Patent Application
20230080265
This instant application contains a Sequence Listing which has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Nov. 23, 2022, is named “131764-0001UT01.txt” and is 88.6 KB bytes in size.
The present application relates to transgenic mouse models and methods for making the same for use in studying viral infections, including those caused by SARS-CoV-2.
The global COVID-19 pandemic has seen more than 4.3 million deaths globally, leading to an unparalleled health and economic crisis worldwide (see, for e.g.: Dong et al. Lancet Infect Dis. 2020; 20(5): 533-41). The disease is caused by a new strain of coronavirus named severe acute respiratory syndrome coronavirus-2 or SARS-CoV-2 (see, for e.g.: Zhou et al. Nature. 2020; 579(7798): 270-3). Many coronaviruses are common human pathogens with low pathogenicity, such as the ones that cause the common cold. A general feature of coronavirus is the spike protein (S), a class I fusion protein that mediates attachment of the virus to cell surface receptors followed by uptake (see, for e.g.: Zhou et al., ibid). Proteolytic cleavage of the S protein by specific proteases and fusion of viral and endosomal membranes triggers the release of viral RNA into the cytosol (see, for e.g.: Fehr and Perlman Methods Mol Biol. 2015; 1282: 1-23). SARS-CoV-2 binds to the extracellular receptor angiotensin-converting enzyme 2 (ACE2) to enter the host's cells, and uses the cellular transmembrane serine protease TMPRSS2 as a cooperating partner for S protein cleavage and priming (see, for e.g.: Hoffmann et al. Cell. 2020. Epub 2020/03/07).
Individuals with COVID-19 manifest a wide range of symptoms, ranging from mild disease to fatal illness. Mild COVID-19 with fever, headache, muscle pain, sore throat, or cough may progress to life-threatening illness with dyspnea and cyanosis. Increasing reports indicate that many severe COVID-19 patients also exhibit signs of liver, heart, and kidney damage, diarrhea, conjunctivitis, stroke, seizure, and encephalitis (see, for e.g.: Wadman et al. Science. 2020; 368(6489): 356-60). Thus there is an urgent and critical need for well-defined, genetically tractable small animal models for SARS-CoV-2 infection that capture a wide spectrum of illnesses ranging from mild to lethal COVID-19 disease.
A suitable mouse model is essential for understanding disease pathogenesis and for evaluating the safety and efficacy of vaccine candidates, antibody candidates, or antiviral compounds. However, mice are not infectable with SARS-CoV-2 due to differences between the human and murine ACE2 (see, for e.g.: Zhou et al. Nature. 2020; 579(7798): 270-3). A few transgenic mouse models expressing the human ACE2 gene are available (see, for e.g.: McCray et al. J Virol. 2007; 81(2): 813-21) but show limited infectability by SARS-CoV-2 and develop mild or overly strong symptoms upon infection (see, for e.g.: Bao et al. The Pathogenicity of SARS-CoV-2 in hACE2 Transgenic Mice. bioRxiv. 2020: 2020.02.07.939389). The weaknesses of the current humanized models have been highlighted in scientific media and call for new models that reproduce key features of the human disease (see, for e.g.: Callaway, E. Nature. 2020; 579(7798): 183). The existing mouse models were created using first-generation transgenic approaches and have significant limitations. First, these mice still express the mouse ACE2 receptor, which can confound data from treatments aimed at disrupting the binding of ACE2 to the virus using small molecules or antibodies. Second, they do not express the human ACE2 gene in the same cellular pattern as the endogenous protein (see, for e.g.: McCray et al. J Virol. 2007; 81(2): 813-21). This is critical, as a wide variety of cell types, including pneumocytes, cardiomyocytes, cardiac fibroblasts, and coronary endothelial cells express high levels of ACE2 and are susceptible to infection with SARS-CoV-2 and may contribute to severe disease (see, for e.g.: Chen et al. Cardiovasc Res. 2020; 116(6): 1097-100). Also, it is likely that the humanization of a single receptor is not sufficient to achieve full infectivity in mice, as evidenced by the mild lung pathology in existing humanized models; thus, the human TMPRSS2 may be needed for efficient infection by SARS-CoV-2 (see, for e.g.: Hoffmann et al. Cell. 2020. Epub 2020/03/07). Lastly, all existing humanized models have been constructed in the Th1-dominant mouse strain C57BL/6; this strain exhibits mild symptoms upon viral and bacterial infections (see, for e.g.: Chen et al. J Virol. 2010; 84(3): 1289-301) and is generally considered a poor strain for respiratory investigations; indeed, Th2-dominant BALB/c mice appear to be better models of COVID-19 than C57BL/6 (see, for e.g.: Dinnon et al. Nature 2020. Epub 2020/08/28). Therefore, existing humanized mouse strains poorly model the wide range of disease severity observed in humans and are not suitable for COVID-19 research.
This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key aspects or essential aspects of the claimed subject matter.
All features of exemplary embodiments which are described in this disclosure and are not mutually exclusive can be combined with one another. Elements of one embodiment can be utilized in the other embodiments without further mention. Other aspects and features of the present invention will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments in conjunction with the accompanying Figures.
As embodied and broadly described herein, an aspect of the present disclosure relates to a construct comprising a human ACE2 cDNA minigene comprising a start codon, and an SV40 mini-intron comprising a splice donor site and a splice acceptor site within the cDNA sequence of human ACE2. In various embodiments, the construct comprises a sequence having at least 80% sequence homology with the sequence of “pSYNB026_57BL6_Ace2_Humanizing Construct” (SEQ ID NO: 1), or any fragment thereof. In various embodiments, the construct comprises a sequence having at least 80% sequence homology with the sequence of “pSYNB028_Ace2_NEO_BalbC” (SEQ ID NO: 2), or any fragment thereof. In various embodiments, the construct further comprises a mouse promotor sequence for the mouse ACE2 gene.
In another aspect, described herein is a genetically-modified mouse comprising in its genome a human ACE2 cDNA minigene comprising a start codon, and an SV40 mini-intron comprising a splice donor site and a splice acceptor site within the cDNA sequence of human ACE2, and a start codon of native mouse ACE2, wherein the start codon of the human ACE2 cDNA minigene is upstream of the start codon of native mouse ACE2, and wherein the human ACE2 cDNA minigene disrupts expression of native mouse ACE2. In various embodiments, no more than 20 amino acids are encoded between the start codon of the human ACE2 cDNA minigene and the start codon of native mouse ACE2. In various embodiments, the mouse is a C57BL/6 mouse. In various embodiments, the mouse is a BALB/c mouse. In various embodiments, the human cDNA minigene is under control of a promotor sequence for the native mouse ACE2 gene.
In another aspect, described herein is a construct comprising a human TMPRSS2 cDNA minigene, the human TMPRSS2 cDNA minigene comprising a start codon, and an SV40 mini-intron comprising a splice donor site and a splice acceptor site within the cDNA sequence of human TMPRSS2. In various embodiments, the construct comprises a sequence having at least 80% sequence homology with the sequence of “pSYNB027_C57BL6_Tmprss2_Humanizing Construct” (SEQ ID NO: 3), or any fragment thereof. In various embodiments, the construct comprises a sequence having at least 80% sequence homology with the sequence of “pSYNB029_BalbC_Tmprss2_NEO Humanizing Construct” (SEQ ID NO: 4), or any fragment thereof. In various embodiments, the construct further comprises a mouse promotor sequence for the mouse TMPRSS2 gene.
