WO2013062134A1 - Method for producing immune-system humanized mouse - Google Patents
Method for producing immune-system humanized mouse Download PDFInfo
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- WO2013062134A1 WO2013062134A1 PCT/JP2012/078267 JP2012078267W WO2013062134A1 WO 2013062134 A1 WO2013062134 A1 WO 2013062134A1 JP 2012078267 W JP2012078267 W JP 2012078267W WO 2013062134 A1 WO2013062134 A1 WO 2013062134A1
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Classifications
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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
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
-
- 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
- A01K2207/00—Modified animals
- A01K2207/12—Animals modified by administration of exogenous cells
-
- 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
-
- 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
- A01K2217/15—Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
-
- 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
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- 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
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
Definitions
- the present invention relates generally to methods for producing a
- immune-system humanized mouse and the immune-system humanized mouse capable of being produced by the methods.
- the humanized mouse system a xenogeneic transplantation and engraftment model for human hematopoietic stem cells (HSCs) and peripheral blood mononuclear cells (MNCs), facilitates the investigation of human hematopoietic and immune systems in vivo 1 ' 2 .
- HSCs human hematopoietic stem cells
- MNCs peripheral blood mononuclear cells
- the present inventors developed a new immune-compromised mouse strain that expresses human membrane bound stem cell factor (SCF) under the control of the phosphoglycerate kinase (PGK) promoter (hSCF Tg NSG).
- SCF human membrane bound stem cell factor
- PGK phosphoglycerate kinase
- hSCF Tg NSG mice by supporting efficient human myeloid development including mast cells, may serve as a novel platform for in vivo investigation of human mast cell development and allergic responses.
- the present invention is the folio wings:
- a method for producing a first immune-system humanized mouse comprising administering human hematopoietic stem cells to a first transgenic immunodeficient mouse whose genome comprises a nucleic acid encoding human Stem Cell Factor operably linked to a promoter,
- the first transgenic immunodeficient mouse expresses the human Stem Cell Factor
- a method for producing an immune-system humanized mouse comprising administering human hematopoietic stem cells to a transgenic immunodeficient mouse whose genome comprises a nucleic acid encoding human Stem Cell Factor operably linked to a promoter,
- transgenic immunodeficient mouse expresses the human Stem Cell Factor
- immune-system humanized mouse is 70% or more
- the frequency of human CD33+ myeloid cells within the total human CD45+ population in the bone marrow of the immune-system humanized mouse is 30% or more, and the frequency of human c-kit+CD203c+ mast cells within the total human CD45+CD33+ myeloid cells in the spleen of the immune-system humanized mouse is 65% or more.
- a transgenic immunodeficient mouse comprising engrafted human hematopoietic cells and human both acquired and innate immune cells differenciated from the hematopoietic cells surviving without being rejected from the mouse;
- immune-system humanized mouse is 70% or more
- the frequency of human CD33+ myeloid cells within the total human CD45+ population in the bone marrow of the immune-system humanized mouse is 30% or more, and the frequency of human c-kit+CD203c+ mast cells within the total human CD45+CD33+ myeloid cells in the spleen of the immune-system humanized mouse is 65% or more; wherein the transgenic immunodeficient mouse comprises a nucleic acid encoding human Stem Cell Factor operably linked to a promoter in the genome, and expresses the human Stem Cell Factor.
- immune/allergic/inflammatory disease relating myeloid cells and/or mast cells comprising applying a test substance to the transgenic immunodeficient mouse of (9) or portion thereof affected with the immune/allergic/inflammatory disease relating myeloid cells and/or mast cells, and evaluating whether or not the test substance improves a condition or symptom of the immune/allergic/inflammatory disease.
- Figures 1 A- IE show that human hematopoietic engraftment is enhanced in hSCF Tg NSG recipients.
- hSCF Tg NSG recipients developed progressive anemia as evidenced by reduced hemoglobin concentration compared with non-Tg NSG mice transplanted with human HSCs from the same donor source.
- B Human CD45+ chimerism was analyzed over time in PB of hSCF Tg and non-Tg NSG recipients.
