humanized non-human animal prepared by the methods described herein, the CD20 gene humanized non-human animal described herein, the double-or multi-humanized non-human animal generated by the methods described herein (or progeny thereof) , a non-human animal expressing the human or humanized CD20 protein, or the tumor-bearing or inflammatory animal models described herein. In some embodiments, the TCR-T, CAR-T, and/or other immunotherapies can treat the CD20-associated diseases described herein. In some embodiments, the TCR-T, CAR-T, and/or other immunotherapies provides an evaluation method for treating the CD20-associated diseases described herein.

Genetically modified animal model with two or more human or chimeric genes

The present disclosure further relates to methods for generating genetically modified animal model with two or more human or chimeric genes. The animal can comprise a human or chimeric CD20 gene and a sequence encoding an additional human or chimeric protein.

In some embodiments, the additional human or chimeric protein can be programmed cell death protein 1 (PD-1) , programmed cell death ligand 1 (PD-L1) , T-Cell immunoreceptor with Ig and ITIM Domains (TIGIT) , Tumour Necrosis Factor alpha (TNF alpha) , tumor necrosis factor receptor superfamily member 9 (4-1BB) , Tumor necrosis factor ligand superfamily member 9 (4-1BBL) , cytotoxic T-lymphocyte-associated protein 4 (CTLA4) , CD47, Signal regulatory protein α (SIRPα) , OX40, T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) , or CD226.

The methods of generating genetically modified animal model with two or more human or chimeric genes (e.g., humanized genes) can include the following steps:

(a) using the methods of introducing human CD20 gene or chimeric CD20 gene as described herein to obtain a genetically modified non-human animal;

(b) mating the genetically modified non-human animal with another genetically modified non-human animal, and then screening the progeny to obtain a genetically modified non-human animal with two or more human or chimeric genes.

In some embodiments, in step (b) of the method, the genetically modified animal can be mated with a genetically modified non-human animal with human or chimeric PD-1, PD-L1, TIGIT, TNFA, 41BB, 41BBL, CTLA4, CD47, SIRPA, OX40, TIM3 or CD226. In some embodiments, the CD20 humanization is directly performed on a genetically modified animal having a human or chimeric PD-1, PD-L1, TIGIT, TNFA, 41BB, 41BBL, CTLA4, CD47, SIRPA, OX40, TIM3 or CD226.

As these proteins may involve different mechanisms, a combination therapy that targets two or more of these proteins thereof may be a more effective treatment. In fact, many related clinical trials are in progress and have shown a good effect. The genetically modified animal model with two or more human or humanized genes can be used for determining effectiveness of a combination therapy that targets two or more of these proteins, e.g., an anti-CD20 antibody and an additional therapeutic agent for the treatment of cancer. The methods include administering the anti-CD20 antibody and the additional therapeutic agent to the animal; and determining the tumor volume of the animal after the combined treatment. In some embodiments, the additional therapeutic agent is an antibody or antigen-binding fragment thereof that specifically binds to PD-1, PD-L1, TIGIT, TNFA, 41BB, 41BBL, CTLA4, CD47, SIRPA, OX40, TIM3 or CD226.

EXAMPLES

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Materials and Methods

The following materials were used in the following examples.

ScaI, NcoI, and EcoRV enzymes were purchased from NEB, and the product numbers are R3122S, R3193S, and R3195S, respectively;

C57BL/6 mice and Flp tool mice were purchased from the National Rodent Laboratory Animal Seed Center of China National Academy of Food and Drug Control;

Brilliant Violet 510 ^TM anti-mouse CD45 was purchased from Biolegend, Cat. No. 103138;

FITC anti-Mouse CD19 was purchased from Biolegend, Cat. No. 115506;

V450 Rat Anti-mouse CD1 lb was purchased from Biolegend, Cat. No. 8232657;

APC anti-mouse CD20 Antibody was purchased from Biolegend, Cat. No. 152107;

PE anti-human CD20 Antibody was purchased from Biolegend, Cat. No. 302305;

Purified anti-mouse CD16/32 was purchased from Biolegend, Cat. No. 101302.