In another aspect, described herein is a genetically-modified mouse comprising a human TMPRSS2 cDNA minigene, the human TMPRSS2 cDNA minigene comprising a start codon, and an SV40 mini-intron comprising a splice donor site and a splice acceptor site within the cDNA sequence of human TMPRSS2, the mouse further comprising in its genome a start codon for native mouse TMPRSS2, wherein the start codon of the human TMPRSS2 cDNA minigene is upstream of the start codon for native mouse TMPRSS2, and wherein the human TMPRSS2 cDNA minigene disrupts expression of native mouse TMPRSS2. In various embodiments, no more than 20 amino acids are encoded between the start codon of the human TMPRSS2 cDNA minigene and the start codon of native mouse TMPRSS2. In various embodiments, the mouse is a C57BL/6 mouse. In various embodiments, the mouse is a BALB/c mouse. In various embodiments, the mouse further comprises in its genome a human ACE2 cDNA minigene, the human ACE2 cDNA minigene comprising a start codon, and an SV40 mini-intron comprising a splice donor site and a splice acceptor site within the cDNA sequence of the human ACE2 cDNA minigene. In various embodiments, the start codon of the human ACE2 cDNA minigene is upstream of the start codon for native mouse ACE2, and the human ACE2 cDNA minigene disrupts expression of native mouse ACE2. In various embodiments, the human TMPRSS2 cDNA minigene is under control of the promotor sequence for the native mouse TMPRSS2 gene.
In another aspect, described herein is a genetically-modified mouse comprising in its genome at least one chromosome comprising a nucleic acid sequence encoding a human cDNA ACE2 minigene, wherein the mouse does not express endogenous ACE2. In various embodiments, the nucleic acid sequence encoding the human ACE2 is operably linked to an endogenous regulatory element at the endogenous ACE2 gene locus in the at least one chromosome.
In another aspect, described herein is a genetically-modified mouse comprising it its genome at least one chromosome comprising a nucleic acid sequence encoding a human cDNA TMPRSS2 minigene, wherein the mouse does not express endogenous TMPRSS2. In various embodiments, the nucleic acid sequence encoding the human TMPRSS2 is operably linked to an endogenous regulatory element at the endogenous TMPRSS2 gene locus in the at least one chromosome.
In another aspect, described herein is a method of making a genetically-modified mouse, the method comprising replacing in at least one cell of the animal, at an endogenous ACE2 gene locus, a sequence encoding a region of an endogenous ACE2 with a sequence encoding a corresponding region of human cDNA ACE2 minigene. In various embodiments, the mouse comprises a start codon of the human ACE2 cDNA minigene and a start codon of native mouse ACE2 gene, and wherein the start codon of the human ACE2 cDNA minigene is less than 20 amino acids from the start codon of native mouse ACE2 gene. In various embodiments, the mouse expresses the human cDNA ACE2 minigene.
In the Present Application:
A detailed description of one or more embodiments of the invention is provided below along with accompanying figures that illustrate the principles of the invention. The invention is described in connection with such embodiments, but the invention is not limited to any embodiment. The scope of the invention is limited only by the claims. Numerous specific details are set forth in the following description in order to provide a thorough understanding of the invention. These details are provided for the purpose of non-limiting examples and the invention may be practiced according to the claims without some or all of these specific details. For the purpose of clarity, technical material that is known in the technical fields related to the invention has not been described in detail so that the invention is not unnecessarily obscured.
The present disclosure describes nucleic acid constructs and methods for generating rodent models expressing human ACE2 and/or TMPRSS2, and not expressing native ACE2 and/or TMPRSS2.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art to which the present invention pertains. As used herein, and unless stated otherwise or required otherwise by context, each of the following terms shall have the definition set forth below.
As may be used herein, the term “animal” refers to living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term “mammal” includes both human and non-human mammals, e.g., bovines, canines, felines, rodents such as rat, murines, simians, equines and humans. Additional examples include adults, juveniles and infants.
The terms “subject,” “host,” “individual,” and “patient” may be used interchangeably herein to refer to animals, typically mammalian animals. Any suitable mammal can be treated by a method, cell or composition described herein. A mammal is a human. A mammal may be any age or at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero). A mammal may be male or female. A mammal may be a pregnant female. In some embodiments a subject may be a human. In some embodiments, a subject may have, has, may be suspected, or is suspected of having a cancer or neoplastic disorder.
As may be used herein, the terms “nucleic acid sequence” and “polynucleotide” are used interchangeably to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
The term “encode” as it is applied to nucleic acid sequences refers to a polynucleotide which is said to “encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, can be transcribed and/or translated to produce the mRNA for the polypeptide and/or a fragment thereof. The antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.
As used herein, the term “vector” refers to a nucleic acid construct deigned for transfer between different hosts, including but not limited to a plasmid, a virus, a cosmid, a phage, a BAC, a YAC, etc. Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo and are commercially available from sources such as Agilent Technologies (Santa Clara, Calif.) and Promega Biotech (Madison, Wis.). The term “construct” refers to a recombinant nucleic acid that has been generated for the purpose of the expression of a specific nucleotide sequence.
It is to be inferred without explicit recitation and unless otherwise intended, that when the present disclosure relates to a polypeptide, protein, polynucleotide, an equivalent or a biologically equivalent of such is intended within the scope of this disclosure. As used herein, the term “biological equivalent thereof” is intended to be synonymous with “equivalent thereof” when referring to a reference protein, polypeptide or nucleic acid, intends those having minimal homology while still maintaining desired structure or functionality. Unless specifically recited herein, it is contemplated that any of the above also includes equivalents thereof. For example, an equivalent intends at least about 70% homology or identity, or at least 80% homology or identity and alternatively, or at least about 85%, or alternatively at least about 90%, or alternatively at least about 95%, or alternatively at least 98% percent homology or identity and/or exhibits substantially equivalent biological activity to the reference protein, polypeptide, or nucleic acid. Alternatively, when referring to polynucleotides, an equivalent thereof is a polynucleotide that hybridizes under stringent conditions to the reference polynucleotide or its complement. Specifically, in some embodiments, the sequence encoding a human ACE2 comprises a sequence encoding an amino acid sequence that is at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to human ACE2 (pSYNB026_C57BL6_Ace2_Humanizing Construct; SEQ ID NO: 1). Specifically, in some embodiments, the sequence encoding a human ACE2 comprises a sequence encoding an amino acid sequence that is at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to human ACE2 (pSYNB028_Ace2_NEO_BalbC; SEQ ID NO: 2). Specifically, in some embodiments, the sequence encoding a human TMPRSS2 comprises a sequence encoding an amino acid sequence that is at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to human TMPRSS2 (pSYNB027_C57BL6_Tmprss2_Humanizing Construct; SEQ ID NO: 3). Specifically, in some embodiments, the sequence encoding a human TMPRSS2 comprises a sequence encoding an amino acid sequence that is at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to human TMPRSS2 (pSYNB029_BalbC_Tmprss2_NEO Humanizing Construct; SEQ ID NO: 4). Stated another way, in one aspect, the disclosure relates to mouse and rodents comprising at least one cell comprising a nucleotide sequence encoding a ACE2 gene wherein the ACE2 gene comprises at least 50 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human ACE2 gene, wherein the animal expresses the human ACE2 gene. Stated another way, in one aspect, the disclosure relates to mouse and rodents comprising at least one cell comprising a nucleotide sequence encoding a TMPRRS2 gene wherein the TMPRRS2 gene comprises at least 50 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human TMPRRS2 gene, wherein the animal expresses the human TMPRRS2 gene.