- C Representative flow cytometry contour plots demonstrating the presence of human CD45+ cells, CD19+ B cells, CD33+ myeloid cells, CD3+ T cells and CD56+CD3- NK cells in recipient BM.
- Figures 2A-2G show that HLA-DR-negative human myeloid cells predominate in hSCF Tg NSG recipient BM.
- A Flow cytometry contour plots demonstrating forward- and side-scatter characteristics of 6 hSCF Tg NSG recipient BM (Sl-1, SI -2, S9-1, S4-1, SI 2-2, S2-1) and 3 non-Tg NSG recipient BM (Nl-1, Nl-2, N9-1) are shown. Polymorphonuclear myeloid cells (red asterisks) are present at high frequencies in hSCF Tg NSG recipient BM.
- B Flow cytometry contour plots demonstrating hCD33 and HLA-DR expression in the same recipients as shown in (A). Consistent with their FSC and SSC characteristics, hSCF Tg NSG recipient BM contained a prominent
- CD33+HLA-DR(-) granulocyte population (red asterisks).
- Nl-1 sacrificed at 21 weeks; Nl-2: sacrificed at 16 weeks; N9-1 : sacrificed at 20 weeks; Sl-1 : sacrificed at 23 weeks; Sl-2: sacrificed at 20 weeks; S9-1 : sacrificed at 16 weeks; S4-1 : sacrificed at 13 weeks; SI 2-2: sacrificed at 8 weeks; S2-1 : sacrificed at 16 weeks)
- C Representative flow cytometry scatter plots of hSCF Tg NSG recipient BM demonstrating the
- Figures 3A-3C show human mast cell development in hSCF Tg NSG recipient BM.
- A Representative flow cytometry scatter plot and histogram demonstrating the identification of human CD45+CD33+CD117+ mast cells.
- B FACS-sorted hCD45+CD33+CD117+CD203c+ human mast cells from a representative non-Tg NSG recipient BM (Nl-1: 0.9% human mast cells within hCD45+CD33+ population) and hSCF Tg NSG recipient BM (Sl-3: 14.6%, S12-3: 8.8%, and S3-2: 7.3% human mast cells within the hCD45+CD33+ population) were examined by MGG staining.
- H&E- and anti-mast cell tryptase antibody- stained bone sections demonstrate hypercellular BM with high frequency of tryptase+ human mast cells in hSCF Tg NSG recipients.
- Non-Tg NSG recipient - Nll-1 70.7% hCD45+.
- hSCF Tg NSG recipients -SI 1-1 98.0% and S9-1: 99.9% hCD45+.
- Nll-1 sacrificed at 20 weeks; SI 1-1: sacrificed at 10 weeks; S9-1:
- Figure 4 Human mast cell development is enhanced in hSCF Tg NSG recipient spleens (S4-1: sacrificed at 13 weeks; S2-1: sacrificed at 16 weeks).
- Figures 5A-5D show human mast cell development in hSCF Tg NSG recipient stomach, small and large intestine. H&E- and anti-mast cell tryptase antibody-stained sections of (A) non-Tg NSG recipient stomach (NSG control: Nl-3), small intestine (N5-1) and large intestine (N9-1) and (B) hSCF Tg NSG recipient stomach (Sl-3), small intestine (SI 2-3) and large intestine (SI 2-3) demonstrating the presence of human mast cells (Nl-3: sacrificed at 24 weeks; N5-1 : sacrificed at 35 weeks; N9-1 : sacrificed at 20 weeks; Sl-3: sacrificed at 21 weeks; SI 2-3: sacrificed at 13 weeks).
- Figures 6A-6B show PB hematological parameters in human HSC-engrafted hSCF Tg NSG and non-Tg NSG recipients and non-irradiated, non-human
- Figure 7 shows human erythroid cell chimerism in BM. Human erythroid chimerism was determined by calculating the frequency of human glycophorin A
- FIGS 8A-8B shows Colony-forming cell (CFC) assay with HSCs and hematopoietic progenitor cells (HPC) derived from hSCF Tg and non-Tg NSG recipients.
- CFC Colony-forming cell
- HPC hematopoietic progenitor cells
- HPC-enriched fraction derived from hSCF Tg and non-Tg NSG recipients HPC-enriched fraction derived from hSCF Tg and non-Tg NSG recipients.
