Non-human animals comprising humanized ace2 and tmprss loci

The non-human animal, non-human animal cell, or non-human animal genome of any one of the preceding claims, wherein

• (I) an endogenous ACE2 locus comprises the humanized ACE2 gene,
• wherein the endogenous ACE2 locus comprises a replacement of the nucleotide sequence encoding the extracellular domain of the endogenous ACE2 protein with the nucleotide sequence encoding the extracellular domain of a human ACE2 protein such that the nucleotide sequence encoding the extracellular domain of a human ACE2 protein is operably linked to an endogenous nucleotide sequence encoding (iii) the transmembrane domain of an endogenous non-human animal ACE2 protein, and (iv) the cytoplasmic domain of an endogenous non-human animal ACE2 protein and/or
• (II) an endogenous TMPRSS locus comprises one of the one or more distinct and humanized TMPRSS genes,
• wherein the endogenous TMPRSS locus comprises a replacement of the nucleotide sequence encoding the extracellular domain of the endogenous TMPRSS protein with the nucleotide sequence encoding the extracellular domain of a human TMPRSS protein such that the nucleotide sequence encoding the extracellular domain of the human TMPRSS protein is operably linked to an endogenous nucleotide sequence encoding (iii) the transmembrane domain of an endogenous TMPRSS protein at the endogenous TMPRSS locus, and (iv) the cytoplasmic domain of the endogenous TMPRSS protein,
• wherein the non-human animal TMPRSS protein and the human TMPRSS protein are orthologous.

The non-human animal, non-human animal cell, or non-human animal genome of any one of the preceding claims, wherein

• (I) the humanized ACE2 gene encodes a recombinant ACE2 protein comprising in operable linkage:
• (i) an ACE2 signal sequence of an endogenous ACE2 protein,
• (ii) an extracellular domain of a human ACE2 protein,
• (iii) a transmembrane domain of an endogenous ACE2 protein, and
• (iv) a cytoplasmic domain of an endogenous mouse ACE2 protein, optionally
• wherein the extracellular domain of a human ACE2 protein is encoded by part of the coding sequence of coding exon 1, all of the coding sequences of coding exon 2 to coding exon 16, inclusive, and part of coding exon 17 of a human ACE2 gene and/or wherein the amino acid sequence of the recombinant ACE2 protein is set forth in SEQ ID NO:20, and
• (II) one of the one or more distinct and humanized TMPRSS genes is:
• (A) a humanized TMPRSS2 gene, optionally wherein the humanized TMPRSS2 gene:
• (i) is at an endogenous TMPRSS2 locus, optionally under control of the endogenous TMPRSS2 promoter at the endogenous TMPRSS2 locus,
• (ii) is formed by a replacement of an endogenous nucleotide sequence of an endogenous TMPRSS2 gene, or portion thereof, with an orthologous nucleotide sequence of a human TMPRSS2 gene,
• (iii) comprises coding exon 4 through the stop codon in coding exon 13 of the human TMPRSS2 gene, and optionally further comprises the 3′ UTR of the human TMPRSS2 gene,
• (iv) comprises coding exons 1-2 of the endogenous TMPRSS2 gene and coding exon 4 through coding exon 13 of the human TMPRSS2 gene, and optionally the 3′ UTR of the human TMPRSS2 gene
• (v) comprises an exon 3 that comprises a 5′ portion of coding exon 3 of the endogenous TMPRSS2 gene and a 3′ portion of coding exon 3 of the human TMPRSS2 gene and/or
• (vi) encodes a humanized TMPRSS2 protein that comprises (i) an extracellular domain of the human TMPRSS2 protein encoded by a human TMPRSS2 gene, and (ii) a cytoplasmic and transmembrane portion encoded by the endogenous TMPRSS2 gene, optionally wherein the extracellular domain of the human TMPRSS2 protein comprises the residues W 106 to G492 of SEQ ID NO:71;
• (B) a humanized TMPRSS4, optionally wherein the humanized TMPRSS4 gene:
• (i) is at an endogenous TMPRSS4 locus, optionally under control of the endogenous TMPRSS4 promoter at the endogenous TMPRSS4 locus,
• (ii) is formed by a replacement of an endogenous nucleotide sequence of an endogenous TMPRSS4 gene, or portion thereof, with an orthologous nucleotide sequence of a human TMPRSS4 gene,
• (iii) comprises coding exon 4 through the stop codon in coding exon 13 of the human TMPRSS4 gene, and optionally further comprises the 3′UTR of the human TMPRSS2 gene,
• (iv) comprises coding exons 1-3 of the endogenous TMPRSS2 gene and coding exon 4 through coding exon 13 of the human TMPRSS4 gene, and optionally the 3′ UTR of the human TMPRSS4 gene and/or
• (v) encodes a humanized TMPRSS4 protein that comprises (i) an extracellular domain of the human TMPRSS4 protein encoded by a human TMPRSS4 gene, and (ii) a cytoplasmic and transmembrane portion encoded by the endogenous TMPRSS4 gene, optionally wherein the extracellular domain of the human TMPRSS4 protein comprises the residues K54-L437 of SEQ ID NO:78;
• and/or
• (C) a humanized TMPRSS11, optionally wherein the humanized TMPRSS11 gene:
• (i) is at an endogenous TMPRSS11 locus, optionally under control of the endogenous TMPRSS11 promoter at the endogenous TMPRSS11 locus,
• (ii) is formed by a replacement of an endogenous nucleotide sequence of an endogenous TMPRSS11 gene, or portion thereof, with an orthologous nucleotide sequence of a human TMPRSS11 gene,
• (iii) comprises coding exon 3 through the stop codon in coding exon 10 of the human TMPRSS11 gene, and optionally further comprises the 3′ UTR of the human TMPRSS11 gene,
• (iv) comprises coding exons 1-2 of the endogenous TMPRSS11 gene and coding exon 3 through coding exon 10 of the human TMPRSS11 gene, and optionally the 3′ UTR of the human TMPRSS11 gene and/or
• (v) encodes a humanized TMPRSS11 protein that comprises (i) an extracellular domain of the human TMPRSS11 protein encoded by a human TMPRSS11 gene, and (ii) a cytoplasmic and transmembrane portion encoded by the endogenous TMPRSS11 gene, optionally wherein the extracellular domain of the human TMPRSS11 protein comprises the residues A42 to 1418 of SEQ ID NO:85.