A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) having a certain percentage (for example, 80%, 85%, 90%, or 95%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. The alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Current Protocols in Molecular Biology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1. Preferably, default parameters are used for alignment. A preferred alignment program is BLAST, using default parameters. In particular, preferred programs are BLASTN and BLASTP, using the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the following Internet address: ncbi.nlm.nih.gov/cgi-bin/BLAST.
As may be used herein, the term “recombinant,” when used to modify “protein,” “peptide,” or “polypeptide,” or any specific protein, peptide, or polypeptide, refers to a protein, peptide, or polypeptide produced by recombinant DNA techniques, wherein generally, DNA encoding the polypeptide is inserted into a suitable expression vector, which is in turn used to transform a host cell to produce the heterologous protein.
As used herein, the term “transgenic” refers to an organism that contains genetic material into which DNA from an unrelated organism has been artificially introduced. As used herein, the term “transgene” refers to a gene that has been transferred from one organism to another.
As used herein, the term “genome editing” refers to insertion, deletion, modification, or replacement of a genome of an organism. The terms “knock-in” and “knock-out”, as used herein, refer to the addition of a gene sequence into a genome and the inactivation or removal of a gene, respectively.
As used herein, the term “allele” refers to one of two or more versions of a gene. “Homozygous” refers to an animal having two alleles for a gene, where both alleles are the same. “Heterozygous” refers to an animal having two alleles for a gene, where the two alleles are different.
As used herein, the term “wild type” refers to a phenotype, a genotype, or gene that predominates in a natural population of organisms or strain of organisms.
As used herein, the term “endogenous” or “native” refers to a gene that originates from within an organism.
As used herein, the term “cassette” or “gene cassette” is a fragment of DNA carrying, and capable of expressing, one or more genes or interest between one or more sets of restriction sites.
As used herein, the term “locus” refers to a specific, fixed position on a chromosome where a particular gene or genetic marker is located.
A “humanized mouse” refers to a mouse carrying functioning human genes, cells, tissues, and/or organs. As used herein a “founder” mouse refers to a mouse which has integrated the transgenic construct.
The term “isolated” as may be used herein refers to molecules or biologicals or cellular materials being substantially free from other materials. In one aspect, the term “isolated” refers to nucleic acid, such as DNA or RNA, or protein or polypeptide, or cell or cellular organelle, or tissue or organ, separated from other DNAs or RNAs, or proteins or polypeptides, or cells or cellular organelles, or tissues or organs, respectively, that are present in the natural source. The term “isolated” also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Moreover, an “isolated nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state. The term “isolated” is also used herein to refer to polypeptides which are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides. The term “isolated” is also used herein to refer to cells or tissues that are isolated from other cells or tissues and is meant to encompass both cultured and engineered cells or tissues.
The term “protein”, “peptide” and “polypeptide” are used interchangeably and in their broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics. The subunits may be linked by peptide bonds. In another aspect, the subunit may be linked by other bonds, e.g., ester, ether, etc. A protein or peptide must contain at least two amino acids and no limitation is placed on the maximum number of amino acids which may comprise a protein's or peptide's sequence. As used herein the term “amino acid” refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics.
The terms “polynucleotide” and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, RNAi, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double and single stranded molecules. Unless otherwise specified or required, any aspect of this technology that is a polynucleotide encompasses both the double stranded form and each of two complementary single stranded forms known or predicted to make up the double stranded form.
As may be used herein, the term “purified” does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified nucleic acid, peptide, protein, biological complexes or other active compound is one that is isolated in whole or in part from proteins or other contaminants. Generally, substantially purified peptides, proteins, biological complexes, or other active compounds for use within the disclosure comprise more than 80% of all macromolecular species present in a preparation prior to admixture or formulation of the peptide, protein, biological complex or other active compound with a pharmaceutical carrier, excipient, buffer, absorption enhancing agent, stabilizer, preservative, adjuvant or other co-ingredient in a complete pharmaceutical formulation for therapeutic administration. More typically, the peptide, protein, biological complex or other active compound is purified to represent greater than 90%, often greater than 95% of all macromolecular species present in a purified preparation prior to admixture with other formulation ingredients. In other cases, the purified preparation may be essentially homogeneous, wherein other macromolecular species are not detectable by conventional techniques.
“Immune cells” includes, e.g., white blood cells (leukocytes) which are derived from hematopoietic stem cells (HSC) produced in the bone marrow, lymphocytes (T cells, B cells, natural killer (NK) cells) and myeloid-derived cells (neutrophil, eosinophil, basophil, monocyte, macrophage, dendritic cells). A “T cell” includes all types of immune cells expressing CD3 including T-helper cells (CD4+ cells), cytotoxic T-cells (CD8+ cells), natural killer T-cells, T-regulatory cells (Treg) and gamma-delta T cells. A “cytotoxic cell” includes CD8+ T cells, natural-killer (NK) cells, and neutrophils, which cells are capable of mediating cytotoxicity responses. Cytokines are small secreted proteins released by immune cells that have a specific effect on the interactions and communications between said immune cells. Cytokines can be pro-inflammatory or anti-inflammatory. A non-limiting example of a cytokine is granulocyte-macrophage colony-stimulating factor (GM-CSF), which stimulates stem cells to produce granulocytes (neutrophils, eosinophils, and basophils) and monocytes.
As used herein, the phrase “immune response” or its equivalent “immunological response” refers to the development of a cell-mediated response (e.g., mediated by antigen-specific T cells or their secretion products). A cellular immune response is elicited by the presentation of polypeptide epitopes in association with Class I or Class II MHC molecules, to treat or prevent a viral infection, expand antigen-specific B-reg cells, TC1, CD4+ T helper cells and/or CD8+ cytotoxic T cells and/or disease generated, autoregulatory T cell and B cell “memory” cells. The response may also involve activation of other components. In some aspect, the term “immune response” may be used to encompass the formation of a regulatory network of immune cells. Thus, the term “regulatory network formation” may refer to an immune response elicited such that an immune cell, preferably a T cell, more preferably a T regulatory cell, triggers further differentiation of other immune cells, such as but not limited to, B cells or antigen-presenting cells—non-limiting examples of which include dendritic cells, monocytes, and macrophages. In certain embodiments, regulatory network formation involves B cells being differentiated into regulatory B cells; in certain embodiments, regulatory network formation involves the formation of tolerogenic antigen-presenting cells.