- Figure 9 shows that human mast cell tryptase+ cells in HSC-engrafted hSCF Tg NSG recipient BM do not co-label with antibodies to human CD 14.
- Confocal immunofluorescence images of BM from hSCF Tg recipient S9-1 demonstrate human mast cell tryptase+ (green) BM cells do not express human CD 14 (red). Low and high magnification images are shown.
- Immunohistochemical labeling of human mast cell tryptase+ cells using the same recipient BM is shown in Figure 3C.
- Figure 10 shows human mast cell development in the lung of hSCF Tg and non-Tg NSG recipients.
- H&E- and anti-mast cell tryptase antibody-stained sections of lung (N5-1, SI 2-3, SI -2) demonstrates the presence of human mast cells.
- Methods for producing an immue-system humanized mouse are provided according to embodiments of the present invention which include administration of human hematopoitetic stem cells (HSC) to a transgenic immunodeficient mouse whose genome comprises a nucleic acid encoding human Stem Cell Factor operably linked to a promoter, wherein the transgenic immunodeficient mouse expresses the human Stem Cell Factor,
- HSC human hematopoitetic stem cells
- Production of the immune-system humanized mouse can be achieved by the engraftment of human hematopoietic stem cells in a human stem cell factor-expressing transgenic immunodeficient mouse whose genome comprises a nucleic acid encoding human Stem Cell Factor operably linked to a promoter, which is characterized by presence of differentiated human hematopoietic cells in the transgenic immunodeficient mouse in which human stem cell factor expressed by the transgenic immunodeficient mouse is delivered to the human hematopoietic stem cells.
- SCF Stem Cell factor
- stem cell factor and "SCF” are used interchangeably herein to refer to a well-known cytokine that binds to the c-Kit receptor (CD117).
- SCF is also known as kit ligand, SF, Kitl, KL- 1 and other names.
- kit ligand kit ligand
- SF c-Kit receptor
- Kitl Kitl
- KL- 1 Kitl
- isoforms of SCF are known including transmembrane (membrane-associated) and soluble isoforms generated by alternative splicing.
- Particular isoforms include human membrane-associated stem cell factor (i.e. human membrane-associated stem cell factor 248 (SCF 248 ), human
- SCF membrane-associated stem cell factor 220
- SCF human soluble stem cell factor
- human SCF along with exemplary nucleic acid sequences encoding human soluble SCF, human SCF 220 or human SCF 248 are shown herein. It will be appreciated by those of ordinary skill in the art that, due to the degenerate nature of the genetic code, alternate nucleic acid sequences encode human soluble SCF, human SCF 220 and human SCF 248 and variants thereof and that such alternate nucleic acids may be used in compositions and methods described herein.
- human SCF encompasses variants of human SCF , human SCF and human sSCF which may be delivered to an immunodeficient mouse according to embodiments of methods of the present invention.
- variant defines either an isolated naturally occurring genetic mutant of a human SCF or a recombinantly prepared variation of a human SCF, each of which contain one or more mutations in its genome compared to the corresponding wild-type human SCF. For example, such mutations can be one or more amino acid substitutions, additions, and/or deletions.
- variant further refers to non-human SCF orthologues.
- wild-type refers to a naturally occurring or unmutated organism, protein or nucleic acid.
- immunodeficient animal according to embodiments of the present invention has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to human SCF 248 , human SCF 220 or human sSCF.
- the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence).
- the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
- the determination of percent identity between two sequences can also be accomplished using a mathematical algorithm.
- a preferred, non limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, PNAS 87:2264 2268, modified as in Karlin and Altschul, 1993, PNAS. 90:5873 5877. Such an algorithm is incorporated into the NBLAST and BLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403.
- Gapped BLAST are utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389 3402.
- PSI BLAST is used to perform an iterated search which detects distant relationships between molecules (Id.).
- the default parameters of the respective programs e.g., of XBLAST and NBLAST
- the default parameters of the respective programs are used (see, e.g., the NCBI website).
- a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11 17. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 is used.
- the percent identity between two sequences is determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.
