NZ709432B2 - Humanized il-6 and il-6 receptor - Google Patents
Humanized il-6 and il-6 receptor Download PDFInfo
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- NZ709432B2 NZ709432B2 NZ709432A NZ70943212A NZ709432B2 NZ 709432 B2 NZ709432 B2 NZ 709432B2 NZ 709432 A NZ709432 A NZ 709432A NZ 70943212 A NZ70943212 A NZ 70943212A NZ 709432 B2 NZ709432 B2 NZ 709432B2
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Classifications
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- A—HUMAN NECESSITIES
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/15—Humanized animals
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
- A01K2217/052—Animals comprising random inserted nucleic acids (transgenic) inducing gain of function
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01K2217/00—Genetically modified animals
- A01K2217/15—Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
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- A—HUMAN NECESSITIES
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- A01K2227/10—Mammal
- A01K2227/105—Murine
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01K2267/03—Animal model, e.g. for test or diseases
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
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- C07K14/52—Cytokines; Lymphokines; Interferons
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Abstract
Disclosed is a genetically modified murine comprising a replacement at an endogenous murine IL-6 locus of a murine gene encoding IL-6 with a human gene encoding human IL-6, wherein the human gene encoding human IL-6 is under control of endogenous murine regulatory elements at the endogenous murine IL-6 locus, and wherein the murine animal is not a mouse. Also disclosed is a genetically modified murine animal, comprising a humanisation of an endogenous murine IL-6R? gene, wherein the humanisation comprises a replacement of murine IL-6R? ectodomain-encoding sequence with human IL-6R? ectodomain-encoding sequence at the endogenous murine IL-6R? locus, and wherein the humanised IL-6R? gene is under control of endogenous murine regulatory elements, and wherein the murine animal is not a mouse. L-6 locus, and wherein the murine animal is not a mouse. Also disclosed is a genetically modified murine animal, comprising a humanisation of an endogenous murine IL-6R? gene, wherein the humanisation comprises a replacement of murine IL-6R? ectodomain-encoding sequence with human IL-6R? ectodomain-encoding sequence at the endogenous murine IL-6R? locus, and wherein the humanised IL-6R? gene is under control of endogenous murine regulatory elements, and wherein the murine animal is not a mouse.
Description
HUMANIZED IL-6 AND IL-6 OR
The present application is a divisional application from New Zealand patent application
number , the entire disclosure of which is incorporated herein by reference.
FIELD OF ION
Non-human animals having a replacement of the endogenous non-human animal
IL-6 and/or IL-6 receptor genes are provided. IL-6 and/or IL-6 receptor genes of the nonhuman
animal are ed, at the endogenous non-human loci, with human IL-6 and/or
humanized IL-6 receptor genes sing human sequence. Non-human animals that have
human IL-6 and/or zed IL-6 or genes, wherein the non-human animals do not
exhibit one or more pathologies that are characteristic of non-human animals transgenic for
human IL-6.
BACKGROUND
Mice transgenic for a human IL-6 gene are known in the art. However, random
insertion of a human IL-6 transgene into the mouse genome results in poorly regulated
sion of the human IL-6 protein, which manifests itself in a variety of pathologies in such
transgenic mice, including, but not limited to, plasmacytosis and glomerulonephritis. As a
result, these mice have limited usefulness.
There is a need for non-human animals, e.g., mice and rats, the express human or
humanized IL-6 and/or human or humanized IL-6 receptor. There is a need for such
humanized mice that do not exhibit one or more ogies exhibited by transgenic hIL-6
mice.
SUMMARY
[0003a] In one aspect, the present invention provides a cally modified murine
animal comprising a replacement at an endogenous murine IL-6 locus of a murine gene
encoding IL-6 with a human gene encoding human IL-6, wherein the human gene encoding
human IL-6 is under control of endogenous murine regulatory elements at the endogenous
murine IL-6 locus, wherein the murine animal is not a mouse.
[0003b] In a further aspect, the present invention provides a genetically modified
murine animal, comprising a humanization of an endogenous murine IL-6Rα gene, wherein
the humanization comprises a replacement of murine IL-6Rα ectodomain-encoding sequence
with human IL-6Rα ectodomain-encoding sequence at the endogenous murine IL-6Rα locus,
and wherein the humanized IL-6Rα gene is under control of nous murine tory
elements, wherein the murine animal is not a mouse.
[0003c] In a further aspect, the present invention provides a method for making a
humanized murine animal, comprising replacing a murine gene sequence encoding murine
IL-6 with a human gene encoding human IL-6 so that the human IL-6 gene is under control of
endogenous murine regulatory elements, wherein the murine animal is not a mouse.
[0003d] In a r aspect, the present ion provides a method for making a
humanized murine animal, comprising replacing all murine exons encoding ectodomain
sequences of murine IL-6Rα with a human genomic nt encoding human IL-6Rα
ectodomain to form a humanized IL-6Rα gene, wherein the humanized IL-6Rα gene is under
control of endogenous murine regulatory ts, wherein the murine animal is not a
mouse.
[0003e] In a further aspect, the present invention provides a genetically modified murine
animal comprising a humanized IL-6Rα gene comprising a replacement of murine
ectodomain-encoding sequence with human ectodomain sequence, wherein the humanized
IL-6Rα gene comprises murine transmembrane and intracellular domain sequences, wherein
the murine animal further comprises a gene encoding a human IL-6, and wherein the genes
encoding human IL-6 and humanized IL-6Rα are under l of nous murine
regulatory elements, wherein the murine animal is not a mouse.
In another aspect, genetically modified non-human animals are provided that
comprise a replacement at an endogenous IL-6 and/or IL-6 or locus of a gene encoding
an endogenous IL-6 and/or IL-6 receptor with a gene encoding a human or humanized IL-6
and/or IL-6 receptor. Murine animals are ed that comprise a replacement of an
endogenous IL-6 gene, at an endogenous murine IL-6 locus, with a human IL-6 gene; and/or
that comprise a replacement of an endogenous IL-6 receptor gene (or nucleotide sequence
encoding an ectodomain thereof) with a human IL-6 receptor gene (or nucleotide sequence
encoding an ectodomain thereof).
In r aspect, genetically modified murine animals are ed that express
a human IL-6 gene under the control of endogenous murine er and/or endogenous
murine regulatory elements, from an endogenous murine IL-6 locus.
In r aspect, genetically modified murine animals are provided that express
a human IL-6 receptor gene (or a gene ng a human ectodomain and murine
embrane and intracellular domains) under the control of endogenous murine
promoter and/or endogenous murine regulatory elements, from an endogenous
murine lL-6 receptor locus.
In one aspect, a genetically ed animal (e.g., a murine animal, e.g., a
mouse or rat) is provided that expresses a human lL-6 protein, n the non-
human animal does not exhibit a pathology selected from plasmacytosis,
glomerulonephritis, glomerulosclerosis, mesangio-proliferative glomerulonephritis,
intestinal lymphoma, kidney lymphoma, splenomegaly, lymph node enlargement,
liver enlargement, megakaryocytes in bone marrow, compacted abnormal plasma
cells, infiltration of plasma cells into lung or liver or kidney, mesangial cell
proliferation in kidney, cerebral pression of lL-6, ramified microglial cells in
white matter, reactive astrocytosis in brain, kidney failure, elevated megakaryocytes
in spleen, muscle wasting (e.g., cnemius muscle wasting), elevated muscle
cathepsins B and B+L (e.g., around 20-fold and ), and a combination f.
In one embodiment, the non-human animal comprises a normal B cell
population. In one embodiment, the normal B cell population is approximately the
same in number and immunophenotype as a wild-type animal, e.g., a wild-type
mouse.
In one embodiment, the non-human animal is murine (e.g., a mouse or rat)
and expresses human lL-6 (hlL-6) in serum at a level below about 800 pg/mL, below
about 700, 600, 500, 400, 300, or 200 pg/mL. In a specific embodiment, the murine
animal expresses hlL-6 in serum at a level of about 50 to about no more than 200
pg/mL, in another embodiment about 75-125 pg/mL, in another embodiment at about
100 pg/mL.
In one aspect, a man animal is provided that expresses hlL-6 and/or
hlL-6R, wherein the non-human animal expresses hlL-6 and/or hlL-6R from an
endogenous non-human lL-6 locus and/or an endogenous non-human hlL-6R locus.
In a specific embodiment, the non-human animal is murine (e.g., mouse or rat).
In one aspect, a genetically modified mouse is ed that expresses hlL-6
from an endogenous mouse lL-6 locus, wherein the endogenous mouse lL-6 gene
has been replaced with a hlL-6 gene.
In one embodiment, the mouse comprises a cell that expresses an lL-6
or ) that ses a human ectodomain on the surface of the cell. In
one embodiment, the cell is a lymphocyte. In one embodiment, the lymphocyte is a
B cell.
In one embodiment, about 6.8 kb at the nous mouse lL-6 locus,
including exons 1 through 5 and a 3’ untranslated sequence, is d and replaced
with about 4.8 kb of human lL-6 gene sequence comprising exons 1 through 5 of the
human lL-6 gene. In a specific embodiment, the human lL-6 gene comprises exons
1 through 5 of the human lL-6 gene of human BAC CTD-2369M23.
In one aspect, a genetically modified mouse is provided that expresses lL-6
from a human lL-6 gene, wherein the mouse ses human lL-6 in its serum.
In one embodiment, the mouse serum exhibits a serum concentration of
human lL-6 of about 25 to about 300 pg/mL, 50 to about 250 pg/mL, 75 to about 200
pg/mL, or 100 to about 150 pg/mL. In a specific embodiment, the level of human lL-6
in the serum of the mouse is about 100 pg/mL.