As may be used herein, the term “antibody” (“Ab”) collectively refers to immunoglobulins (or “Ig”) or immunoglobulin-like molecules including but not limited to antibodies of the following isotypes: IgM, IgA, IgD, IgE, IgG, and combinations thereof. Immunoglobulin-like molecules include but are not limited to similar molecules produced during an immune response in a vertebrate, for example, in mammals such as humans, rats, goats, rabbits and mice. Unless specifically noted otherwise, the term “antibody” includes intact immunoglobulins and “antibody fragments” or “antigen binding fragments” that specifically bind to a molecule of interest (or a group of highly similar molecules of interest) to the substantial exclusion of binding to other molecules (for example, antibodies and antibody fragments that have a binding constant for the molecule of interest that is at least 103 M−1 greater, at least 104M−1 greater or at least 105 M−1 greater than a binding constant for other molecules in a biological sample). The term “antibody” also includes genetically engineered forms such as chimeric antibodies (for example, humanized murine antibodies), heteroconjugate antibodies (such as, bispecific antibodies). See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, Immunology, 3rd Ed., W. H. Freeman & Co., New York, 1997.
As may be used herein, the term “monoclonal antibody” refers to an antibody produced by a cell into which the light and heavy chain genes of a single antibody have been transfected or, more traditionally, by a single clone of B-lymphocytes. Monoclonal antibodies generally have affinity for a single epitope (i.e., they are monovalent) but may be engineered to be specific for two or more epitopes (e.g., bispecific). Methods of producing monoclonal antibodies are known to those of skill in the art, for example by creating a hybridoma through fusion of myeloma cells with immune spleen cells, phage display, single cell amplification from B-cell populations, single plasma cell interrogation technologies, and single B-cell culture. Monoclonal antibodies include recombinant antibodies, chimeric antibodies, humanized antibodies, and human antibodies.
As may be used herein, the term “antigen” refers to a compound, composition, or substance that may be specifically bound and/or recognized by the products of specific humoral or cellular immunity and antigen recognition molecules, including but not limited to an antibody molecule, single-chain variable fragment (scFv), cell surface immunoglobulin receptor, B-cell receptor (BCR), T-cell receptor (TCR), engineered TCR, modified TCR, or CAR. The term “epitope” refers to an antigen or a fragment, region, site, or domain of an antigen that is recognized by an antigen recognition molecule. Antigens can be any type of molecule including but not limited to peptides, proteins, lipids, phospholipids haptens, simple intermediary metabolites, sugars (e.g., monosaccharides or oligosaccharides), hormones, and macromolecules such as complex carbo-hydrates (e.g., polysaccharides). Some non-limiting examples of antigens include antigens involved in autoimmune disease (including autoantigens), allergy, and graft rejection, tumor antigens, toxins, and other miscellaneous antigens. Non-limiting examples of tumor antigens include mesothelin, ROR1 and EGFRvIII, ephrin type-A receptor 2 (EphA2), interleukin (IL)-13r alpha 2, an EGFR VIII, a PSMA, an EpCAM, a GD3, a fucosyl GM1, a PSCA, a PLAC1, a sarcoma breakpoint, a Wilms Tumor 1, a hematologic differentiation antigen, a surface glycoprotein, a gangliosides (GM2), a growth factor receptor, a stromal antigen, a vascular antigen, or a combination thereof. Antigens expressed by pathogens include, but are not limited to, microbial antigens such as viral antigens, bacterial antigens, fungal antigens, protozoa, and other parasitic antigens.
As may be used herein, “homology” or “identical”, percent “identity” or “similarity”, when used in the context of two or more nucleic acids or polypeptide sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, e.g., at least 60% identity, preferably at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region (e.g., nucleotide sequence encoding the chimeric PVX described herein). Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. The alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Current Protocols in Molecular Biology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1. Preferably, default parameters are used for alignment. A preferred alignment program is BLAST, using default parameters. In particular, preferred programs are BLASTN and BLASTP, using the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the following Internet address: ncbi.nlm.nih.gov/cgi-bin/BLAST. The terms “homology” or “identical,” percent “identity” or “similarity” also refer to, or can be applied to, the complement of a test sequence. The terms also include sequences that have deletions and/or additions, as well as those that have substitutions. As described herein, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is at least 50-100 amino acids or nucleotides in length. An “unrelated” or “non-homologous” sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences disclosed herein.
As used herein, “treating” or “treatment” of a disease in a subject refers to: (1) preventing the symptoms or disease from occurring in a subject that is predisposed or does not yet display symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression of the disease or the symptoms of the disease. As understood in the art, “treatment” is an approach for obtaining beneficial or desired results, including clinical results. For the purposes of the present technology, beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of a condition (including a disease), stabilized (i.e., not worsening) state of a condition (including disease), delay or slowing of condition (including disease), progression, amelioration or palliation of the condition (including disease), states and remission (whether partial or total), whether detectable or undetectable. When the disease is cancer, the following clinical end points are non-limiting examples of treatment: reduction in tumor burden, slowing of tumor growth, longer overall survival, longer time to tumor progression, inhibition of metastasis or a reduction in metastasis of the tumor. In one aspect, treatment excludes prophylaxis.
As used herein, the term “vaccine” refers to a biological preparation that provides active acquired immunity to a particular infectious disease. As used herein, the term “antiviral therapy” refers to a treatment with a drug(s) that inhibit the ability of a virus(es) to multiply in the body of a subject.
As may be used herein, the term “expression” refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. The expression level of a gene may be determined by measuring the amount of mRNA or protein in a cell or tissue sample. In one aspect, the expression level of a gene from one sample may be directly compared to the expression level of that gene from a control or reference sample. In another aspect, the expression level of a gene from one sample may be directly compared to the expression level of that gene from the same sample following administration of a compound.
As may be used herein, the term “introduce” as applied to methods of producing modified cells refers to the process whereby a foreign (i.e., extrinsic or extracellular) agent is introduced into a host cell thereby producing a cell comprising the foreign agent. Methods of introducing nucleic acids include but are not limited to transduction, retroviral gene transfer, transfection, electroporation, transformation, viral infection, microinjection, and other recombinant DNA techniques known in the art. Transduction may be done via a vector (e.g., a viral vector). Transfection may be done via a chemical carrier, DNA/liposome complex, or micelle (e.g., Lipofectamine (Invitrogen)) or microinjection, ballistic-assisted transfection, electric-assisted transfection, or sonic-assisted transfection. Viral infection may be done via infecting the cells with a viral particle comprising the polynucleotide of interest (e.g., AAV). Introduction may comprise CRISPR mediated gene editing or Transcription activator-like effector nuclease (TALEN) mediated gene editing. Methods of introducing non-nucleic acid foreign agents (e.g., soluble factors, cytokines, proteins, peptides, enzymes, growth factors, signaling molecules, small molecule inhibitors) include but are not limited to culturing the cells in the presence of the foreign agent, contacting the cells with the agent, contacting the cells with a composition comprising the agent and an excipient, and contacting the cells with vesicles or viral particles comprising the agent.