- Mutations can be introduced using standard molecular biology techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
- site-directed mutagenesis and PCR-mediated mutagenesis.
- one or more amino acid mutations can be introduced without altering the functional properties of human SCF proteins.
- Conservative amino acid substitutions can be made in human SCF proteins to produce human SCF protein variants.
- Conservative amino acid substitutions are art recognized substitutions of one amino acid for another amino acid having similar characteristics.
- each amino acid may be described as having one or more of the following characteristics: electropositive, electronegative, aliphatic, aromatic, polar, hydrophobic and hydrophilic.
- a conservative substitution is a substitution of one amino acid having a specified structural or functional characteristic for another amino acid having the same characteristic.
- Acidic amino acids include aspartate, glutamate; basic amino acids include histidine, lysine, arginine; aliphatic amino acids include isoleucine, leucine and valine; aromatic amino acids include phenylalanine, glycine, tyrosine and tryptophan; polar amino acids include aspartate, glutamate, histidine, lysine, asparagine, glutamine, arginine, serine, threonine and tyrosine; and hydrophobic amino acids include alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, proline, valine and tryptophan; and conservative substitutions include substitution among amino acids within each group. Amino acids may also be described in terms of relative size, alanine, cysteine, aspartate, glycine, asparagine, proline, threonine, serine, valine, all typically considered to be small.
- Human SCF variants can include synthetic amino acid analogs, amino acid derivatives and/or non-standard amino acids, illustratively including, without limitation, alpha-aminobutyric acid, citrulline, canavanine, cyanoalanine, diaminobutyric acid, diaminopimelic acid, dihydroxy-phenylalanine, djenkolic acid, homoarginine, hydroxyproline, norleucine, norvaline, 3-phosphoserine, homoserine,
- 5-hydroxytryptophan 1 -methylhistidine, methylhistidine, and ornithine.
- Human SCF variants are encoded by nucleic acids having a high degree of identity with a nucleic acid encoding a wild-type human SCF.
- the complement of a nucleic acid encoding a human SCF variant specifically hybridizes with a nucleic acid encoding a wild-type human SCF under high stringency conditions.
- nucleic acid refers to R A or DNA molecules having more than one nucleotide in any form including single-stranded, double-stranded, oligonucleotide or polynucleotide.
- nucleotide sequence refers to the ordering of nucleotides in an oligonucleotide or polynucleotide in a single- stranded form of nucleic acid.
- nucleic acid refers to Watson-Crick base pairing between nucleotides and specifically refers to nucleotides hydrogen bonded to one another with thymine or uracil residues linked to adenine residues by two hydrogen bonds and cytosine and guanine residues linked by three hydrogen bonds.
- a nucleic acid includes a nucleotide sequence described as having a "percent complementarity" to a specified second nucleotide sequence.
- a nucleotide sequence may have 80%, 90%, or 100% complementarity to a specified second nucleotide sequence, indicating that 8 of 10, 9 of 10 or 10 of 10 nucleotides of a sequence are complementary to the specified second nucleotide sequence.
- nucleotide sequence 3'-TCGA-5' is 100% complementary to the nucleotide sequence 5'-AGCT-3'. Further, the nucleotide sequence 3'-TCGA-5' is 100% complementary to a region of the nucleotide sequence 5'-TTAGCTGG-3'.
- hybridization and “hybridizes” refer to pairing and binding of complementary nucleic acids. Hybridization occurs to varying extents between two nucleic acids depending on factors such as the degree of complementarity of the nucleic acids, the melting temperature, Tm, of the nucleic acids and the stringency of
- hybridization conditions as is well known in the art.
- stringency of hybridization conditions refers to conditions of temperature, ionic strength, and composition of a hybridization medium with respect to particular common additives such as formamide and Denhardt's solution. Determination of particular hybridization conditions relating to a specified nucleic acid is routine and is well known in the art, for instance, as described in J. Sambrook and D. W. Russell, Molecular Cloning: A
- High stringency hybridization conditions are those which only allow hybridization of substantially complementary nucleic acids. Typically, nucleic acids having about 85-100% complementarity are considered highly complementary and hybridize under high stringency conditions. Intermediate stringency conditions are exemplified by conditions under which nucleic acids having intermediate complementarity, about 50-84% complementarity, as well as those having a high degree of complementarity, hybridize. In contrast, low stringency hybridization conditions are those in which nucleic acids having a low degree of complementarity hybridize.