EXAMPLE 1: Preparation of humanized mice with CD20 gene

Mouse CD20 gene (NCBI Gene ID: 12482, Primary source: MGI: 88321, UniProt: P19437, located at positions 11227043-112435137 of chromosome 19 NC_000085.7, based on transcript NM_007641.6 and its encoded protein NP_031667.1 (SEQ ID NO: 1) ) and human CD20 gene (NCBI Gene ID: 931, Primary source: HGNC: 7315, UniProt ID: A0A024R507, located at positions 60455847-60470752 of chromosome 11 NC_000011.10, based on transcript NM_021950.4 and its encoded protein NP_068769.2 (SEQ ID NO: 2) ) are used. FIG. 1 is a schematic diagram of the comparison between human CD20 gene and mouse CD20 gene.

To prepare the CD20 gene humanized mice, a nucleotide sequence encoding human CD20 protein was introduced into the mouse endogenous CD20 locus, so that the mouse expresses a human CD20 protein. Specifically, using gene editing technology, under the control of the regulatory element of the mouse CD20 gene, the coding region of the mouse CD20 gene was replaced with the coding region of the human CD20 gene. Alternatively, a partial sequence from exon 2 to exon 7 of the human CD20 gene (about 6.1 kb) was used to replace a partial sequence from exon 2 to exon 7 of the mouse CD20 gene (about 7.2kb) . A schematic diagram of the humanized CD20 locus is shown in FIG. 2.

A targeting strategy is shown in FIG. 3. The figure shows that the targeting vector contains the homology arm sequences upstream and downstream of the mouse CD20 gene, and the A fragment containing the human CD20 sequence. Among them, the upstream homology arm sequence (5′homology arm, SEQ ID NO: 3) is the same as the nucleotide sequence from 11236186 to 11240359 of the NCBI accession number NC_000085.7, and the downstream homology arm sequence (3′homology arm sequence, SEQ ID NO: 4) is identical to the nucleotide sequence from 11227043 to 11229028 of NCBI Accession No. NC_000085.7. The human CD20 nucleotide sequence in the A fragment (SEQ ID NO: 5) is the same as the nucleotide sequence from 60462375 to 60468468 of the NCBI accession number NC_000011.10. The connection between the human CD20 sequence and the upstream mouse sequence is designed as: 5'-CTTATTTTCAGGCGTTTGAAAA

CAACACCCAGAAATTCAGTAAA-3' (SEQ ID NO: 6) , wherein "A" in the sequence

is the last nucleotide of the mosue sequence, and the first "A" in the sequence " ATGA" is the first nucleotide of the human sequence. The connection between the human CD20 sequence and the downstream mouse sequence is designed as:5’-AAAATGACAGCTCTCC

TTTTCTTTTCT-3' (SEQ ID NO: 7) , where the last "A" in the sequence

is the last nucleotide of the human sequence, and the “A” in the sequence " ACTC" is the first nucleotide of the mouse sequence.

The targeting vector also includes a resistance gene for positive clone selection, namely the coding sequence of neomycin phosphotransferase (Neo) , and two site-specific recombination systems (Frt) flanking the resistance gene, forming a Neo cassette. The connection between the 5′ end of the Neo cassette and the upstream human sequence is designed as 5'-CAGAGTTATATTCACTAA

ACGGTATCGATAAGCTTGATATCGAATTCCGAA GTTCCTATTCTCTAGAAAG-3' (SEQ ID NO: 8) , wherein "C" in the sequence

is the last nucleotide of the upstream human sequence, and the first "G" in the sequence " GTCG" is the first nucleotide of the Neo cassette. The connection between the 3′ end of the Neo cassette with the downstream human sequence is designed as 5'-AGTATAGGAACTTCATCAGTCAGGTACATAATGGTGGATC CACTAGTTCTAGAGCGGCCGCGT