In the context of a nucleic acid or amino acid sequence, the term “chimeric” intends that the sequence contains is comprised of at least one substituent unit (e.g., fragment, region, portion, domain, polynucleotide, or polypeptide) that is derived from, obtained or isolated from, or based upon other distinct physical or chemical entities. For example, a chimera of two or more different proteins may comprise the sequence of a variable region domain from an antibody fused to the transmembrane domain of a cell signaling molecule. In some aspects, a chimera intends that the sequence is comprised of sequences from at least two distinct species.
Other examples of implementations will become apparent to the person skilled in the art in view of the teachings of the present description and as such, will not be further described here.
Note that titles or subtitles may be used throughout the present disclosure for convenience of a reader, but in no way should these limit the scope of aspects or embodiments of the invention(s) detailed herein. Moreover, certain theories may be proposed and disclosed herein; however, in no way they, whether they are right or wrong, should limit the scope of the invention so long as the invention is practiced according to the present disclosure without regard for any particular theory or scheme of action.
Any and all references cited throughout the specification are hereby incorporated by reference in their entirety for all purposes.
It will be understood by those of skill in the art that throughout the present specification, the term “a” used before a term encompasses embodiments containing one or more to what the term refers. It will also be understood by those of skill in the art that throughout the present specification, the term “comprising”, which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, un-recited elements or method steps.
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 pertains. In the case of conflict, the present document, including definitions will control.
As used in the present disclosure, the terms “around”, “about” or “approximately” shall generally mean within the error margin generally accepted in the art. Hence, numerical quantities given herein generally include such error margin such that the terms “around”, “about” or “approximately” can be inferred if not expressly stated.
Disclosed herein are mouse models for evaluating COVID-19 disease, and methods for generating them. Existing mouse models of COVID-19 disease have humanized ACE2 and were created using traditional transgenic approaches that have several limitations as follows: 1) multiple copies of a human gene are inserted randomly in the genome; 2) expression of the human ACE2 transgene does not have a normal expression pattern across tissues (see, for e.g.: McCray et al. J Virol. 2007; 81(2): 813-21 and Yang et al. Comp Med. 2007; 57(5): 450-9); and 3) the mouse ACE2 gene is not knocked out, potentially leading to confounding effects when testing therapeutics. The approaches described herein are innovative from several perspectives. While, exemplified and described herein, aspects and embodiments of the invention(s) are not limited to mouse models. The methods and principles described herein may be applied to any rodent, including but not limited to, rat, hamster, guinea pig, etc.
First, the models disclosed herein address the limitations in the previous models by using an innovative CRISPR/Cas9 assisted Homology Directed Repair (HDR) technology to simultaneously knock out the endogenous mouse gene while inserting a human cDNA minigene counterpart in the mouse genome under control of the mouse promoter. The general mouse humanization strategy is illustrated in
Second, humanized mice were generated on two different genetic backgrounds: C57BL/6 with a predominantly Th1 response, and BALB/c with a Th2-dominant response. This approach, in combination with the generation of singly and doubly humanized ACE2/TMPRSS2 mice, allowed for the modeling of a wide spectrum of illnesses caused by SARS-CoV-2 infection, ranging from mild to severe COVID-19 disease and different sequelae.
The mouse models provided herein represent a critical tool that allows for testing the safety and efficacy of SARS-CoV-2 antibodies in a human-like setting by addressing practical concerns. There is a concern with vaccines and antibodies that there may be an unwanted antibody-dependent enhancement (ADE) of disease caused by antibody Fc coupling to virus via Fc receptors (FcRs) on immune cells and which facilitates virus uptake and replication. This unwanted action is better modeled in the humanized FcRn mice proposed. Additionally, the clinical success of an antibody-based therapeutic against SARS-CoV-2 is tied to longevity, half-life, which is related to its affinity for the FcRn (encoded by FCGRT) that is expressed on endothelial cell membranes and constantly endocytoses IgG from the plasma and recycles it back to the plasma. Mouse FcRn has a different affinity for human IgG than human FcRn, and transgenic mice expressing human FcRn, but not wild type mice, exhibit antibody PK profiles highly correlated with humans and non-human primates (see, for e.g.: Avery et al. MAbs 2018; 10(2): 244-55, and Tam et al. MAbs 2013; 5(3): 397-405). A humanized FCGRT strain, therefore, provides an accurate modeling of antibody half-lives, virus neutralization, and likelihood of therapeutic antibody-FcRn interactions and immune complex formation. Thus, it is contemplated that the singly and doubly humanized ACE2/TMPRSS2 mouse models provided herein may be triply humanized with human FCGRT for further testing of SARS-CoV-2 antibodies. While the description and examples provided herein include generation of transgenic mice in C57BL/6 and BALB/c backgrounds, other mice and rodents of any background can be used. The invention also contemplates the combination of hACE2 and hTMPRSS2 strains developed herein with other genetically engineered mouse strains, such as strains containing knockouts of other mouse genes, strains containing other humanized loci, or strains containing other transgenes.
As BALB/c mice can develop severe SARS-like disease, it is expected that one or more humanized mice in the BALB/c background serve as models for severe COVID-19, whereas the humanized C57BL/6 mice are likely to serve as models of asymptomatic SARS-CoV-2 infection or mild COVID-19. Mouse models that reproduce a spectrum of SARS-CoV-2-induced illnesses are critical tools for deciphering mechanisms of SARS-CoV-2 pathogenesis and immunity and for developing and testing vaccines and treatments for COVID-19. A thorough comparison of SARS-CoV-2 infection in different humanized mouse strains will identify the most relevant models of the human disease and define key endpoints for evaluation of antiviral and vaccine candidates. Additionally, results obtained from our studies with single hACE2 vs single hTMPRSS2 vs double hACE2/hTMPRSS2 mice may inform the precise role of ACE2 and TMPRSS2 in SARS-CoV-2 infection.
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 the native 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 usually integrates at random into the genome, often in multiple loci. 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 generally found to be sufficient to provide suitable support for expression and/or regulation of the transgene.
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 ACE2 or TMPRRS2 gene(s) 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 efficacy of these human therapeutics in the animal models.
In some embodiments, the genetically modified animals can be used for determining effectiveness of vaccines, the development of a new diagnostic strategy and/or a therapeutic strategy. The methods involve administering a vaccine and/or a therapeutic agent to treat SARS-CoV-2 to the animal as described herein; and determining the effects of the vaccine, diagnostic strategy and/or a therapeutic agent. The effects that can be determined include, e.g., a whether the vaccine/diagnostic strategy/therapeutic agent can stimulate an immune response, whether the vaccine/diagnostic strategy/agent can upregulate the immune response or downregulate immune response, decrease of symptoms, decrease the risk of developing complications from infection, increase the survival rate, increase life expectancy, etc. 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. 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 vaccine, or the model system for a research in pharmacology, immunology, microbiology and medicine. 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 hACE2 or hTMPRRS2 genes function, drugs for human hACE2 or hTMPRRS2 targeting sites, the drugs for immune-related diseases.