- specific hybridization and “specifically hybridizes” refer to hybridization of a particular nucleic acid to a target nucleic acid without substantial hybridization to nucleic acids other than the target nucleic acid in a sample.
- Hybridization and conditions to achieve a desired hybridization stringency are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001; and Ausubel, F. et al., (Eds.), Short Protocols in Molecular Biology, Wiley, 2002.
- An example of high stringency hybridization conditions is hybridization of nucleic acids over about 100 nucleotides in length in a solution containing 6*SSC, 5*Denhardt's solution, 30% formamide, and 100 micrograms/ml denatured salmon sperm DNA at 37 °C overnight followed by washing in a solution of 0.1 * SSC and 0.1% SDS at 60 °C for 15 minutes.
- SSC is 0.15M NaCl/0.015M Na citrate.
- Denhardt's solution is 0.02% bovine serum albumin/0.02% FICOLL/0.02% polyvinylpyrrolidone.
- Nucleic acids encoding human SCF or an human SCF variant can be isolated or generated recombinantly or synthetically using well-known methodology.
- Kinds of immunodeficient mouse used in the present method are not particulary limited as long as the immune-system humanized mouse can be produced by the present method.
- the immunodeficient mouse used in the present invention is a mouse having severe combined immune deficiency.
- severe combined immune deficiency refers to a condition characterized by absence of T cells and lack of B cell function.
- SCID Common forms include: X-linked SCID which is characterized by gamma chain gene mutations in the IL2RG gene and the lymphocyte phenotype T(-) B(+) NK(-); and autosomal recessive SCID characterized by Jak3 gene mutations and the lymphocyte phenotype T(-) B(+) NK(-), ADA gene mutations and the lymphocyte phenotype T(-) B(-) NK(-), IL-7R alpha-chain mutations and the lymphocyte phenotype T(-) B(+) NK(+), CD3 delta or epsilon mutations and the lymphocyte phenotype T(-) B(+) NK(+), RAG1/RAG2 mutations and the lymphocyte phenotype T(-) B(-) NK(+), Artemis gene mutations and the lymphocyte phenotype T(-) B(-) NK(+), CD45 gene mutations and the lymphocyte phenotype T(-
- the immunodeficient mouse used in the present invention is a mouse having the severe combined immunodeficiency mutation (Prkdc scid ), commonly referred to as the scid mutation.
- the scid mutation is well-known and located on mouse chromosome 16 as described in Bosma, et al., Immunogenetics 29:54-56, 1989. Mice homozygous for the scid mutation are characterized by an absence of functional T cells and B cells, lymphopenia, hypoglobulinemia and a normal hematopoetic microenvironment.
- the scid mutation can be detected, for example, by detection of markers for the scid mutation using well-known methods.
- the immunodeficient mouse used in the present invention is a mouse having an IL2 receptor gamma chain deficiency in combination with the severe combined immunodeficiency (scid) mutation.
- IL2 receptor gamma chain deficiency refers to decreased IL2 receptor gamma chain. Decreased IL2 receptor gamma chain can be due to gene deletion or mutation. Decreased IL2 receptor gamma chain can be detected, for example, by detection of IL2 receptor gamma chain gene deletion or mutation and/or detection of decreased IL2 receptor gamma chain expression using well-known methods.
- the immunodeficient mouse used in the present invention is a mouse having a NOD background.
- NOD background is a well-known background of a diabetes susceptible nonobese diabetic (NOD) mouse (Diabetes. 1993
- NOD background includes NOD/ShiLtJ background and NOD/Shi background (http://jaxmice.jax.org/strain/001976.html). NOD background is preferably NOD/ShiLtJ background.
- the immunodeficient mouse used in the present invention is a mouse having the NOD background, the severe combined immune deficiency (scid) mutation, and a complete knockout of the interleukin-2 receptor gamma chain.