In one embodiment, the level of a pan B cell-specific marker in bone marrow
of the mouse is about the same as that of a wild-type mouse. In one embodiment,
the level of a pan B cell-specific marker in spleen is about the same as that of a wild-
type mouse. In one embodiment, the pan B cell-specific marker is selected from
B220, CD19, CD20, CD22, CD79a, CD79b, L26, and Pax-5 (BSAP).
In one aspect, a genetically modified mouse is provided that expresses hlL6,
wherein the mouse does not exhibit a feature selected from plasmacytosis,
megaly, lymph node enlargement, compacted abnormal plasma cells, and a
ation thereof.
In one embodiment, the mouse comprises a spleen that is about the same
weight (per body weight) as a wild-type mouse. In one embodiment, the lymph
nodes of the mouse are about the same weight (per body weight) as a wild-type
mouse. In one embodiment, plasma cells of the mouse do not exhibit plasmocytosis
characteristic of mice that overexpress human lL-6.
In one embodiment, the mouse does not exhibit glomerulonephritis.
In one embodiment, the mouse exhibits a mesangial cell level comparable to
a wild-type mouse.
In one aspect, a genetically modified mouse is provided that expresses hlL6
from an endogenous mouse lL-6 locus, wherein the endogenous mouse lL-6 gene
has been replaced with a hlL-6 gene, n the mouse does not exhibit a feature
selected from a morphologically detectable neuropathology, a reactive astrocytosis,
and a combination thereof. In one embodiment, the mouse comprises a brain that is
morphologically indistinct from a ype mouse brain. In one embodiment, the
mouse comprises brain tissue that ts a level of reactive astrocytosis that is no
higher than that of a wild-type mouse.
In one embodiment, the mouse does not express human lL-6 in neurons. In
one embodiment, the mouse comprises activated astrocyte levels that are
comparable to activated yte levels in a wild-type mouse.
In one embodiment, the mouse comprises ramified microglial cells in its white
, wherein the ed microglial cells are present in an amount equivalent to
an amount of ramified microglial cells in a wild-type mouse.
In one embodiment, the mouse does not exhibit a reactive atrocytosis. In one
embodiment, the white matter of the mouse is logically indistinct from the
white matter of a wild-type mouse. In one embodiment, the white matter of the
mouse is ogically indistinct from a wild-type mouse white matter with respect to
histochemical staining of reactive astrocytes.
In one embodiment, the mouse comprises a brain that is morphologically
inct from a wild-type mouse brain. In one embodiment, the mouse comprises
brain tissue that exhibits a level of reactive astrocytosis that is no higher than that of
a wild-type mouse.
In one aspect, a cally modified mouse is provided that expresses hlL6
from an endogenous mouse lL-6 locus, wherein the endogenous mouse lL-6 gene
has been replaced with a hlL-6 gene, wherein the mouse does not exhibit a feature
selected from a life span ned by about 50% or more, kidney failure,
hypergammaglobulinemia, elevated megakaryocytes in spleen, elevated
megakaryocytes in bone marrow, plasmacytosis of spleen, plasmacytosis of thymus,
plasmacytosis of lymph nodes, ulonephritis, glomerulosclerosis, and a
combination thereof.
In one embodiment, the mice have a life span that exceeds 20 weeks. In one
embodiment, the mice have a life span that exceeds 30 weeks, 40 weeks, or 50
weeks. In one embodiment, the mice exhibit a life span about equal to that of a wild-
type mouse of the same strain.
In one embodiment, the mice exhibit a level of megakaryocytes in spleen that
is no more than about the splenic ryocyte level of a wild-type mouse
In one embodiment, the mice comprise lymphoid organs that are essentially
devoid of abnormal and compactly arranged plasmacytoid cells.
In one embodiment, the mice exhibit gamma globulin serum levels equivalent
to gamma globulin serum levels in wild-type mice. In one embodiment, the levels of
(11- and B-globulin in serum of the mice are equivalent to (11- and ulin serum
levels of wild-type mice of the same strain.
In one aspect, a genetically modified mouse is provided that expresses
human lL-6 from an endogenous mouse lL-6 locus, wherein the endogenous mouse
lL-6 gene has been replaced with a hlL-6 gene, wherein the mouse does not exhibit
a feature selected from muscle wasting, an elevated cathepsin B level as ed
with a wild-type mouse of the same strain, an elevated cathepsin A+B level as
compared with a wild-type mouse of the same strain, an increased liver weight as
compared with a wild-type mouse of the same strain, and a combination thereof.
In one embodiment, the weight of the liver of the mouse is about 800-900 mg
at 12 weeks.
In one embodiment, the mouse exhibits a cathepsin B level throughout its life
span that is no more than about the level observed in a wild-type mouse. In one
embodiment, the mouse exhibits a cathepsin A+B level throughout its life span that is
no more than about the level ed in a wild-type mouse.
In one embodiment, the mouse as an adult exhibits a gastrocnemus muscle
weight that is within about 10% of the weight of a wild-type mouse of the same strain.
In one embodiment, the mouse as an adult exhibits a gastrocnemus muscle weight
that is about the same as that of a ype mouse.
In one aspect a mouse is provided that comprises a nucleotide sequence
encoding a human lL-6 protein, wherein the nucleotide sequence encoding the
human lL-6 protein replaces in whole or in part an endogenous nucleotide sequence
encoding and endogenous mouse lL-6 protein.
In one aspect, a mouse is provided that comprises a replacement at an
endogenous mouse lL-6 receptor locus of mouse lL-6Ra ectodomain with an
ectodomain sequence of a human lL-6Rq to form a chimeric human/mouse lL-6Ra
gene.
In one embodiment, the chimeric lL-6Ra gene is under the control of a mouse
er and/or mouse regulatory elements at the nous mouse lL-6Rq locus.
In one embodiment, about 35.4 kb of mouse lL-6Rq ectodomain-encoding
sequence is replaced with about 45.5 kb of human lL-6R main-encoding
sequence.
In one ment, the human lL-6R ectodomain-encoding sequence
encompasses the first (ATG) codon in exon 1 h exon 8.
In one embodiment, the mouse lL-6Rq sequence that is replaced includes a
contiguous ce that encompasses exons 1 through 8. In a specific
embodiment, exons 1 through 8 and a portion of intron 8 is deleted.
WO 63556
In one , a cally modified mouse is provided, comprising a
replacement at an endogenous mouse lL-6 locus of a mouse gene encoding lL-6
with a human gene encoding human lL-6, wherein the human gene encoding human
lL-6 is under control of endogenous mouse regulatory elements at the endogenous
mouse lL-6 locus.
In one embodiment, the human gene encoding human lL-6 is a human lL-6
gene of BAC ID CTD-2369M23.
In one embodiment, the mouse expresses a mouse lL-6Rq. In one
embodiment, the mouse expresses a human lL-6Rq. In one embodiment, the
humanized lL-6Ror ses a human ectodomain. In one embodiment, the
humanized lL-6Rq comprises a mouse transmembrane domain and a mouse
cytoplasmic domain. In one embodiment, the mouse expresses a humanized lL-6Rq
that comprises a humanization of ectodomain but not transmembrane and/or
cytosolic domain.
In one embodiment, the mouse does not exhibit a feature selected from
plasmocytosis, glomerulosclerosis, glomerulonephritis, kidney failure,
hypergammaglobulinemia, elevated megakaryocytes in spleen, elevated
megakaryocytes in bone marrow, splenomegaly, lymph node enlargement,
compacted abnormal plasma cells, and a combination thereof.
In one aspect, a genetically ed mouse is provided, comprising a
zation of an endogenous mouse lL-6Ror gene, wherein the humanization
comprises a replacement of mouse lL-6Rq ectodomain-encoding sequence with
human lL-6Rq ectodomain-encoding sequence at the endogenous mouse lL-6Rq
locus.
In one embodiment, a uous mouse sequence sing mouse exons
1 h 8 is replaced with a contiguous genomic fragment of human lL-6Ror
sequence encoding a human lL-6Rq ectodomain. In one embodiment, the
contiguous genomic fragment of human lL-6Rq sequence encoding the ectodomain
is from BAC 92J23.
In one embodiment, the mouse further comprises a zed lL-6 gene. In
one embodiment, the mouse comprises a replacement at an endogenous mouse lL-6
locus of a mouse lL-6 gene with a human lL-6 gene. In one embodiment, the
humanized lL-6 gene is under control of endogenous mouse regulatory elements.
In one aspect, a method is provided for making a humanized mouse,
comprising replacing a mouse gene ce encoding mouse lL-6 with a human
gene encoding human lL-6.
WO 63556
In one embodiment, the replacement is at an endogenous mouse lL-6 locus,
and the human gene encoding human lL-6 is operably linked to endogenous mouse
regulatory sequences.
In one aspect, a method for making a humanized mouse is provided,
comprising replacing mouse exons encoding ectodomain sequences of mouse |L-
6Rq with a human genomic fragment encoding ectodomain sequences of human |L-
6Rq to form a humanized lL-6Rq gene.
In one embodiment, the replacement is at an nous mouse lL-6Rq
locus, and the humanized lL-6Rq gene is operably linked to endogenous mouse
regulatory sequences.
In one aspect, a genetically modified mouse is provided, comprising a
humanized lL-6Rq gene comprising a replacement of mouse ectodomain-encoding
sequence with human main sequence, wherein the humanized lL-6Rq gene
comprises a mouse transmembrane sequence and a mouse cytoplasmic sequence;
wherein the mouse further comprises a gene encoding a human lL-6, wherein the
gene encoding a human lL-6 is under control of endogenous mouse lL-6 regulatory
elements.
In one embodiment, the mouse is incapable of expressing a fully mouse lL-
6Rq and incapable of expressing a mouse lL-6.