In addition, the mice described in the present disclosure can be mated with the mice containing other human or chimeric genes, so as to obtain a mouse expressing two or more human genes. For example, in some embodiments, the mice express hACE2 and hTMPRRS2.
Three genes were humanized; ACE2, TMPRSS2, and FCGRT. ACE2 is the main extracellular receptor that binds the viral S protein and is required for viral entry in target cells. TMPRSS2 is an extracellular serine protease required for S protein priming and subsequent fusion of viral and cellular membranes. FCGRT is a neonatal Fc receptor (also FcRN, IgG receptor FcRN large subunit p51) is the main regulator of IgG pharmacokinetics and is important for testing human antibodies in mice. Triply humanized mouse models (ACE2/TMPRSS2/FCGRT) in C57BL/6J and BALB/c backgrounds were generated, as well as doubly and singly humanized mouse strains. The mouse products generated are shown in Table 1. C57BL/6 and BALB/c mice that are prototypical Th1- and Th2-type mouse strains, respectively. The two backgrounds were used with the goal of observing the range of symptoms in individuals with COVID-19, from mild to fatal illness. It was hypothesized that one or more of the humanized mice in the BALB/c background would model severe COVID-19, whereas the humanized C57BL/6J mice were likely to serve as models of asymptomatic or rapid recovery from infection.
The Ace2 locus on the mouse X-chromosome was modified to express the human ACE2 protein. The humanized ACE2 allele was also combined with the humanized FCGRT allele on the C57BL/6J background through breeding.
Guide RNA
The Ace2-crRNA_32 guide RNA was developed and tested. The protospacer sequence is 5′-AAGGCTGAGAAGGAGCCAGG-3′ (SEQ ID NO: 5), targeting Cas9 cleavage 13 bp downstream of the mouse Ace2 start codon in exon 2. The synthetic Ace2-crRNA_32 crRNA was purchased from Integrated DNA Technologies (IDT) and annealed to synthetic tracrRNA (IDT) to produce active guide RNA for genome editing.
Donor Plasmid and Knock-In Allele Design
The donor plasmid “pSYNB026_C57BL6_Ace2_Humanizing Construct” (SEQ ID NO: 1) was designed and constructed with a total insert size (region between homology arms) of 4037 bp. The plasmid, shown in
The donor vector, in glycerol stock, was streaked on an ampicillin plate and a single colony was used to start a maxiprep culture. Maxiprep DNA was analyzed by restriction digestion to confirm the structure and absence of rearrangements prior to microinjection.
The microinjection session was performed with embryos from C57BL/6J mice as summarized in Table 2. The process consisted of several steps: superovulation and donor embryo harvest, pronuclear microinjection, and reimplantation into pseudopregnant females. At least two microinjections of 100 embryos will be done per DNA construct. The session was performed with enhanced specificity eSpCas9, and yielded 29 pups, with two females having correct integration of the donor vector and 6 additional animals having integration events that scored positive for vector backbone PCRs, suggesting tandem donor integrations.
Founder Genotyping Assays
Twenty-six potential founder animals were genotyped with the assays depicted in
5′ and 3′ vector backbone (BB) assays were performed using one primer targeting the homology arm and a second primer targeting the donor vector backbone sequence. BB assays identify random donor integration at off-target sites in the genome as well as tandem integration of multiple donor copies at the target site. Correct targeted homologous integration events exclude the donor vector backbone sequences.
Founder Genotyping Data
Founder Breeding
Two vector backbone-negative female founders (#2, #26) were mated to males homozygous for the FCGRT humanized knock-in allele (Fcgrt KI/KI). Both females transmitted the hACE2 knock-in event to offspring. However, founder #26 only gave one viable litter while founder #2 produced multiple litters with positive offspring. Offspring from founder #2 were used for subsequent breeding and characterization.
Knock-in Allele Validation
DNA samples from F1 animals were assessed for sequence confirmation of the knock-in allele in founder line 2. Samples were assessed from females ACE2-B6 2.4 and ACE2-B6 2.12 (genotype Ace2 KI/+; Fcgrt KI/+) and males ACE2-B6 2.5 and ACE2-B6 2.14 (genotype Ace2 KI/Y; Fcgrt KI/+). The knock-in allele was PCR amplified and amplicons were sequenced.
Off-Target Mutation Analysis
The IDT CRISPR-Cas9 Design Checker online tool was used to identify the top five potential off-target cut sites in the mouse genome. Primers were designed to amplify the target regions for sequencing. The off-target sites and primer sequences are shown in
DNA samples from animals ACE2-B6 2.12 and ACE2-B6 2.14 were used for PCR amplification and sequencing across potential off-target sites. Wild-type control animals were included in the analysis. As shown in
Colony Expansion Breeding
F1 animals harboring the ACE2 and FCGRT knock-in alleles were intercrossed to generate ACE2 homozygous (KI/KI) females and hemizygous (KI/Y) males with and without the FCGRT knock-in allele. Tissue samples were harvested from animal ACE2-B6 2.46 (Ace2 KI/Y; Fcgrt KI/+) according to the following procedure.
Expression Analysis
Animals are euthanized by CO2 inhalation followed by cervical dislocation.
Tissues pieces are placed immediately in 500 μl RNAlater (aqueous solution) at room temperature, then moved to 4° C. Tissues in RNAlater are held at 4° C. at least overnight, and frozen at −80° C. until ready for analysis.
Tissue Samples for Harvesting
The Tmprss2 locus on the mouse chromosome 16 was modified to express the human TMPRSS2 protein. The humanized TMPRSS2 allele was also combined with the humanized FCGRT allele on the C57BL/6J background through breeding.
Guide RNA
The Tmprss2-crRNA_5 guide RNA was developed and tested. The protospacer sequence is 5′-TGTGCTCAGACTGATACCCG-3′ (SEQ ID NO: 17), targeting Cas9 cleavage at codon 19 in exon 3 of the mouse Tmprss2 gene. The synthetic Tmprss2-crRNA_5 crRNA was purchased from IDT and annealed to synthetic tracrRNA (IDT) to produce active guide RNA for genome editing.
Donor Plasmid and Knock-in Allele
The donor plasmid “pSYNB027_C57BL6_Tmprss2_Humanizing Construct” (SEQ ID NO: 3) was designed and constructed with a total insert size (region between homology arms) of 4161 bp. The plasmid, shown in
The donor vector, in glycerol stock, was streaked on an ampicillin plate and a single colony was used to start a maxiprep culture. Maxiprep DNA was analyzed by restriction digestion to confirm the structure and absence of rearrangements prior to microinjection.
One microinjection session was performed with embryos from C57BL/6J mice as summarized in Table 4. Fourteen pups were born from 173 injected embryos. One female and one male pup had the correct integration of the donor vector and 4 additional animals had integration events that scored positive with vector backbone PCRs, suggesting tandem donor integrations.
Founder Genotyping Assays
Fourteen potential founder animals were genotyped with the assays depicted in
5′ and 3′ vector backbone (BB) assays were performed using one primer targeting the homology arm and a second primer targeting the donor vector backbone sequence. BB assays identify random donor integration at off-target sites in the genome as well as tandem integration of multiple donor copies at the target site. Correct targeted homologous integration events exclude the donor vector backbone sequences.