CAGAACGAAAATCTAAACTCCTCTATTACTG-3' (SEQ ID NO: 9) , where the "C" in the sequence

is the last nucleotide of the Neo cassette, and the first "A" in the sequence " AAAT" is the first nucleotide in the downstream human sequence. In addition, a negative selection marker (the encoding gene for diphtheria toxin A subunit (DTA) ) was also included downstream of the 3′ homology arm of the targeting vector. The mRNA sequence of the modified humanized mouse CD20 is shown in SEQ ID NO: 10, and the expressed protein sequence is shown in SEQ ID NO: 2.

Given that human CD20 has multiple isoforms or transcripts, the methods described herein can be applied to other isoforms or transcripts.

The construction of the vector can be carried out by enzyme digestion and ligation. The constructed targeting vector is preliminarily verified by enzyme digestion, and then sent to a sequencing company for sequencing verification. The targeting vector verified by sequencing was electroporated into embryonic stem cells of C57BL/6 mice, and the obtained cells were screened with the positive clone selection marker. PCR and Southern Blot technology were used to detect and confirm the integration of exogenous genes. Specifically, the correct positive clones were screened, identified with PCR, and then confirmed with Southern Blot (the cellular DNA was digested with NcoI, ScaI, or EcoRV enzymes and 3 probes were used for hybridization, the lengths of the target fragments are shown in the table below) . The positive clones that were confirmed by Southern Blot, and confirmed to be positive (with no random insertion) by sequencing and were selected.

Table 3: Specific probes and target fragment lengths

Endonuclease	Probe	Wildtype fragment size	Recombination fragment size
NcoI	5’Probe	8.0kb	6.4kb
ScaI	3’Probe	9.2kb	12kb
EcoRV	NeoProbe	-	12kb

The PCR assay includes the following primers:

PCR-F1: 5′-TGGAGGAAGCCTCAAGTGTCTCA-3′ (SEQ ID NO: 11)

PCR-RI: 5′-CCTATACCGCATCAGCTTCTGTCA-3′ (SEQ ID NO: 12) ;

The Southern Blot detection includes the following probe primers:

5′Probe:

5′Probe-F: 5′-ACTGTGCAGAAAAGGCAACAGGCTA-3′ (SEQ ID NO: 13) ,

5′Probe-R: 5′-TCCTGGGACCTACTCTCTCCTTGTG-3′ (SEQ ID NO: 14) ;

3′Probe:

3′Probe-F: 5′-TTTACATGGCATGCCCACAATGGTT-3′ (SEQ ID NO: 15) ,

3′Probe-R: 5′-ATTCCACCTTCACTGAGTTCCCTCC-3′ (SEQ ID NO: 16) ;

NeoProbe:

NeoProbe-F: 5′-GGATCGGCCATTGAACAAGAT-3′ (SEQ ID NO: 17) ,

NeoProbe-R: 5′-CAGAAGAACTCGTCAAGAAGGC-3′ (SEQ ID NO: 18) .

The positive clone cells (black mice) were introduced into the isolated blastocysts (white mice) according to techniques known in the art, and the obtained chimeric blastocysts were transferred to cell culture medium for short-term culture and then transplanted to the fallopian tubes of recipient female mice (white mice) to produce F0 generation chimeric mice (black and white) . The F0 generation chimeric mice were backcrossed with the wild-type mice to obtain the F1 generation mice, and then the F1 generation heterozygous mice were mated with each other to obtain the F2 generation homozygous mice. The mice can also be mated with the Flp tool mice to remove the positive clone selection marker gene (see FIG. 4 for a schematic diagram of the process) , and then mated with each other to obtain CD20 gene humanized homozygous mice. The genotype of the offspring mouse somatic cells can be determined by PCR (primers are shown in the table below) , and the identification results of an exemplary F1 generation mouse (the Neo marker gene has been removed) are shown in FIG. 5, where the F1-01, F1-02 and F1- 03 mice were all positive heterozygous mice. This shows that a CD20 gene humanized mouse that can be stably passaged without random insertion can be obtained.