In various s, the genetically modified mice described herein comprise
the c modifications in their germline.
In one aspect, a tissue, cell, or membrane fragment from a mouse as
described herein is provided.
In one embodiment, the tissue or cell is from a mouse that ses a
human lL-6 protein, but that does not express a mouse lL-6 protein. In one
embodiment, the tissue or cell is from a mouse that expresses a humanized lL-6Rq
n, but not a mouse lL-6Rq protein. In one embodiment, the humanized lL-6Rq
n comprises a human ectodomain and a mouse transmembrane domain and a
mouse cytosolic domain. In one embodiment, the tissue or cell is from a mouse that
ses a human lL-6, a humanized lL-6Rq, and that does not express a mouse
lL-6 and does not express an lL-6Rq that comprises a mouse ectodomain.
In one , an ex vivo complex of a mouse cell bearing a humanized lL-
6Rq (human ectodomain and mouse transmembrane and mouse asmic
domain) and a human lL-6 is provided.
In one aspect, a mouse embryo comprising a genetic modification as
described herein is provided.
In one aspect, a mouse host embryo is provided that comprises a donor cell
that comprises a genetic cation as described herein.
In one aspect, a pluripotent or totipotent non-human animal cell comprising a
genetic modification as described herein is provided. In one embodiment, the cell is
a murine cell. In one embodiment, the cell is an E8 cell.
In one aspect, a mouse egg is ed, wherein the mouse egg comprises
an ectopic mouse chromosome, wherein the ectopic mouse chromosome comprises
a genetic modification as described .
In one aspect, the mouse, embryo, egg, or cell that is genetically ed to
se a human lL-6 gene or human or humanized lL-6Ra gene is of a mouse that
is of a C57BL strain selected from C57BL/A, C57BL/An, GrFa,
C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10,
C57BL/10808n, C57BL/10Cr, and C57BL/Ola. In another embodiment, the mouse is
a 129 strain selected from the group consisting of a strain that is 129P1, 129P2,
129P3, 129X1, 12981 (e.g., 12981/8V, 12981/8vlm), 12982, 12984, 12985,
12989/8vaH, 12986 (129/8vaTac), 12987, 12988, 129T1, 129T2 (see, e.g.,
Festing et al. (1999) Revised nomenclature for strain 129 mice, Mammalian Genome
, see also, Auerbach et al (2000) Establishment and Chimera Analysis of
129/8va- and C57BL/6-Derived Mouse Embryonic Stem Cell Lines). In a specific
embodiment, the genetically modified mouse is a mix of an aforementioned 129
strain and an aforementioned C57BL/6 strain. In another specific embodiment, the
mouse is a mix of aforementioned 129 strains, or a mix of entioned BL/6
strains. In a specific embodiment, the 129 strain of the mix is a 12986
(129/8vaTac) strain. In r embodiment, the mouse is a BALB strain, e.g.,
BALB/c strain. In yet another embodiment, the mouse is a mix of a BALB strain and
another aforementioned strain. In one embodiment, the mouse is 8wiss or 8wiss
Webster mouse.
Each of the s and ments described herein are e of being
used together, unless excluded either explicitly or clearly from the context of the
embodiment or aspect.
BRIEF DESCRIPTION OF THE DRAWINGS
provides an illustration, not to scale, of the human (top) and mouse
(bottom) lL-6 genomic loci. Exons I, II, III, IV, and V (in both human and mouse) are
indicated by closed boxes to the right in the figure. 8elected ve regulatory
regions are indicated by open boxes to the left in the figure.
shows acute phase response (mSAA level) in the presence or
absence of turpentine in wild-type mice, humanized main IL-6R mice, and
mice with humanized IL-6 and IL-6R genes.
shows turpentine-dependent acute phase se (8AA) in wild-type
mice the absence or presence of anti-mouse IL-6R antibody (left); and turpentine-
dependent acute phase response in humanized L-6R mice in the absence or
present of anti-human IL-6R antibody (right).
shows FACS analysis for splenic B cells of wild-type and humanized
IL-6 mice; pan B cell marker.
shows FACS analysis for splenic T cells of wild-type an humanized IL-
6 mice; T helper cells and cytotoxic T cells.
shows FACS analysis for splenic cells of wild-type and humanized IL-6
mice; Ly6G/C(Gr1).
shows FACS analysis for splenic cells of wild-type and humanized IL-6
mice; NK cells and granulocytes (Ly6Ghi+/CD11bhi+).
shows FACS analysis for blood B cells of ype and humanized IL-
6 mice; pan B cell marker.
shows FACS analysis for blood T cells of wild-type and humanized IL-
6 mice; T helper cells and cytotoxic T cells.
shows FACS analysis for blood myeloid cells of wild-type and
humanized IL-6 mice; Gr1+ cells.
shows FACS analysis for blood myeloid cells of wild-type and
humanized IL-6 mice; CD11b vs. Ly6G/C(Gr1).
shows FACS analysis for blood d cells of ype and
humanized IL-6 mice; DX5 vs CD11b cells.
shows FACS analysis of bone marrow IgM/CD24/B220 for wild-type
and humanized IL-6 mice. Top: normal progression in bone marrow. Bottom: FACS
analysis for wild-type, hlL-6 heterozygotes, and hlL-6 homozygotes (IgM staining).
shows FACS analysis of bone marrow IgM/CD24/B220 for ype
and humanized IL-6 mice. Top: normal progression in bone marrow. : FACS
analysis for wild-type, hlL-6 heterozygotes, and hlL-6 gotes (CD24 staining).
shows FACS analysis of bone marrow CD43 and B220 for wild-type
and humanized IL-6 mice. Top: normal progression in bone marrow. Bottom: FACS
analysis for wild-type, hlL-6 heterozygotes, and hlL-6 homozygotes (CD43 staining).
DETAILED DESCRIPTION
lL-6 and lL-6R
The |L-6 receptor (IL-6R) was long characterized as a receptor for a B cell
stimulatory factor (BSF-2, or B cell Stimulatory Factor 2; also, BCDF, or B Cell
entiation Factor) responsible for inducing B cells to size immunoglobulin
(Yamasaki et al. (1988) Cloning and Expression of the Human Interleukin-6(BSF-
2/lFNB 2) or, Science 241 :825-828). lL-6 was first described as interferon-[52
as the result of its discovery during a search for a virally-induced protein termed
interfereon-B, by ng human fibroblasts with dsRNA poly(|)poly(C) to induce an
iral response enbach et al. (1980) Two interferon mRNAs in human
fibroblasts: In vitro translation and Escherichia coli cloning s, Proc. Natl Acad.
Sci. USA 77(12):?152-7156; Keller et al. (1996) Molecular and Cellular Biology of
Interleukin-6 and Its Receptor, Frontiers in Bioscience 1:d340-357).
The human cDNA encodes a 468 amino acid protein having a 19-mer signal
sequence and a cytoplasmic domain of about 82 amino acids that lacks a tyrosine
kinase domain (see, Id.). The N-terminal (ectodomain) of the protein has an lg
superfamily domain of about 90 amino acids, a 250-amino acid domain between the
Ig superfamily domain and the membrane, a transmembrane span of about 28 amino
acids (see, Id.). The ectodomain of the receptor binds its ligand lL-6, which triggers
association with gp130 in the membrane and it is this complex that ts signal
transduction; the cytoplasmic domain edly does not transduce signal (Taga et
al. (1989) Interleukin-6 Triggers the Association of Its Receptor with a le
Signal Transducer, gp130, Cell 58:573-581). Indeed, a soluble form of lL-6R lacking
a cytoplasmic domain can associate with |L-6 and bind gp130 on the surface of a cell
and effectively transduce signal (Id.).
The homology of hlL-6R and mlL-6R at the protein level is only about 54%;
the transmembrane domain has a homology of about 79%, whereas the cytoplasmic
domain has a homology of about 54% (Sugito et al. (1990)).
The l ligand for the |L-6R, |L-6, was first isolated from cultures of HTLV-
1-transformed T cells (see, Hirano et al. (1985) Purification to homogeneity and
characterization of human B cell entiation factor (BCDF or BSFp-2), Proc. Natl.
Acad. Sci. USA 82:5490-5494). A human cDNA for the |L-6 gene was cloned at
least twice, once as BSF-2 (see, Hirano et al. (1086) Complementary DNA fro a
novel human interleukin (BSF-2) that induces B lymphocytes to produce
immunoglobulin, Nature 324:73-76) and once as lFNB 2 (see, Zilberstein et al. (1986)
Structure and expression of cDNA and genes for human interferon-beta-2, a distinct
species inducible by growth-stimulatory cytokines, EMBO 5:2529-2537), gh it
has since been demonstrated that inant human lL-6 exhibits no detectable
IFN activity.
Human lL-6 is a 184-amino acid n that exhibits only about 42%
homology with mouse lL-6, although the c organization of the human and
mouse genes are basically the same, and the er regions of the human and
mouse genes share a 400-bp stretch that is highly conserved (see, Tanabe et al.
(1988) Genomic Structure of the Murine lL-6 Gene: High Degree Conservation of
Potential Regulatory Sequences between Mouse and Human, J. l.
141(11):3875-3881).
The human lL-6 gene is about 5 kb (Yasukawa et al. (1987) Structure and
expression of human B cell stimulatory factor-2 (BSC-2/lL-6) gene, EMBO J.
6(10):2939-2945), whereas the mouse lL-6 gene is about 7 kb (Tanabe et al. (1988)
Genomic Structure of the Murine lL-6 Gene: High Degree Conservation of Potential
Regulatory Sequences between Mouse and Human, J. lmmunol. 141(11):3875-
3881). The mouse and human lL-6 genes reportedly share highly ved 5'-
flanking sequence important to regulation. A schematic diagram of the human and
mouse lL-6 genomic loci is shown in (not to scale). Exons I, II, III, IV, and V
(in both human and mouse) are indicated by closed boxes to the right in the figure.