Founder Genotyping Data
Founder Breeding
Vector backbone-negative founders #1 (female) and #8 (male) were mated to animals homozygous for the FCGRT humanized knock-in allele (Fcgrt KI/KI). Both founders transmitted the hTMPRSS2 knock-in event to offspring. However, male #8 unexpectedly gave a high percentage of female offspring (6/6 females), all of which were positive for the hTMPRSS2 knock-in allele. Male #8 was then mated to wild-type C57BL/6J females and produced a litter of 5 females and 2 males, all of which were again positive for the knock-in event. Female #1 displayed a normal offspring sex distribution (9 females, 5 males), with transmission of the hTMPRSS2 knock-in allele to 50% of offspring. Offspring from founder #1 were selected for further breeding and characterization.
Knock-in Allele Validation
DNA samples from Tmprss2 KI animals were assessed for sequence confirmation of the knock-in allele in founder line 1. Samples from females TMPRSS2-B6 1.7, 1.8 and 1.15 and males TMPRSS2-B6 1.16, 1.60 and 1.61 (genotypes all Tmprss2 KI/+; Fcgrt KI/+) were assessed. The knock-in allele was PCR amplified and amplicons were sequenced. The knock-in allele showed the expected sequence in tested animals, as shown in
Off-Target Mutation Analysis
The IDT CRISPR-Cas9 Design Checker online tool was used to identify the top five potential off-target cut sites in the mouse genome. Primers were designed to amplify the target regions for sequencing. The off-target sites and primer sequences are shown in
DNA samples from animals TMPRSS2-B6 1.60 and 1.61 were used for PCR amplification and sequencing across potential off-target sites. Wild-type control animals were included in the analysis. As shown in Table 6, no mutations were detected in either animal for the 5 sites analyzed.
Colony Expansion Breeding
F1 animals harboring the hTMPRSS2 and hFCGRT knock-in alleles were intercrossed to generate hTMPRSS2/hFCGRT double homozygous (KI/KI; KI/KI) animals. Compound heterozygous animals (KI+, KI/+) were later crossed back to C57BL/6J wild-type animals to remove the hFCGRT knock-in allele for a separate colony of animals with only the hTMPRSS2 knock-in allele. Tissue samples were harvested from Tmprss2 KI/KI; Fcgrt KI/KI animals TMPRSS2-B6 1.87 (M, 7 wks old), 1.97 (F, 5 wks old), 1.99 (M, 5 wks old) and 1.103 (F, 3 wks old).
Expression Analysis
Animals are euthanized by CO2 inhalation followed by cervical dislocation. Tissues pieces are placed immediately in 500 μl RNAlater (aqueous solution) at room temperature, then moved to 4° C. Tissues in RNAlater are held at 4° C. at least overnight, and frozen at −80° C. until ready for harvesting.
Tissue Samples for Harvesting
The Ace2 locus on the mouse X-chromosome was modified to express the human ACE2 protein.
Guide RNA
The Ace2-crRNA_32 guide RNA was developed and tested. The protospacer sequence is 5′-AAGGCTGAGAAGGAGCCAGG-3′ (SEQ ID NO: 28), targeting Cas9 cleavage 13 bp downstream of the mouse Ace2 gene. The synthetic Ace2-crRNA_32 crRNA was purchased from IDT and annealed to synthetic tracrRNA (IDT) to produce active guide RNA for genome editing.
Donor Plasmid and Knock-in Allele
The donor plasmid “pSYNB028_Ace2_NEO_BalbC” (SEQ ID NO: 2) was designed and constructed with a total insert size (region between homology arms) of 5831 bp. The plasmid, shown in
The donor vector, in glycerol stock, was streaked on an ampicillin plate and a single colony was used to start a maxiprep culture. Maxiprep DNA was analyzed by restriction digestion to confirm the structure and absence of rearrangements prior to gene targeting in BALB/cJ embryonic stem cells.
BALB/cJ embryonic stem cells (from TransViragen) were nucleofected with 1 μM SpCas9 protein, 1.2 μM Ace2-crRNA_32/tracrRNA and 4 μg supercoiled donor vector. Cells were selected with G418 and individual clones were picked and screened by PCR for integration of the hACE2 knock-in cassette.
ES Cell Screening Assays
ES cell clones were screened with the PCR assays depicted in
5′ LRPCR and 3′ LRPCR: Long-range PCR assays with one primer that anneals in native mouse sequence outside the donor vector homology arm and one primer that anneals in sequence unique to the donor vector. Positive amplification by these primers indicates insertion of the transgene at the target locus.
5′ and 3′ vector backbone (BB) assays were performed using one primer targeting the homology arm and a second primer targeting the donor vector backbone sequence. BB assays identify random donor integration at random non-targeted sites in the genome as well as tandem integration of multiple donor copies at the target site. Correct targeted homologous integration events exclude the donor vector backbone sequences. BB assays are performed after a subset of ES cell clones are expanded.
ES Cell Southern Blot Data
Six PCR-positive ES cell clones were selected for clonal expansion and Southern blot analysis of the hACE2 knock-in allele.
Chimera Production
hACE2 targeted ES cell clones B2, B3, B4 and B5 were individually injected into BALB/cJ blastocyst embryos for chimera production. Clones B2 and B3 were each injected into 32 blastocysts, resulting in 15 and 11 pups respectively. Clone B2 only gave 1 low-contribution female chimera and B3 gave 5 low-contribution chimeras (2 females and 3 males). Clones B4 and B5 were injected in 48 and 57 blastocysts, yielding 27 and 7 pups respectively. Clone B4 gave 17 chimeras, including 1 high-contribution, 7 medium-contribution and 8 low-contribution males. Clone B5 gave 7 chimeras including 1 high-contribution and 5 low-contribution males.
Chimera Breeding for Germline Transmission of the hACE Allele
Male chimeras from ES cell clones B3, B4 and B5 were mated to BALB/cJ or transgenic Flpo-BALB/cJ females for germline transmission of the hACE2 allele. Mating to Flpo-BALB/cJ allowed removal of the neomycin cassette concurrent with germline transmission of the targeted allele. One chimera from clone B3 produced a single germline transmission event. Clone B5 produced multiple germline transmission events. Chimeras from clone B4 produced the highest percentage of germline transmission events, and this line was selected for colony expansion and characterization.