Table 4: Primer names and specific sequences

Further, the CRISPR/Cas system was used for gene editing, and a schematic diagram of the targeting strategy is shown in FIG. 6. FIG. 6 shows the targeting vector containing the upstream and downstream homology arm sequences, and the human CD20 sequence (SEQ ID NO: 5) . The upstream homology arm sequence (5′ homology arm, SEQ ID NO: 22) is identical to the nucleotide sequence at positions 11236186-11237594 of NCBI accession number NC_000085.7, and the downstream homology arm sequence (3′ homology arm, SEQ ID NO: 23) is identical to the nucleotide sequence at positions 11227619-11229028 of NCBI accession number NC_000085.7. The construction of the targeting vector can be carried out by conventional methods, such as enzyme digestion and ligation, direct synthesis and the like. The constructed targeting vector is preliminarily verified by enzyme digestion, and then sent to a sequencing company for sequencing verification. Sequencing-verified correct vectors were used for subsequent experiments.

The target sequence determines the targeting specificity of the sgRNA and the efficiency of the induced cleavage by Cas9. Therefore, target sequence selection is the prerequisite for constructing sgRNA expression vectors. A total of 14 sgRNA sequences (sgRNA1-sgRNA14) were designed and synthesized. The UCA kit was used to detect the sgRNA activity. From the results, it can be seen that sgRNAs have different activities. sgRNA-3 and sgRNA-10 were selected. The target site sequences are as follows:

sgRNA-3 target site sequence (SEQ ID NO: 24) : 5′-TGGAGCAGGTTGCATGGCGAGGG-3′

sgRNA-10 target site sequence (SEQ ID NO: 25) : 5′-GGAGCGATCTCATTTTCCACTGG-3′

The forward oligonucleotide and the reverse oligonucleotide were obtained by adding restriction sites on the 5′ end and on the complementary strand (see the below table for the sequences) . After annealing, the annealed products were ligated to the pT7-sgRNA plasmid (the plasmids were first linearized with BbsI) to obtain expression vectors pT7-CD20-3 and pT7- CD20-10.

Table 5: Sequences of sgRNA-3 and sgRNA-10

The clones were randomly selected and sent to a sequencing company for sequencing verification, and the correct expression vectors pT7-CD20-3 and pT7-CD20-10 were selected for subsequent experiments.

pT7-sgRNA plasmid source: The plasmid backbone of the pT7-sgRNA vector comes from Takara, Cat. No. 3299. The DNA containing the T7 promoter and sgRNA scaffold was synthesized by a plasmid synthesis company, and then connected to the backbone vector by enzyme digestion and ligation (EcoRI and BamHI) in turn. After sequencing verification by a sequencing company, the results showed that the target plasmid was obtained. Fragment DNA containing T7 promoter and sgRNA scaffold (SEQ ID NO: 32) :

Premixed in vitro transcription products of the pT7-CD20-3 and pT7-CD20-10 plasmids (us ing Ambion in vitro transcription kits, according to the instructions) and Cas9 mRNA were inject ed into mouse fertilized eggs (C57BL/6 mice) using microinjection. Similarly, targeting vector pl asmids were injected into the cytoplasm or nucleus of mouse fertilized eggs. Microinjection of e mbryos was carried out according to the method in the "Experimental Manual for Mouse Embry o Operation (Third Edition) . " The injected fertilized eggs were transferred to cell culture medium for a short-term culture, and then transplanted into the fallopian tubes of recipient mice to produ ce humanized mice (F0 mice) . The mRNA sequence of the humanized mouse CD20 is shown in SEQ ID NO: 10, and the expressed protein sequence is shown in SEQ ID NO: 2.