Selected putative regulatory regions are ted by open boxes to the left in the
figure. The putative regulatory regions for humans are, from left to right, a
glucocorticoid element from -557 to -552; an IFN enhancer core sequence from -472
to -468; a glucocorticoid element from -466 to -461; an AT-rich region from -395 to -
334, a sus AP-1 binding site from -383 to -277; an IFN enhancer core
sequence from -253 to -248; a GGAAA-containing motif from -205 to -192; a c-fos
SRE homology sequence from -169 to -82 containing an IFN enhancer core
sequence, a cAMP-response t, a GGAAA motif, a CCAAT box, and a GC-rich
region; and AP-1 g site from -61 to -55; and a CCAAT box from -34 to -30. The
putative regulatory regions for mouse are, from left to right, a GC rich region from -
553 to -536, a glucocorticoid element from -521 to -516 and from -500 to -495; a Z-
DNA stretch from -447 to -396; an AP-1 binding site overlapping an IFN enhancer
core sequence from -277 to -288, a GGAAA motif overlapping an IFN enhancer core
sequence from -210 to -195; a c-fos SRE homology region from -171 to -82
containing a cAMP se element, a GGAAA motif overlapping an IFN enhancer
core sequence, and a GC-rich region; and, an AP-1 binding site from -61 to -55.
Mouse codons l-V have lengths 19, 185, 114, 150, and 165, respectively. Mouse
intron lengths are: l-ll, 162 bp; ll-lll, 1253 bp; lll-IV, 2981 bp; |V-V, 1281 bp. Human
codons l-V have s 19, 191, 114, 147, and 165. Human intron lengths are l-ll,
154; ll-lll, 1047; lll-lV, 706; lV-V, 1737. Genomic organization data are from Tanabe
et al. (1988), and wa et al. (1987) Structure and expression of human B cell
stimulatory factor-2 (BSF-2/lL-6) gene, EMBO J. 9(10):2939-2945.
It might be reasonable to presume that the mouse and human |L-6 genes
appear to be similarly regulated based on the similarity of their 5'-flanking sequence.
A y cell types exhibit enhanced |L-6 expression in response to |L-1, TNF,
PDGF, IFNB, serum, poly(l)poly(C), and cycloheximide (see, Tanabe etal. (1988).
lL-6 in humans mediates the acute phase se, hematopoiesis, B cell
differentiation, T cell activation, growth and/or differentiation and/or activation of a
variety of cell types (e.g., hepatocytes, fibroblasts, endothelial cells, neurons,
pituitary cells, mas, myelomas, breast carcinomas, NK cells, macrophages,
osteoclasts, etc.) (reviewed in, e.g., Heinrich et al. (1990), Kishimoto et al. (1989),
and Keller et al. (1996); Sugita et al. (1990) Functional Murine Interleukin Receptor
with lntracisternal A le Gene Product at its Cytoplasmic Domain, J. Exp. Med.
171:2001-2009).
In practice, however, mice transgenic for human lL-6 exhibit a panoply of
substantial and debilitating pathologies, reflecting a significant pleiotropy of the |L-6
gene. Transgenic mice comprising a 6.6-kb fragment containing the human |L-6
gene and a p enhancer (Eu) produce high concentrations of hlL-6 and extremely
high lgG1 levels (120- to 400-fold over wild-type mice), reflecting an |L-6
deregulation that is accompanied by plasmacytosis, mesangio-proliferative
glomerulonephritis, and high bone marrow megakaryocyte levels (Suematsu et al.
(1989) lgG1 plasmacytosis in interleukin 6 transgenic mice, Proc. Natl Acad. Sci.
USA 86:7547-7551). Aberrant regulation of |L-6 and/or |L-6R is associated with
as, plastocytomas, rheumatoid arthritis, Castleman's disease, mesangial
proliferative glomerulonephritis, cardiac myxoma, plams cell sias, sis,
and other disorders (see, Kishimoto, T. (1989) The Biology of eukin-6, Blood
74(1):1-10; Sugita et al. ; also, Hirano et al. (1990) Biological and clinical
aspects of interleukin 6, Immunology Today 11(12):443-449)). |L-6 is also implicated
in sustaining levels of intra-prostatic androgens during androgen deprivation
treatment of te cancer patients by a paracrine and/or autocrine mechanism,
potentially providing tion-resistant prostate tumor growth (Chun et al. (2009)
Interleukin-6 Regulates Androgen Synthesis in Prostate Cancer Cells, Clin. Cancer
Res. 15:4815-4822).
The human protein is d as a 212 amino acid n, in mature form a
184 amino acid protein following cleavage of a 28 amino acid signal sequence. It
contains two N-glycosylation and two O-glycosylation sites, and human |L-6 is
phosphorylated in some cells. The mouse protein is encoded as a 211 amino acid
protein, in mature form a 187 amino acid protein following cleavage of a 23 amino
acid signal sequence. osylation sites are present, but not N-glycosylation
sites. (See reviews on lL-6, e.g., Heinrich et al. (1990) Interleukin-6 and the acute
phase response, Biochem. J. 265:621-636.)
lL-6 function is pleiotropic. The lL-6 receptor is found on activated B cells but
reportedly not on resting B cells. In contrast, lL-6R is found on resting T cells and
can reportedly promote T cell differentiation, activation, and proliferation, including
the differentiation of T cells into cytotoxic T lymphocytes in the presence of |L-2.
Humanized lL-6/lL-6R Ectodomain Mice and lLMediated Acute Phase
Response
In humans, lL-6 induces the acute phase response. Early studies with human
hepatocytes established that lL-6 induces acute phase proteins such as, e.g., C-
reactive protein (CRP) and serum amyloid A (SAA) in a dose-dependent and time-
dependent manner (reviewed in ch et al. (1990) Interleukin-6 and the acute
phase se, m. J. 265:621-636). Non-human animals, e.g., mice or rats,
comprising humanized lL-6 and lL-6R genes are therefore useful systems for
measuring the acute phase response mediated by human lL-6. Such animals are
also useful for determining whether a nce induces an lLmediated acute
phase response, by exposing a humanized L-6R animal as described herein to
the substance, and measuring a level of one or more acute phase response proteins
(or RNAs). In one embodiment, the humanized animal is exposed to the nce
in the presence of an antagonist of a human lL-6R, and a level of one or more acute
phase response proteins (or RNAs) is measured, wherein a reduction in a level of an
acute phase response protein (or RNA) in the ce of the human lL-6R
antagonist indicates a human lL-6R-mediated acute phase response.
Human lL-6 can bind both human lL-6R and mouse lL-6R; mouse lL-6 binds
mouse lL-6R but not human lL-6R (no binding of mlL-6 to hlL-6R able,
whereas hlL-6 can compete with mlL-6 for binding mlL-6R; Coulie et al. (1989) High-
and low-affinity receptors for murine eukin 6. Distinct distribution on B and T
cells, Eur. J. l. 19:2107-211); see also, e.g., Peters etal. (1996) The
Function of the Soluble Interleukin 6 (IL-6) Receptor In Vivo: Sensitization of Human
Soluble IL-6 Receptor Transgenic Mice Towards IL-6 and Prolongation of the Plasma
Half-life of IL-6, J. Exp. Med. 183:1399-1406). Thus, human cells that bear hlL-6R in
a mouse (e.g., in a xenogenic transplant) cannot rely on endogenous mlL-6 to carry
out ILmediated functions, including but not limited to the role of IL-6 blood cell or
lymphocyte development (e.g., hematopoiesis, B cell activation, T cell activation,
etc.)
In a mixed in vivo system comprising a wild-type mouse IL-6 gene and a
human IL-6R gene (but no mouse IL-6R gene), an acute phase response inducer is
not expected to induce detectable levels of acute phase proteins that would indicate
an acute phase response. However, a humanized mouse as described herein,
comprising a humanized IL-6 gene and an IL-6R gene comprising a humanized
ectodomain sequence will d to an acute phase se inducer and exhibit
acute phase response proteins in serum. Mice wild-type for IL-6/IL-6R tested for
acute phase proteins in the ce or absence of the acute phase inducer
turpentine showed a turpentine-dependent increase in acute phase ns. Mice
with humanized IL-6 gene, but not IL-6R, showed no acute phase se in the
presence of tine. But mice bearing both a human IL-6 gene and an IL-6R
gene with a zed ectodomain exhibited a strong acute phase response (. The ILmediated acute phase response was IL-6 dependent in both wild-type
mice ( top) and in humanized IL-6/IL-6R ectodomain mice ( bottom), as
evidenced by the ability of the appropriate anti-IL-6R antibody to abrogate the acute
phase response at a iently high antibody dose. Thus, a double humanization of
IL-6 and IL-6R recapitulates the wild-type ILmediated acute phase response with
respect to serum acute phase proteins.
Genetically Modified Mice
Genetically modified mice are provided that s a human IL-6 and/or a
humanized IL-6 orfrom endogenous mouse loci, wherein the endogenous
mouse IL-6 gene and/or the endogenous mouse IL-6 receptor gene have been
replaced with a human IL-6 gene and/or a human sequence comprising a sequence
that encodes an ectodomain of a human IL-6 receptor. The genetically modified
mice express the human IL-6 and/or humanized IL-6 receptor from humanized
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 IL-6 and a zed IL-6 receptor in a manner that
does not result in the y of substantial ogies observed in IL-6 transgenic
mice known in the art.
enic mice that express human lL-6 are known in the art. However,
they generally suffer from icant pathologies that severely limit their usefulness.