Knock-in Allele Validation
DNA samples from ACE2-C ES cell clone B4 and from two F1 germline transmission animals from clone B4 chimeras were assessed for sequence confirmation of the knock-in allele, as shown in
Off-Target Mutation Analysis
The IDT CRISPR-Cas9 Design Checker online tool was used to identify the top five predicted off-target cut sites in the mouse genome. Primers were designed to amplify the target regions for sequencing. The off-target sites and primer sequences are shown in
DNA samples from animals ACE2-C B4.13 and B4.18 were used for PCR amplification and sequencing across potential off-target sites. A wild-type control animal was included in the analysis. ACE2-C B4.13 was heterozygous for a 24 bp deletion at off-target site #1, and animal ACE2-C B4.18 was heterozygous for a 14 bp deletion at off-target site 1, as shown in
Colony Expansion Breeding
Male chimeras only transmitted the X-linked ACE2 knock-in allele to female offspring. F1 females harboring the ACE2 knock-in allele (neo-removed) and Flpo transgene were crossed back to chimeras in some cases to generate homozygous KI/KI females and hemizygous KI/Y males with the hACE2 allele. Females harboring the hACE2-NEO allele (from chimeras crossed to BALB/cJ females) were crossed to Flpo-BALB/c males for production of offspring with the hACE2 allele with neo removed by cotransmission of the Flpo transgene with the hACE2-NEO allele. After homozygous females and hemizygous males were produced, they were intercrossed for further line expansion and, where applicable, to segregate the Flpo transgene away from the hACE2 knock-in allele. Intercross animals are also being screened for mutations at off-target site 1 to identify animals with wild-type sequence for removal of the off-target mutations from the colony. Tissue samples were harvested from ACE2-C B4.127 (Ace2 KIN; R26 Flpo/+).
Expression Analysis
Animals are euthanized by CO2 inhalation followed by cervical dislocation.
Tissues pieces are placed immediately in 500 μl RNAlater (aqueous solution) at room temperature, then moved to 4° C. Tissues in RNAlater are held at 4° C. at least overnight, and frozen at −80° C. until ready for analysis.
Tissue Samples for Harvesting
The Tmprss2 locus on mouse chromosome 16 was modified to express the human TMPRSS2 protein.
Guide RNA
The Tmprss2-crRNA_5 guide RNA was developed and tested. The protospacer sequence is 5′-TGTGCTCAGACTGATACCCG-3′ (SEQ ID NO: 17), targeting Cas9 cleavage at codon 19 in exon 3 of the mouse Tmprss2 gene. The synthetic Tmprss2-crRNA_32 crRNA was purchased from IDT and annealed to synthetic tracrRNA (IDT) to produce active guide RNA for genome editing.
Donor Plasmid and Knock-in Allele
The donor plasmid “pSYNB029_BalbC_Tmprss2_NEO Humanizing Construct” (SEQ ID NO: 4) was designed and constructed with a total insert size (region between homology arms) of 5954 bp. The plasmid, shown in
The donor vector, in glycerol stock, was streaked on an ampicillin plate and a single colony was used to start a maxiprep culture. Maxiprep DNA was analyzed by restriction digestion to confirm the structure and absence of rearrangements prior to gene targeting in BALB/cJ embryonic stem cells.
BALB/cJ embryonic stem cells (TransViragen) were nucleofected with 1 μM SpCas9 protein, 1.2 μM Tmprss2-crRNA_5/tracrRNA and 4 μg supercoiled donor vector. Cells were selected with G418 and individual clones were picked and screened by PCR for integration of the hTMPRSS2 knock-in cassette.
ES Cell Screening Assays
ES cell clones were screened with the PCR assays depicted in
5′ LRPCR and 3′ LRPCR: Long-range PCR assays with one primer that anneals in native mouse sequence outside the donor vector homology arm and one primer that anneals in sequence unique to the donor vector. Positive amplification by these primers indicates insertion of the transgene at the target locus.
5′ and 3′ vector backbone (BB) assays were performed using one primer targeting the homology arm and a second primer targeting the donor vector backbone sequence. BB assays identify random donor integration at random non-targeted sites in the genome as well as tandem integration of multiple donor copies at the target site. Correct targeted homologous integration events exclude the donor vector backbone sequences. BB assays are performed after a subset of ES cell clones are expanded.
ES Cell Southern Blot Data
Eleven PCR-positive ES cell clones were selected for clonal expansion and Southern blot analysis of the hTMPRSS2 knock-in allele.
Based on the Southern blot results, clones D10 (biallelic targeted, no tandem integration) and G4 (biallelic targeted, tandem integration) were selected for blastocyst injection to produce chimeras for germline transmission of the targeted allele. In the case of tandem integration, the crosses to Flp recombinase animals required for neomycin cassette removal also collapse tandem integration events to single-copy.
Chimera Production
hTMPRSS2 targeted ES cell clones D10 and G4 were individually injected into C57BL/6J blastocyst embryos for chimera production. Clone D10 was injected into 20 blastocysts, resulting in 12 pups, with 5 chimeras (1 medium-contribution male, 2 each low-contribution males and females). Clone G4 was injected in 16 blastocysts and yielded 9 pups, with 7 chimeras (1 high-contribution male, 1 each medium contribution male and female, 3 low-contribution males and 1 low-contribution female).
Chimera Breeding for Germline Transmission of the hTMPRSS2 Allele
Chimeras from both D10 and G4 ES cell clones were mated to BALB/cJ or transgenic Flpo-BALB/cJ animals for germline transmission of the hTMPRSS2 allele. Mating to Flpo-BALB/cJ allowed removal of the neomycin cassette concurrent with germline transmission of the targeted allele and removal of tandem multimers. Chimeras from clone D10 failed to transmit the hTMPRSS2 allele through the germline. Three chimeras from clone G4 each produced multiple germline transmission events. The G4 line was selected for colony expansion and characterization.
Knock-in Allele Validation
DNA samples from TMPRSS2-C ES cell clone G4 and animals TMPRSS2-C animals G4.32 (female, Tmprss2 KI/+) and G4.33 (male, Tmprss2 KI/+; R26 Flpo/+) were assessed for sequence confirmation of the knock-in allele. The knock-in allele was PCR amplified and amplicons were sequenced (
Off-Target Mutation Analysis
The IDT CRISPR-Cas9 Design Checker online tool was used to identify the top five predicted off-target cut sites in the mouse genome. Primers were designed to amplify the target regions for sequencing. The off-target sites and primer sequences are shown in
DNA samples from animal TMPRSS2-C G4.33 and from ES cell clone G4 were used for PCR amplification and sequencing across potential off-target sites. Wild-type control DNA was included in the analysis. No mutations were detected in the animal or ES cell clone for the 5 sites analyzed as shown in Table 9.
Colony Expansion Breeding
Animals harboring the hTMPRSS2 knock-in allele with neo cassette removed (KI/+) with or without the Flpo transgene are intercrossed to produce hTMPRSS2 homozygous (KI/KI) animals for rapid colony expansion and characterization. As the colony expands, breeding strategies are adjusted to remove the Flpo transgene and produce animals with only the knock-in allele. Tmprss2 KI/KI; R26 Flpo/+animals TMPRSS2-C G4.164 (female, 9 wks old) and G4. 192 (male, 5 wks old) are analyzed for gene expression.
Sequences
Sequences for the constructs described herein are provided below. The sequences are all given in 5′—>3′.
This application claims priority to and the benefit of U.S. Provisional Patent Application Ser. No. 63/237,445, filed Aug. 26, 2021, and entitled “Humanized Mouse Models for Study of COVID-19”, the disclosure of which is incorporated herein by reference in its entirety.
This invention was made with government support under grant number 1 R44 A1157900-01A1, awarded by the NIH-NIAID-Small Business Innovation Research (SBIR) program. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63237445 | Aug 2021 | US |