The genotype of F0 mouse somatic cells can be identified by conventional detection methods (such as PCR analysis) . PCR analysis includes the following primers: WT-F (SEQ ID NO: 19) and WT-R (SEQ ID NO: 20) , and WT-F (SEQ ID NO: 19) and Mut-R (SEQ ID NO: 21) . Positive F0 CD20 humanized mice were mated with C57BL/6 mice to obtain F1 generation mice. The same PCR method was used to determine the genotype of the F1 generation mice, and the F1 mice identified as positive by PCR were subjected to Southern blot detection to confirm whether there was random insertion. For Southern blot detection, genomic DNA was taken from mouse tail, and NeoI enzyme or AseI enzyme was used to digest the DNA.

5′ Probe Synthesis Primer:

5′ Probe-F (SEQ ID NO: 33) : 5′-ACTGTGCAGAAAAGGCAACAGGCTA-3′

5′ Probe-R (SEQ ID NO: 34) : 5′-TCCTGGGACCTACTCTCTCCTTGTG-3′

3′ Probe Synthesis Primer:

3′ Probe-F (SEQ ID NO: 35) : 5′-TTTACATGGCATGCCCACAATGGTT-3′

3′ Probe-R (SEQ ID NO: 36) : 5′-ATTCCACCTTCACTGAGTTCCCTCC-3′

The results of Southern blot detection are shown in FIG. 7. The results showed that there were no random insertions in 3 of the 4 mice tested. These 3 mice (1EA61-0001, 1EA61-0009 and 1EA61-0010) were thus confirmed to be positive heterozygous mice absent of random insertions. This indicates that CD20 humanized mice that can be stably passaged without random insertion can be constructed using this method.

The expression of CD20 mRNA in mice was further detected by RT-PCR. Specifically, a 9-week-old female C57BL/6 wild-type mouse and a CD20 gene humanized homozygous mouse were taken, and the spleen tissue was collected (extracted according to the instructions of the Trizol kit) after euthanasia by cervical dislocation. Cellular RNA was extracted and reverse transcribed into eDNA to perform RT-PCR detection (see the table below for primers) . The detection results (shown in FIG. 8) showed that only mouse CD20 mRNA was detected in C57BL/6 wild-type mice (+/+) (human CD20 mRNA was not detected) . Also, only human CD20 mRNA was detected in CD20 gene humanized homozygous mice (H/H) (mouse CD20 mRNA was not detected) .

The level of CD20 mRNA in mice was further detected by RT-PCR. Specifically, a 9-week-old female C57BL/6 wild-type mouse and a CD20 gene humanized homozygous mouse were taken, and the spleen tissue was collected (extracted according to the instructions of the Trizol kit) after euthanasia by cervical dislocation. Cellular RNA was extracted and reverse transcribed into cDNA to perform RT-PCR detection (see the table below for primers) . The detection results (shown in FIG. 8) showed that only mouse CD20 mRNA was detected in C57BL/6 wild-type mice (+/+) (human CD20 mRNA was not detected) . Also, only human CD20 mRNA was detected in CD20 gene humanized homozygous mice (H/H) (mouse CD20 mRNA was not detected) .

Table 6: RT-PCR primers and sequences

The expression of human or humanized CD20 protein in CD20 humanized mice was determined by conventional detection methods, such as flow cytometry. Specifically, 9-week-old female C57BL/6 wild-type mice and 10-week-old female CD20 gene humanized homozygous mice were taken, and spleen tissue was collected after cervical euthanasia. Flow cytometry was performed using APC anti-mouse CD20 Antibody (mCD20-APC) , PE anti-human CD20 Antibody (hCD20-PE) , Brilliant Violet 510 ^TM anti-mouse CD45, FITC anti-Mouse CD19 (mCD19-BV605) , Purified anti-mouse CD16/32, etc. The results showed that B cells (characterized as mCD45+mCD19+) in the spleen of C57BL/6 mice had 48.1%mCD20 positive cells (characterized by mCD45+mCD19+mCD20+) and 0.17%hCD20 positive cells (characterized by mCD45+mCD19+hCD20+) . B cells in the spleen of CD20 humanized homozygous mice had 0.11%mCD20 positive cells (characterized by mCD45+mCD19+mCD20+) and 57.4%hCD20 positive cells (characterized by mCD45+mCD19+hCD20+) . It shows that the spleen cells of C57BL/6 mice do not express human CD20 protein, and the spleen cells of CD20 humanized mice can successfully express human CD20 protein.