Humanized mice as described herein express a human lL-6 and/or zed lL-6
receptor under the control of endogenous mouse regulatory elements at endogenous
mouse lL-6 and lL-6Rq loci. These mice, in contrast, exhibit sion ns
with respect to these genes that are different from transgenic mice known in the art.
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 nous 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 ably
ates 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 onal assay. Inclusion in the
human transgene of upstream and/or downstream human sequences are apparently
presumed to be sufficient to provide le support for expression and/or regulation
of the transgene no matter where in the animal's genome the transgene winds up.
But in many cases the transgene with human regulatory ts expresses in a
manner that is unphysiological or othenNise unsatisfactory, and can be ly
detrimental to the . In contrast, the inventors demonstrate 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 and t of the humanized
animal's physiology.
ized mouse eggs injected with a construct having the MHC class I
promoter H2 and a B-globin intron driving expression of a 695-bp mouse lL-6 gene
reportedly produce mice that constitutively express mouse lL-6 at relatively high
levels (as compared with wild-type mice) (see, Woodrofe et al. (1992) Long-Term
Consequences of Interleukin-6 pression in Transgenic Mice, DNA and Cell
Biology 11(8):587-592). But these mice are prone to develop lymphomas associated
with the intestines, lymph nodes, and kidney, as well as splenic amyloid deposits.
They also exhibit abnormal B cell maturation (see, Woodrofe et al., ld.), so that
studies of B cell function are compromised. In contrast, mice as described herein
that comprise a replacement of the mouse lL-6 gene with a human lL-6 gene at the
mouse lL-6 locus are not prone to develop these lymphomas, and the mice exhibit
apparently normal B cell populations.
Mice (C57BL/6) transgenic for hlL-6 due to a random insertion of a 6.6-kb
(BamHl-Pvu ll nt) length of human DNA containing the hlL-6 gene coupled
with an lgM enhancer have been reported (see, Suematsu et al. (1989) lgG1
plasmocytosis in interleukin 6 transgenic mice, Proc. Natl. Acad. Sci. USA 86:7547-
7551). The mice express hlL-6 at n 800 pg/mL and 20,000 pg/mL in serum,
where wild-type mice typically express only about 100 pg/mL lL-6. The mice exhibit
an increase in serum lg (120 to 400-fold over wild-type mice) and a decrease in
albumin as they age. The mice suffer from a massive plasmacytosis, exhibit
splenomegaly and lymph node enlargement, as well as exhibiting plasma cells and
increased megakaryocytes in bone marrow. Upon inspection, what appear to be
enlarged lymph nodes are instead massed of compacted abnormal plasma cells.
Both spleen and thymus exhibit e proliferation of plasma cells, which also
infiltrate portions of the lung, liver, and kidney. Kidney in these mice also exhibits |L-
6—stimulated mesangial cell proliferation typical of mesangio-proliferative
glomerulonephritis. Similarly, mice (BALB/c) transgenic for a trimmed hlL-6 cDNA
driven by a mouse H-2Ld promoter randomly inserted into the genome display severe
cytosis (see, su et al. (1992) Generation of plasmacytomas with the
chromosomal translocation t(12;15) in interleukin 6 transgenic mice, Proc. Natl.
Acad. Sci. USA 89:232-235). gh C57BL/6 mice that overexpress hlL-6 do not
develop transplantable plasmacytomas (they do exhibit plasmacytosis), transgenic
BL/6 mice back-crossed into BALB/c mice reportedly do.
Random transgenesis of a hlL-6 cDNA driven by a glial fibrillary acidic protein
(GFAP) gene promoter reportedly results in hlL-6 overexpression in the mouse
central s system, which also leads to icant pathologies (see, ll et
al. (1993) Neurologic disease induced in transgenic mice by cerebral overexpression
of interleukin 6, Proc. Natl. Acad. Sci. USA 90:10061-10065). These mice exhibit
extensive neuropathology and reactive astrocytosis resulting from lL-6 expression in
the CNS due to loss of control as the result of random integration of an lL-6
ene at an ntly CNS-permissive transcriptional locus. Although
expression of hlL-6 cDNA linked to a B-globin 3’-UTR and driven by a neuron-specific
enolase promoter njected into fertilized mouse eggs (F1 C57BL/6 x BALB/c)
produced mice with a normal an and without apparent neurological defects that
expressed hlL-6 in neurons but not elsewhere (see, Fattor et al. (1994) lL-6
Expression in Neurons of Transgenic Mice Causes Reactive Astrocytosis and
se in Ramified lial Cells But No Neuronal Damage, Eur. J. Neuroscience
7:2441-2449), the mice exhibited high levels (20- to 30-fold higher than wild-type) of
activated and enlarged astrocytes with increased processes throughout the brain, as
well as a 10- to 15-fold se in ramified microglial cells in white matter. Thus,
brain expression of IL-6 edly leads to conditions that range from reactive
astrocytosis to frank and profound neuropathology.
Microinjection into fertilized eggs of an F1 cross of C57BL/6x”DBAII” mice of
a 639-bp hlL-6 cDNA linked to a B-globin 3’-UTR and a mouse MT-1 promoter
reportedly produced a transgenic mouse in which the hlL-6 gene was randomly
integrated produced a weakened and diseased mouse that dies young of kidney
e (see Fattori et al. (1994) Blood, Development of Progressive Kidney Damage
and Myeloma Kidney in Interleukin-6 Transgenic Mice, Blood 63(9):2570-2579).
Transgenic mice expired at 12-20 weeks and exhibited elevated levels of a1 and [5-
globulins in plasma, hypergammaglobulinemia, ed megakaryocytes in spleen
(3-fold higher than wild-type) and bone marrow, plasmacytosis of lymphoid organs
(spleen, thymus, and lymph nodes) characterized by abnormal and tly
arranged plasmocytoid cells, and glomerulonephritis leading to glomerulosclerosis
similar to multiple myeloma.
Microinjection into fertilized eggs of a C57BL/6J mouse of a H-2Ld-driven hlL-
6 cDNA caused ILdependent muscle wasting in mice, characterized in part by a
significantly lower gastrocnemius muscle weight in transgenic mice as compared to
weight-matched controls, a difference that was ameliorated by treatment with an IL-6
nist (see, Tsujinaka et al. (1996) Interleukin 6 or Antibody Inhibits
Muscle Atrophy and Modulates Proteolytic Systems in Interleukin 6 Transgenic Mice,
J. Clin. Invest. 97(1):244-249). At 12 weeks these mice yed serum hlL-6 levels
of more than 600,000 pg/mL. The transgenic mice also had livers that weighed
about 1,242 mg, as compared to control livers that were about 862 mg. enic
mice treated with IL-6 antagonist had livers that weighed about 888 mg. Muscle
cathepsins B and B+L were icantly higher (20-fold and 62-fold) in transgenic
mice than in controls, a phenomenon that was eliminated in transgenic mice treated
with an IL-6 antagonist. cathepsin B and L mRNAs were estimated to be about
277% and 257%, respectively, as compared with wild-type mice; the difference was
significantly reduced with IL-6 antagonist treatment.
Mice sing a hlL-6 minigene driven by a mouse MHC class I H-
2Ld promoter and a hlL-6R minigene driven by a chicken B-actin promoter, and a
gp130 gene, exhibited pathologies typical of hlL-6 transgenic mice (e.g.,
hepergammaglobulinemia, splenomegaly, mesangial proliferative ulonephritis,
lung lymphoid infiltration) as well as ventricular hypertrophy (Hirota et al. (1995)
Continuous activation of gp130, a signal-transducing receptor component for
interleukin 6—related cytokines, causes myocardial hypertrophy in mice, Proc. Natl
Acad. Sci. USA 92:4862—4866). The ventricular rophy is believed to be
mediated by a continuous activation of gp130 (Id.). The role of lL-6 is reportedly to
help strengthen the cytokine receptor complex and induce dimerization of gp130,
which is the signal transducing component responsible for transducing the lL-6 signal
(Paonessa et al. (1995) Two distinct and independent sites on lL-6 trigger gp130
dimer formation and signalling, EMBO J. 14(9):1942—1951). The activated complex
is believed to be a hexamer composed of two lL-6, each lL-6 bound to one lL-6Ra
and two gp130 (each lL-6 contains two independent binding sites) exhibiting a
2:2:2 iometry, wherein the dimerization of gp130 causes activation of JAK—Tyk
ne kinases, phosphorylation of gp130 and STAT family transcription factors and
other intracellular substrates (Id.; Stahl, N. (1994) Association and Activation of Jak-
Tyk Kinases by CNTF-LlF-OSM-lL-6 [5 Receptor Components, Science 263:92—95),
consistent with a general model of cytokine receptor complex formation (see, Stahl,
N. and Yancopoulos, G. (1993) The Alphas, Betas, and Kinases of Cytokine
Receptor Complexes, Cell 74:587-590; Davis et al. (1993) LIFRB and gp130 as
Heterodimerizing Signal Transducers of the Tripartite CNTF Receptor, Science
260:1805-1808; mi et al. (1993) lLlnduced Homodimerization ofgp130 and
Associated Activation of a Tyrosine Kinase, e 2601808—1810).
Mice transgenic for human le-6R driven by a rat PEP ykinase
er and human lL-6 driven by a mouse metallothionein-1 promoter are
reportedly markedly smaller that mice transgenic for human lL-6 alone or human le-
6R alone (Peters et al. (1997) Extramedullary Expansion of poietic Progenitor
Cells in |nterleukin(|L-)—6—le-6R Double Transgenic Mice, J. Exp. Med. :?55-
766), reflected in d body fat and reduced weight (20-25 g vs. 40 g). Double
transgenic mice reportedly also exhibit spleen (5-fold) and liver (2—fold) ement
as compared with reportedly normal organ weights for single transgenic mice,
apparently due to extramedullary proliferation of hematopoeitic cells of spleena and
liver but not bone marrow, as well as elevated megakaryocytes in spleen and
plasmacellular infiltrates in all parenchymal organs (Id.). Double transgenics also
exhibit livers with an increase of about 200- to about 300-fold in granulocytes,
macrophages, itor cells, and B cells as compared with single transgenics; in
contrast, lL-6 single transgenic mice ted lesser increases in macrophages (15-
fold) and B cells (45-fold) (Id.). The extraordinary findings are presumably due to
stimulation of growth and differentiation of hematopoietic progenitor cells by
activating gp130 signal transduction (Id.).