A method for making a non-human animal, non-human animal cell, or non-human animal genome comprising a humanized ACE2 and a humanized TMPRSS gene, the method comprising:

• (A) generating a genetically modified non-human embryonic stem (ES) cell comprising:
• (I) humanized ACE2 gene encoding a recombinant ACE2 protein, wherein the recombinant ACE2 protein comprises in operable linkage:
• (a) an ACE2 signal sequence of a non-human animal ACE2 protein or an ACE2 signal sequence of a human ACE2 protein,
• (b) an extracellular domain substantially identical to an extracellular domain of a human ACE2 protein,
• (c) a transmembrane domain of a non-human animal ACE2 protein or a transmembrane domain of a human ACE2 protein, and
• (d) a cytoplasmic domain of a non-human animal ACE2 protein or a cytoplasmic domain of a human ACE2 protein, and
• (II) one or more distinct and humanized TMPRSS genes, each encoding a distinct recombinant TMPRSS protein that comprises in operable likage:
• (a) a TMPRSS signal sequence of a non-human animal TMPRSS protein or TMPRSS signal sequence of a human TMPRSS protein,
• (b) an extracellular domain substantially identical to the extracellular domain of the human TMPRSS protein, and
• (c) a transmembrane domain of the non-human animal TMPRSS protein or a transmembrane domain of the human TMPRSS protein, and
• (d) a cytoplasmic domain of the non-human TMPRSS protein or a cytoplasmic domain of the human TMPRSS protein,
• wherein the non-human animal TMPRSS protein and the human TMPRSS protein are orthologous
• (B) introducing the modified non-human animal ES cell into a host embryo of non-human animal to form a donor cell-non-human animal embryo complex;
• and
• (C) gestating the donor cell-non-human animal embryo in a surrogate non-human animal mother, wherein the surrogate non-human animal mother produces rodent progeny that express the humanized ACE2 and TMPRSS proteins.