EXAMPLE 2. Generation of double-or multi-gene humanized mice

The CD20 gene humanized mice generated using the methods described herein can also be used to generate double-or multi-gene humanized mouse models. For example, in Example 1, the embryonic stem (ES) cells for blastocyst microinjection can be selected from mice comprising other genetic modifications such as modified (e.g., human or humanized) PD-1, PD-L1, TIGIT, TNFA, 41BB, 41BBL, CTLA4, CD47, SIRPA, OX40, TIM3 and/or CD226. Alternatively, embryonic stem cells from humanized CD20 mice described herein can be isolated, and gene recombination targeting technology can be used to obtain double-gene or multi-gene-modified mouse models of CD20 and other gene modifications. In addition, it is also possible to breed the homozygous or heterozygous CD20 gene humanized mice obtained by the methods described herein with other genetically modified homozygous or heterozygous mice. According to Mendel's law, it is possible to generate double-gene or multi-gene modified heterozygous mice comprising a modified (e.g., human or humanized) CD20 gene and other genetic modifications. Then the heterozygous mice can be bred with each other to obtain homozygous double-gene or multi-gene modified mice. These double-gene or multi-gene modified mice can be used for in vivo validation of gene regulators targeting human CD20 and other genes.

EXAMPLE 3. In vivo efficacy verification

The CD20 humanized mice prepared by this method can be used to evaluate the efficacy of drugs targeting human CD20. For example, CD20 humanized homozygous mice are subcutaneously inoculated with mouse colon cancer cell MC38 (or mouse lymphoma cell EL4) . When the tumor volume grew to about 100 mm3, the mice are divided into a control group and a treatment group based on the tumor volum. The treatment group mice receive drugs targeting human CD20, and the control group mice receive an equal volume of normal saline or PBS. The tumor volume and the body weight of the mice are determined, and the in vivo safety and efficacy of the drug can be effectively assessed based on the changes in the body weight and the tumor sizes.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

A genetically-modified, non-human animal whose genome comprises at least one chromosome comprising a sequence encoding a human or chimeric CD20.
The animal of claim 1, wherein the sequence encoding the human or chimeric CD20 is operably linked to an endogenous regulatory element at the endogenous CD20 gene locus in the at least one chromosome.
The animal of claim 1 or 2, wherein the sequence encoding a human or chimeric CD20 comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to human CD20 (NP_068769.2 (SEQ ID NO: 2) ) .
The animal of any one of claims 1-3, wherein the animal is a mammal, e.g., a monkey, a rodent, a mouse, or a rat.
The animal of any one of claims 1-4, wherein the animal is a mouse.
The animal of any one of claims 1-5, wherein the animal does not express endogenous CD20 or expresses a decreased level of endogenous CD20.
The animal of any one of claims 1-6, wherein the animal has one or more cells expressing human or chimeric CD20.
The animal of claim 7, wherein the expressed human or chimeric CD20 can regulate B cell development.
The animal of claim 7, wherein the expressed human or chimeric CD20 can maintain normal B cell development.
A genetically-modified, non-human animal, wherein the genome of the animal comprises a replacement of a sequence encoding a region of endogenous CD20 with a sequence encoding a corresponding region of human CD20 at an endogenous CD20 gene locus.
The animal of claim 10, wherein the sequence encoding the corresponding region of human CD20 is operably linked to an endogenous regulatory element at the endogenous CD20 locus, and one or more cells of the animal expresses a human or chimeric CD20.
The animal of claim 10 or 11, wherein the sequence encoding the corresponding region of human CD20 is immediately after endogenous 5’-UTR.
The animal of any one of claims 10-12, wherein the sequence that is replaced comprises the full-length coding sequence of endogenous CD20 (e.g., a nucleic acid sequence encoding amino acids 1-291 of SEQ ID NO: 1) .
The animal of any one of claims 10-12, wherein the sequence that is replaced comprises exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7, or a part thereof, of the endogenous CD20 gene.
The animal of any one of claims 10-14, wherein the sequence that is replaced starts with the start codon and ends with the stop codon of the endogenous mouse CD20 gene.
The animal of any one of claims 10-15, wherein the animal is heterozygous with respect to the replacement at the endogenous CD20 gene locus.
The animal of any one of claims 10-15, wherein the animal is homozygous with respect to the replacement at the endogenous CD20 gene locus.
A method for making a genetically-modified, non-human animal, comprising:

replacing in at least one cell of the animal, at an endogenous CD20 gene locus, a sequence encoding a region of endogenous CD20 with a sequence encoding a corresponding region of human CD20.
The method of claim 18, wherein the sequence encoding the corresponding region of human CD20 comprises exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7, or a part thereof, of a human CD20 gene.
The method of claim 18 or 19, wherein the sequence encoding the corresponding region of human CD20 comprises a portion of exon 2, exon 3, exon 4, exon 5, exon 6, and a portion of exon 7 of a human CD20 gene.
The method of any one of claims 18-20, wherein the sequence encoding the corresponding region of human CD20 encodes amino acids 1-297 of SEQ ID NO: 2.
The method of any one of claims 18-21, wherein the sequence encoding a region of endogenous CD20 comprises exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7, or a part thereof, of the endogenous CD20 gene.
The method of any one of claims 18-22, wherein the animal is a mouse, and the sequence encoding a region of endogenous CD20 starts within exon 2 and ends within exon 7 of the endogenous mouse CD20 gene.
A method of making a genetically-modified animal cell that expresses a human or chimeric CD20, the method comprising:

replacing at an endogenous CD20 gene locus, a nucleotide sequence encoding a region of endogenous CD20 with a nucleotide sequence encoding a corresponding region of human CD20, thereby generating a genetically-modified animal cell that includes a nucleotide sequence that encodes the human or chimeric CD20, wherein the animal cell expresses the human or chimeric CD20.
The method of claim 24, wherein the animal is a mouse.
The method of claim 24 or 25, wherein the nucleotide sequence encoding the human or chimeric CD20 is operably linked to an endogenous CD20 regulatory region, e.g., promoter.
The animal of any one of claims 1-17, wherein the animal further comprises a sequence encoding an additional human or chimeric protein.
The animal of claim 27, wherein the additional human or chimeric protein is programmed cell death protein 1 (PD-1) , programmed cell death ligand 1 (PD-L1) , T-Cell immunoreceptor with Ig and ITIM Domains (TIGIT) , Tumour Necrosis Factor alpha (TNF alpha) , tumor necrosis factor receptor superfamily member 9 (4-1BB) , Tumor necrosis factor ligand superfamily member 9 (4-1BBL) , cytotoxic T-lymphocyte-associated protein 4 (CTLA4) , CD47, Signal regulatory protein α (SIRPα) , OX40, T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) , or CD226.
The method of any one of claims 18-26, wherein the animal or mouse further comprises a sequence encoding an additional human or chimeric protein.
The method of claim 29, wherein the additional human or chimeric protein is PD-1, PD-L1, TIGIT, TNFA, 41BB, 41BBL, CTLA4, CD47, SIRPA, OX40, TIM3 or CD226.
A method for making a genetically-modified mouse, comprising:

(1) providing a plasmid comprising a human CD20 gene fragment, flanked by a 5’ homology arm and a 3’ homology arm, wherein the 5’ and 3’ homology arms target an endogenous CD20 gene;

(2) providing two small guide RNAs (sgRNAs) that target the endogenous CD20 gene;

(3) modifying genome of a fertilized egg or an embryonic stem cell by using the plasmid of step (1) , the sgRNAs of step (2) , and Cas9; and

(4) transplanting the fertilized egg obtained in step (3) into the oviduct of a pseudopregnant female mouse or transplanting the embryonic stem cell obtained in step (3) into a blastocyst which is then transplanted into the oviduct of a pseudopregnant female mouse to produce a child mouse that functionally expresses a humanized CD20 protein,

(5) mating the mouse obtained in step (2) to obtain a homozygote mouse

.
The method of claim 31, wherein the fertilized egg is modified by CRISPR with sgRNAs that target a 5’-terminal targeting site comprising SEQ ID NO: 24 and a 3’-terminal targeting site comprising SEQ ID NO: 25.
The method of claim 31, wherein the sequence encoding the humanized CD20 protein is operably linked to an endogenous regulatory element at the endogenous CD20 gene locus.
The method of claim 31, wherein the genetically-modified mouse does not express an endogenous CD20 protein.
The method of claim 31, wherein the 5’ homology arm comprises SEQ ID NO: 22 and the 3’ homology arm comprises SEQ ID NO: 23.
The method of claim 31, wherein the mouse can be used to test effectiveness of an anti-human CD20 antibody for treating cancer.
A method of determining effectiveness of an anti-CD20 antibody for the treatment of cancer, comprising:

administering the anti-CD20 antibody to the animal of any one of claims 1-17, wherein the animal has a cancer; and

determining the inhibitory effects of the anti-CD20 antibody to the cancer.
The method of claim 37, wherein the cancer comprises one or more cells that express CD20.
The method of claim 37 or 38, wherein the cancer comprises one or more cancer cells that are injected into the animal.
The method of any one of claims 37-39, wherein determining the inhibitory effects of the anti-CD20 antibody to the cancer involves measuring the tumor volume in the animal or measuring fluorescence intensity.
The method of claim 37, wherein the cancer is chronic lymphocytic leukemia (CLL) , follicular lymphoma, diffuse large B-cell lymphoma (DLBCL) , Non-Hodgkin lymphoma, Burkitt lymphoma, mantle cell lymphoma, marginal zone lymphoma, lymphoma lymphoblastic, Hodgkin lymphoma, or melanoma.
A method of determining toxicity of an anti-CD20 antibody, the method comprising administering the anti-CD20 antibody to the animal of any one of claims 1-17; and determining weight change of the animal.
The method of claim 42, the method further comprising performing a blood test (e.g., determining red blood cell count) .
A protein comprising an amino acid sequence, wherein the amino acid sequence is one of the following:

(a) an amino acid sequence set forth in SEQ ID NO: 1 or 2;

(b) an amino acid sequence that is at least 90%identical to SEQ ID NO: 1 or 2;

(c) an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 1 or 2;

(d) an amino acid sequence that is different from the amino acid sequence set forth in SEQ ID NO: 1 or 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid; and

(e) an amino acid sequence that comprises a substitution, a deletion and/or insertion of one, two, three, four, five or more amino acids to the amino acid sequence set forth in SEQ ID NO: 1 or 2.
A nucleic acid comprising a nucleotide sequence, wherein the nucleotide sequence is one of the following:

(a) a sequence that encodes the protein of claim 44;

(b) SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 22 or 23;

(c) a sequence that is at least 90%identical to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 22 or 23;

(d) a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 22 or 23; and

(e) a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 22 or 23.
A cell comprising the protein of claim 44 and/or the nucleic acid of claim 45.
The cell of claim 46, wherein the cell expresses a human or chimeric CD20.
An animal comprising the protein of claim 44 and/or the nucleic acid of claim 45.