Further, double transgenic (mouse metallothionine er-driven
hlL-6/rat PEP carboxykinase promoter-driven hlL-6R) mice t a hepatocellular
hyperplasia that is reportedly identical to human nodular regenerative lasia
with sustained hepatocyte proliferation that strongly suggests that |L-6 is responsible
for both hepatocyte proliferation and pathogenic hepatocellular transformation
(Maione et al. (1998) Coexrpession of |L-6 and soluble lL-6R causes nodular
regenerative lasia and adenomas of the liver, EMBO J. 17(19):5588—5597).
Because hepatocellular hyperplasia is edly not observed in single transgenic
hlL-6 mice and hlL-6 can bind mlL-6R, the finding may appear paradoxical until it is
considered that the double transgenic may result in higher levels of hlL-6 complexed
to soluble |L-6R (here, soluble hlL-6R), which complex is a more potent inhibitor that
|L-6 alone (Id.).
In st to mice that are transgenic for human |L-6, humanized |L-6
mice that comprise a replacement at an endogenous mouse |L-6 locus, which retain
mouse regulatory elements but comprise a humanization of |Lencoding sequence,
do not exhibit the severe pathologies of prior art mice. Genetically modified mice that
were heterozygous or homozygous for hlL-6 were grossly .
Mice with a humanized lL-6 gene (MAID 760) as described in the
Examples were immunophenotyped and found to have normal B cell s in
FACS analyses (lymphocyte-gated) of spleen B cells using a pan B cell marker
(CD445R(B220)) (. For spleen, wild-type mice exhibited 63% B cells; hlL-6
zygote mice exhibited 63% B cells; and mice homozygous for hlL-6 at the
endogeous mouse locus exhibited 63% B cells. B cell numbers for gous hlL-
6 mice immunized with TNP-KLH were also normal (65% for wild-type, and 61% for
hlL-6 homozygotes).
Splenic T cells were also about the same as wild-type (.
Percentages of c T cells for Thelper/Tcytoxic were, for wild-type %
(ratio of 1.4:1); for hlL-6 heterozygotes 23%/14% (ratio of 16:1); for hlL-6
homozygotes 21%/15% (ratio of 1.4:1) (markers were CD8a-APC; CD4-FITC).
Homozygous hlL-6 mice zed with TNP-KLH exhibited similar splenic T cell
numbers to wild-type mice, i.e., Thelper/Tcytotoxic were 22%/20% (ratio of 1.1:1) as
compared with 21 %/19% for wild-type (also a ratio of 1.1 :1 ).
Humanized |L-6 mice also exhibited about normal levels of splenic NK
cells on FACS analysis (CD11b and DX5) (. hlL-6 heterozygotes exhibited
2.2% NK cells, and hlL-6 homozygotes ted 1.8% NK cells, whereas wild-type
mice exhibited 2.4% NK cells. ing immunization with TN P-KLH, homozygotes
exhibited 1.6% splenic NK cells, whereas wild-type mice exhibited 2.1% splenic NK
cells.
Humanized |L-6 mice also exhibited normal levels of splenic
Ly6G/C(Gr1) cells (. hlL-6 heterozygotes exhibited 7.0% GR1+ cells (1.3%
Gr1hi); homozygotes exhibited 6.8% Gr1+ cells (0.9% Gr1hi), whereas ype mice
ted 8.0% Gr1+ cells (1 .8%Gr1hi). Immunized |L-6 homozygotes (immunized
with TNP-KLH) exhibited 11% Gr1+ cells (4.0% Gr1hi), whereas wild-type mice
exhibited 10% Gr1+ cells (30% GM“).
] Humanized |L-6 mice also exhibited normal blood B and T cell
numbers in FACS analysis (and . FACs with a pan B cell marker
R(B220)) revealed that homozygous hlL-6 mice ed 52% B cell as
compared with wild-type 53%; heterozygotes exhibited 38% (an average of two
different stainings of 29% and 47%). Homozygous hlL-6 mice immunized with TNP-
KLH gave similar B cell numbers (43%, as compared with 45% for wild-type mice).
Humanized |L-6 mice ted normal blood T cell numbers in FACS
analysis as measured by CD8a and CD4 staining. Heterozygous hlL-6 mice
exhibited Thelper/Tcytotoxic numbers of 39%/26% (ratio of ; homozygous hlL-
6 mice exhibited Th/Tc numbers of 24%/20% (ratio of , whereas wild-type mice
exhibited Th/Tc numbers of 26%/20% (ratio of 1.3:1). Homozygous hlL-6 mice
immunized with TNP-KLH had Th/Tc numbers of 29%/21% (ratio of 1.4:1), whereas
wild-type immunized mice had Th/Tc numbers of 28%/23% (1 .2:1).
zed lL-6 mice also exhibited myeloid cell numbers in blood
that were similar to wild-type mice as measured by FACS is of naive and
immunized mouse blood stained with Ly6G/C(Gr1) and CD11b, as well as CD11b
and DX5 (, , and FIG 12). Heterozygous hlL-6 mice exhibited %Gr+
cells of 10.8%, homozygotes 6.9%, whereas wild-type mice exhibited 9.7%.
Immunized hlL-6 homozygotes exhibited M1(Ly6G/C(Gr) of 101-104)/
G/C(Gr) staining of about 102-103) numbers of 43%/34%, whereas wild-type
mice had numbers of 45%/38%. FACS plots of CD11b (vertical axis) vs. Ly6G/C
(horizontal axis) for immunized homozygous hlL-6 mice showed cell percentage in
nts (upper left/upper right/lower right) of 16%/8%/3%, which were identical to
immunized wild-type quadrant numbers.
] Homozygous H-immunized humanized IL-6 mice exhibited
CD11b vs. DX5(NK) staining FACS plots that were similar to zed wild-type
mice. Quadrant is blood FACS plots (CD11b vertical axis, DX5(NK) horizontal
axis) revealed upper left/upper right/lower right numbers of 9.5%/17%/10% for hlL-6
homozygotes and 6.5%/17.3%/14% for wild-type mice.
Humanized IL-6 mice ted an isotype response that was
essentially the same as observed in wild-type mice. Early and final lgG1, IgG2a,
IgG2b, IgG3, lgA, IgE, and IgM levels were about the same as observed in wild-type
mice. In one experiment, final IgM was slightly higher in humanized mice; final lgG3
was also elevated in humanized mice.
B cell development in naive hlL-6 mice was essentially
indistinguishable from development in wild-type mice based on FACS analysis of
bone marrow lgM/CD24/B220 staining (). phenotyping of immune
mice revealed that marker populations for various cell types in the B cell
development progression were essentially normal in hlL-6 mice. Progression of cells
from hematopoietic stem cells, common lymphoid itors, ProB cells, PreB cells,
and immature and mature B cells is normal in hlL-6 mice ( and )
EXAMPLES
e 1: Replacement of Endogenous Mouse lL-6 Gene with hlL-6 Gene
The 4.8-kb human IL-6 gene containing exons 1 through 4 of the
human IL-6 gene ed 6.8 kb of the murine IL-6 gene locus.
A targeting construct for replacing the mouse with the human IL-6
gene in a single targeting step was constructed using VELOCIGENE® genetic
engineering technology (see, Valenzuela et al. (2003) High-throughput engineering
of the mouse genome coupled with high-resolution expression analysis, Nature
Biotech, 21(6):652-659). Mouse and human IL-6 DNA were obtained from bacterial
artificial chromosome (BAC) RPCI-23 clone 368C3, and from BAC CTD clone
2369M23, tively. Briefly, a Notl linearized targeting construct ted by
gap repair cloning containing mouse IL-6 am and downstream homology arms
flanking a 4.8 kb human IL-6 sequence extending from ATG in exon 1 to exon 5 with
16 nucleotides of 3’ downstream sequence (genomic coordinates: NCBIh37.1:
ch7:22,766,882 to 22,771,637) and a floxed neo selection cassette, was
electroporated into F1 H4 mouse embryonic stem (ES) cells (C57BL/6 x 129 F1
hybrid). Correctly targeted ES cells (MAID 790) were further electroporated with a
transient Cre-expressing vector to remove the drug selection cassette. Targeted ES
cell clones t drug cassette (MAID 1428) were introduced into an 8-cell stage
mouse embryo by the VELOCIMOUSE® method (see, US Pat. No. 7,294,754,
7,576,259, 7,659,442, and irou et al. (2007) F0 generation mice that are
essentially fully derived from the donor gene-targeted ES cells allowing immediate
phenotypic analyses Nature Biotech. 91-99). VELOCIMICE® (F0 mice fully
derived from the donor ES cell) bearing the zed IL-6 gene were identified by
genotyping for loss of mouse allele and gain of human allele using a modification of
allele assay (see, Valenzuela et al. (2003)).
Correctly targeted ES cell clones were identified by a Ioss-of—native-
allele (LONA) assay (Valenzuela et al. 2003) in which the number of copies of the
native, unmodified [[6 gene were determined by two TaqManTM quantitative
polymerase chain reactions ) ic for sequences in the mouse ”6 gene
that were targeted for deletion. The qPCR assays comprised the following primer-
probe sets (written 5’ to 3’): upstream fonNard primer, TTGCCGGTTT TCCCTTTTCT
C (SEQ ID NO:1); upstream reverse primer, AGGGAAGGCC GTGGTTGTC (SEQ ID
NO:2); am probe, FAM-CCAGCATCAG TCCCAAGAAG GCAACT-BHQ (SEQ
ID NO:3); downstream forward primer, TCAGAGTGTG GGCGAACAAA G (SEQ ID
NO:4); ream reverse primer, GTGGCAAAAG CAGCCTTAGC (SEQ ID NO:5);
downstream probe, FAM-TCATTCCAGG CCCTTCTTAT TGCATCTG-BHQ (SEQ ID
NO:6); in which FAM refers to the 5-carboxyfluorescein fluorescent probe and BHQ
refers to the fluorescence quencher of the black hole quencher type (Biosearch
Technologies). DNA purified from ES cell clones that that have taken up the targeting
vector and incorporated in their s was combined with TaqManTM Gene
Expression Master Mix (Life Technologies) according to the manufacturer’s
suggestions in a 384-well PCR plate (MicroAmpTM Optical 384-Well on Plate,
Life Technologies) and cycled in an d Biosystems Prism 7900HT, which
collects fluorescence data during the course of the PCRs and determines a threshold
cycle (Ct), the fractional PCR cycle at which the accumulated fluorescence reaches a
pre-set threshold. The upstream and downstream l/6-specific qPCRs and two qPCRs
for non-targeted reference genes were run for each DNA sample. The differences in
the Ct values (ACt) between each l/6-specific qPCR and each reference gene qPCR
were calculated, and then the difference between each ACt and the median ACt for
all s assayed was calculated to obtain AACt values for each sample. The copy
number of the ”6 gene in each sample was calculated from the following formula:
copy number = 2 . 2-AACt. A correctly targeted clone, having lost one of its native
copies, will has an ”6 gene copy number equal to one. Confirmation that the human
IL6 gene sequence replaced the deleted mouse II6 gene sequence in the humanized
allele was confirmed by a TaqManTM qPCR assay that comprises the ing
primer-probe sets (written 5’ to 3’): the human d primer,
CCCCACTCCACTGGAATTTG (SEQ ID NO:7); the human reverse primer,
GTTCAACCACAGCCAGGAAAG (SEQ ID NO:8); and the human probe, FAM-
AGCTACAACTCATTGGCATCCTGGCAA-BHQ (SEQ ID NO:9).
The same LONA assay was used to assay DNA purified from tail
biopsies for mice derived from the targeted ES cells to determine their II6 genotypes
and confirm that the humanized II6 allele had transmitted through the germline. Two
pups heterozygous for the replacement are bred to generate a mouse that is
gous for the replacement of the endogenous mouse lL-6 gene by the human
IL-6 gene. Pups that are homozygous for the replacement are used for phenotyping.
The am junction of the murine locus and the sequence
containing the hlL-6 gene is designed to be within 5’-AATTAGAGAG TTGACTCCTA
ATAAATATGA GACTGGGGAT AGCT CATTCTGCTC CCAC
CAAGAACGAT AGTCAATTCC AGAAACCGCT ATGAACTCCT TCTCCACAAG
CAGG AAATCCTTAG CCCTGGAACT GGCG GTCGAGCCCT
GTGTGAGGGA GGGGTGTGTG GCCCAGG (SEQ ID NO:10), wherein the final
mouse nucleotide priorto the first nucleotide of the human gene is the “T” in CCGCT,
and the first nucleotide of the human sequence is the first “A” in ATGAA. The
downstream junction of the sequence ning the hlL-6 gene and the murine locus
is designed to be within 5’-TTTTAAAGAA ATATTTATAT TGTATTTATA
TAATGTATAA ATGGTTTTTA TACCAATAAA TGGCATTTTA AAAAATTCAG
TGAG TGTGTCACGC TCCCGGGCTC GATAACTATA ACGGTCCTAA
GGTAGCGACT CGAGATAACT T-3’ (SEQ ID NO:11), wherein the final nucleotide of
the human sequence is with the final “G” in TCACG and the first nucleotide of the
mouse sequence is the first “C” in CTCCC; the ream junction region also
contained a onP site at the 3’ end (the beginning of which is shown) for removal of a
floxed ubiquitin promoter-driven neo cassette. The junction of the neo cassette with
the mouse lL-6 locus is designed to be within 5’-TATACGAAGT TATCCTAGGT
TGGAGCTCCT AAGTTACATC CAAACATCCT CCCCCAAATC AATAATTAAG
CACTTTTTAT TAAA GTTAAATAAG AAGTGAAAGC TGCAGATGGT
GAGTGAGA (SEQ ID NO:12), where the final “C” of AGCTC is the final nucleotide of
the neo cassette; the first nucleotide of the mouse genome following the te is
the initial “C” of CTAAG.
WO 63556
Example 2: lmmunophenotyping of Naive and lmmunized hlL-6 Mice: B Cells
] Mice homozygous for the h|L-6 gene replacement were analyzed for B
cells (DC445R(B220). Lymphocyte-gated fractions from spleen cell preparations of
naive and immunized (TNP-KLH) h|L-6 mice were stained and immunophenotyped
using flow cytometry. FACS analysis showed that the percentage of B cells of the
spleen cell preparation as measured by CD45R(8220)-FITC staining were about the
same (63% of cells) for preparations from naive wild-type, hlL-6 heterozygotes, and
hlL-6 homozygotes. For immunized mice, B cells accounted for about 65% of total
cells of the spleen cell preparation in wild-type mice, and about 61% of total cells in
h|L-6 homozygotes. Spleens of h|L-6 mice (both naive and immunized) contain a
population of B cells that is about the same size as the splenic B cell population in
wild-type mice.
Bone marrow of wild-type, h|L-6 heterozygotes, and h|L-6
homozygotes was stained with B cell s (CD45R(B220)-APC, CD24(HSA)-PE,
or CD43 conjugated to a dye and/or lgM (lgM-FITC). B cell development in bone
marrow of normal mice will be ted in surface markers as cells progress from
stem cells to early pro-B cells to late pro-B cells, to large pre-B cells to small pre-B
cells to immature B cells and finally, to mature B cells. Common cyte
progenitor pro-B cells will express CD45R, and in later stages will express lgM as
immature and later as mature B cells. Thus, stained and anti-lgM-stained B
cells should reveal a pattern characteristic of B cell development. Bone marrow of
h|L-6 heterozygotes and gotes yed a pattern of CD45R(8220)-APC
and gM-FITC staining that was essentially indistinguishable from wild-type bone
marrow, showing populations of B cells that stained positive for CD45R(B220) and
lgM, or CD45R(B220) alone. B cell sub-populations in bone marrow of h|L-6 mice
revealed by FACS staining were similar to those in wild-type mice (Table 1; see also,
).
Table 1. B Cells in Bone Marrow of Naive Mice
hlL-6 Mouse
Wild-type Mouse
(%) Heterozygote Homozygote
(%) (%)
CLP-ProB
PreB-lmmatureB
Staining for CD24 (see ) ed the (normal) n shown
in Table 2, indicating normal development in bone marrow.
Table 2. B Cells in Bone Marrow of Naive Mice
hlL-6 Mouse
Wild-type Mouse_
(0/0) Heterozygote Homozygote
(%) (%)
Developing HSC-
46.6 46 43
Mature CLP/early
-2 9-0 10-1
ProB
Late ProB, PreB,
7-2 11.6 10-7
Immature B
Mature B 14.1 14.9 17
Staining for CD43 (see ) revealed the (normal) pattern shown
in Table 3, indicating normal development in bone marrow.
Table 3. B Cells in Bone Marrow of Naive Mice
hlL-6 Mouse
Wild-type Mouse
(°/o) Heterozygote Homozygote
(%) (%)
PreBll-lmmature
28.4 21.4 21.2
B cells
Mature B cells 8.1
Thus, immunophenotyping of nai've hlL-6 mice ed that B cell
development in such mice is essentially normal.
Example 3: Replacement of Endogenous Mouse lL-6Ra Ectodomain Gene
ce with hlL-6Rq main Gene Sequence
The 45 kb human lL-6Ra gene containing exons 1 through 8 of the
human lL-6Ra gene replaced 35.4 kb of the murine lL-6Rq gene locus. Mouse
exons 9 and 10 were retained; only exons 1-8 were humanized. In total, 35,384 nt of
mouse sequence was ed by 45,047 nt of human sequence.
A targeting construct for replacing the mouse with the human lL-6Rd
gene in a single targeting step was constructed using VELOCIGENE® genetic
engineering technology (see, Valenzuela et al. (2003) High-throughput engineering
of the mouse genome coupled with high-resolution expression analysis, Nature
Biotech, 21(6):652-659). Mouse and human lL-6Rq DNA were obtained from
bacterial artificial chromosome (BAC) RPCl-23 clone 125J8, and from BAC CTD
clone 2192J23, respectively. Briefly, a Notl linearized targeting construct generated
by gap repair cloning containing mouse lL-6Rq upstream and downstream homology
arms flanking a 45 kb human lL-6Rq sequence extending from ATG in exon 1 to
exon 8 with 69 nucleotides of 3’ downstream ce and a floxed neo selection
cassette, was electroporated into F1H4 mouse embryonic stem (ES) cells (C57BL/6
x 129 F1 hybrid). Correctly targeted ES cells (MAID 794) were further electroporated
with a transient Cre-expressing vector to remove the drug selection cassette.
Targeted ES cell clones without drug cassette (MAID 1442) were introduced into an
8-cell stage mouse embryo by the VELOCIMOUSE® method (see, US Pat. No.
7,294,754, 7,576,259, 7,659,442, and irou et al. (2007) F0 generation mice
that are essentially fully derived from the donor gene-targeted ES cells allowing
immediate ypic analyses Nature Biotech. 25(1):91-99). VELOCIMICE® (F0
mice fully derived from the donor ES cell) bearing the humanized lL-6Rq gene were
identified by genotyping for loss of mouse allele and gain of human allele using a
modification of allele assay (see, Valenzuela et al. (2003)).
Correctly targeted ES cell clones were identified by a Ioss-of—native-
allele (LONA) assay (Valenzuela et al. 2003) in which the number of copies of the
native, fied ”6 gene were determined by two TaqManTM quantitative
polymerase chain reactions (qPCRs) specific for ces in the mouse ”6 gene
that were targeted for deletion. The qPCR assays comprised the following primer-
probe sets en 5’ to 3’): upstream forward primer, GCCCTAGCAT GCAGAATGC
(SEQ ID ; upstream reverse , AAGAGGTCCC TTTG C (SEQ
ID NO:14); upstream probe, CCCACATCCA TCCT GTGAG (SEQ ID
NO:15); downstream forward primer, GAGCTTGCCC CCAGAAAGG (SEQ ID
NO:16); downstream reverse primer, CGGCCACATC TCTGGAAGAC (SEQ ID
NO:17); downstream probe, CATGCACTGC CCCAAGTCTG GTTTCAGT (SEQ ID
NO:18). DNA purified from ES cell clones that that have taken up the targeting vector
and incorporated in their genomes was ed with TaqManTM Gene Expression
Master Mix (Life Technologies) according to the cturer’s suggestions in a
384-well PCR plate (MicroAmpTM Optical 384-Well Reaction Plate, Life
Technologies) and cycled in an d Biosystems Prism 7900HT, which collects
fluorescence data during the course of the PCRs and determines a threshold cycle
(Ct), the fractional PCR cycle at which the accumulated fluorescence reaches a pre-
set threshold. The upstream and ream lL-6Rq-specific qPCRs and two
qPCRs for non-targeted reference genes were run for each DNA sample. The
differences in the Ct values (ACt) between each IL-6Rq-specific qPCR and each
reference gene qPCR were ated, and then the difference between each ACt
and the median ACt for all samples assayed was calculated to obtain AACt values for
each sample. The copy number of the ”6 gene in each sample was calculated from
the following formula: copy number = 2 - . A correctly targeted clone, having
lost one of its native copies, will have an IL-6Rq gene copy number equal to one.
Confirmation that the human IL-6Ra gene sequence replaced the deleted mouse IL-
6Rq gene sequence in the humanized allele was med by a TaqManTM qPCR
assay that comprises the ing primer-probe sets (written 5’ to 3’): the human
fonNard primer, GGAGAGGGCA GAGGCACTTA C (SEQ ID NO:19); the human
reverse primer, GGCCAGAGCC CAAGAAAAG (SEQ ID N020); and the human
probe, CCCGTTGACT GTAATCTGCC CCTGG (SEQ ID NO:21).
The same LONA assay was used to assay DNA purified from tail
es for mice derived from the targeted ES cells to determine their IL-6Ra
genotypes and confirm that the humanized IL-6Rq allele had transmitted through the
germline. Pups zygous for the replacement are bred to generate a mouse that
is homozygous for the replacement of the endogenous mouse IL-6Rq gene by the
human IL-6Ra (ectodomain) gene. Pups that are homozygous for the replacement
are used for phenotyping.
The upstream junction of the murine locus and the sequence
containing the hlL-6Rq gene is designed to be within 5’-CGAGGGCGAC
TGCTCTCGCT GCCCCAGTCT CGCC CGGCCCCGGC GCCG
CTCTGCCGCC CGCCGTCCCG CGTAGAAGGA AGCATGCTGG CCGTCGGCTG
CGCGCTGCTG GCTGCCCTGC TGGCCGCGCC GGGAGCGGCG CTGGCCCCAA
GGCGCTGCCC TGCGCAGGGT AAGGGCTTCG G (SEQ ID NO:22), n the
final mouse nucleotide prior to the first nucleotide of the human gene is the “C” in
GAAGC, and the first nucleotide of the human ce is the first “A” in ATGCT.
The downstream junction of the sequence containing the hlL-6 gene and the murine
locus is designed to be within 5’-CAAGATTATT GGAGTCTGAA TACC
TGTTGAGGGA AATCTTTATT TTGGGAGCCC TTGATTTCAA TGCTTTTGAT
TCCCTATCCC TGCAAGACCC GGGCTCGATA ACTATAACGG TCCTAAGGTA
GCGACTCGAG ATAACTTC-3’ (SEQ ID NO:23), wherein the final nucleotide of the
human sequence is with the final “A” in CAAGA and the first nucleotide of the mouse
sequence is the first “C” in CCCGG; the ream junction region also contained a
onP site at the 3’ end for removal of a floxed tin promoter-driven neo cassette.
The first nucleotide of the loxp site is the first “A” in ATAAC. The junction of the neo
te with the mouse IL-6Rα locus is designed to be within 5’-TATACGAAGT
TATCCTAGGT TGGAGCTCTA CTCCATATGC TCACTTGCCG GCTA
CGATACGGTG AGGCCCGTGC GAAGAGTGGC ACAGATCAGG AGGCTTATGT
GGTCAGTCCA CAGTATGGC (SEQ ID NO:24), where the final “C” of AGCTC is the
final nucleotide of the neo cassette; the first nucleotide of the mouse genome
following the cassette is the initial “T” of TACTC.
] Where the terms “comprise”, “comprises”, “comprised” or “comprising” are
used in this specification ding the claims) they are to be reted as specifying the
presence of the stated features, integers, steps or components, but not precluding the
presence of one or more other features, integers, steps or components, or group thereof.
The discussion of documents, acts, materials, devices, articles and the like is
included in this specification solely for the purpose of providing a context for the present
invention. It is not suggested or represented that any or all of these matters formed part of
the prior art base or were common general knowledge in the field relevant to the t
invention as it existed before the priority date of each claim of this application.
Claims (11)
1. A genetically ed murine animal comprising a replacement at an endogenous murine IL-6 locus of a murine gene encoding IL-6 with a human gene encoding human IL-6, 5 wherein the human gene encoding human IL-6 is under control of endogenous murine regulatory ts at the endogenous murine IL-6 locus, and wherein the murine animal is not a mouse.
2. The genetically modified murine animal of claim 1, n the human gene encoding 10 human IL-6 comprises exons 1 through 5 of the human IL-6 gene found in the CTD-2369M23 bacterial artificial chromosome.
3. The genetically ed murine animal of claim 1, wherein the murine animal expresses a humanized IL-6Rα wherein the endogenous murine IL-6Rα gene has been 15 replaced with a human sequence comprising a sequence that encodes an main of a human IL-6Rα.
4. The genetically modified murine animal of claim 1, wherein the murine animal does not exhibit a feature selected from plasmocytosis, glomerulosclerosis, ulonephritis, 20 kidney failure, hypergammaglobulinemia, elevated ryocytes in spleen, elevated megakaryocytes in bone marrow, splenomegaly, lymph node enlargement, compacted abnormal plasma cells, and a combination thereof.
5. The genetically modified murine animal of claim 3, wherein the humanized IL-6Rα 25 comprises murine transmembrane and intracellular domains.
6. A cally modified murine animal, comprising a humanization of an endogenous murine IL-6Rα gene, wherein the humanization comprises a replacement of murine IL-6Rα ectodomain-encoding sequence with human IL-6Rα ectodomain-encoding ce at the 30 endogenous murine IL-6Rα locus, and wherein the humanized IL-6Rα gene is under control of endogenous murine regulatory elements, and wherein the murine animal is not a mouse.
7. The genetically ed murine animal of claim 6, further comprising a humanized IL-6 gene comprising a replacement at an endogenous murine IL-6 locus of a murine gene 35 encoding IL-6 with a human gene encoding human IL-6.
8. A method for making a humanized murine animal, comprising replacing a murine gene sequence encoding murine IL-6 with a human gene encoding human IL-6 so that the human IL-6 gene is under control of endogenous murine regulatory elements, and wherein the murine animal is not a mouse.
9. A method for making a humanized murine animal, comprising replacing all murine exons encoding ectodomain ces of murine IL-6Rα with a human genomic fragment ng human IL-6Rα main to form a humanized IL-6Rα gene, wherein the humanized IL-6Rα gene is under control of endogenous murine regulatory elements, and 10 wherein the murine animal is not a mouse.
10. A genetically modified murine animal comprising a humanized IL-6Rα gene comprising a replacement of murine ectodomain-encoding sequence with human ectodomain ce, wherein the humanized IL-6Rα gene comprises murine transmembrane and 15 intracellular domain sequences, wherein the murine animal further comprises a gene encoding a human IL-6, and wherein the genes encoding human IL-6 and humanized IL-6Rα are under control of nous murine regulatory elements, and wherein the murine animal is not a mouse. 20
11. The genetically modified murine animal according to claim 1, 6, or 10, or the method according to claim 8 or 9, substantially as herein bed with reference to any of the Examples and/or
Applications Claiming Priority (5)
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US201161552900P | 2011-10-28 | 2011-10-28 | |
US61/552,900 | 2011-10-28 | ||
US201161556579P | 2011-11-07 | 2011-11-07 | |
US61/556,579 | 2011-11-07 | ||
NZ623145A NZ623145B2 (en) | 2011-10-28 | 2012-10-29 | Humanized il-6 and il-6 receptor |
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NZ709432A NZ709432A (en) | 2015-12-24 |
NZ709432B2 true NZ709432B2 (en) | 2016-03-30 |
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