- Such mouse include NOD/ShiLtJ- Prkdc scid -I12rg null (NSG) (The Journal of Immunology, 1995, 154: 180-191 ; Nature Reviews, 2007, 7: 118-130) and
- NSG mice are preferable. NSG mice is also known as NOO.Cg-Prkdc scid Il2rg tml Wjl ISzi mice, NOD/LtSz-sc d Il2rg-I- mice and other names. NOG mice is also known as Il2rg tm,Sug /iic. NSG mice combine multiple immune deficits from the NOD/ShiLtJ background, the severe combined immune deficiency (scid) mutation, and a complete knockout of the interleukin-2 receptor gamma chain.
- NSG mice lack mature T, B and NK cells, and are deficient in cytokine signaling. NSG mice are characterized by lack of IL2R/y (gamma c) expression, no detectable serum immunoglobulin, no hemolytic complement, no mature T lymphocytes, and no mature natural killer cells.
- IL2R/y gamma c
- the immunodeficient mouse used in the present invention is Rag2 and I12rg double knockout mice such as BALB/c-Rag2-/-fl2rg-/- (C.Cg-Rag2' mdFwa Il2rg tmlSug IYic), md-Rag2-AIl2rg-/- (Stock
- mice strain ( d)-Rag2 tmlFwa Il2rg lmlKrf /B ) and other mice strain have the same immunodeficient phenotype like NSG.
- a transgenic immunodeficient mouse whose genome . comprises a nucleic acid encoding human SCF operably linked to a promoter, wherein the mouse expresses the encoded human SCF, are used.
- the genome of the transgenic immunodeficient mouse comprises an expression cassette including a nucleic acid encoding human SCF, wherein the nucleic acid is operably linked to a promoter and a polyadenylation signal and further contains an intron, and the mouse expresses the encoded human SCF.
- transgenic mouse Any of various methods can be used to introduce a human SCF transgene into an immunodeficient mouse to produce a transgenic immunodeficient mouse expressing human SCF. Such techniques are well-known in the art and include, but are not limited to, pronuclear microinjection and transformation of embryonic stem cells. Methods for generating transgenic mouse that can be used include, but are not limited to, those described in J. P. Sundberg and T. Ichiki, Eds., Genetically Engineered Mice Handbook, CRC Press; 2006; M. H. Hofker and J. van Deursen, Eds., Transgenic Mouse Methods and Protocols, Humana Press, 2002; A. L. Joyner, Gene Targeting: A Practical Approach, Oxford University Press, 2000; Manipulating the Mouse Embryo: A Laboratory Manual, 3 ⁇ rd >edition, Cold Spring Harbor Laboratory Press; Dec. 15, 2002, ISBN- 10:
- a transgenic mouse expressing human SCF can be achieved by methods such as DNA injection of an expression construct into a preimplantation embryo or by use of stem cells, such as embryonic stem (ES) cells or induced pluripotent stem (iPS) cells.
- stem cells such as embryonic stem (ES) cells or induced pluripotent stem (iPS) cells.
- expression construct and "expression cassette” are used herein to refer to a double-stranded recombinant DNA molecule containing a desired nucleic acid coding for human SCF sequence and containing one or more regulatory elements necessary or desirable for the expression of the operably linked coding sequence.
- the expression construct is an expression vector comprising a nucleic acid encoding human SCF operably linked to a promoter, wherein the human SCF can be expressed in a mouse when transferred into a mouse cell.
- the expression construct is an artificial chromosome comprising human genome fragment encoding human SCF, wherein the human SCF can be expressed when the artificial chromosome is integrated in mouse genome.
- regulatory element refers to a nucleotide sequence which controls some aspect of the expression of nucleic acid sequences.
- exemplary regulatory elements illustratively include an enhancer, an internal ribosome entry site (IRES), an intron; an origin of replication, a polyadenylation signal (pA), a promoter, a transcription termination sequence, and an upstream regulatory domain, which contribute to the replication, transcription, post-transcriptional processing of a nucleic acid sequence.
- IRS internal ribosome entry site
- pA polyadenylation signal
- promoter a transcription termination sequence
- upstream regulatory domain which contribute to the replication, transcription, post-transcriptional processing of a nucleic acid sequence.
- operably linked refers to a nucleic acid in functional relationship with a second nucleic acid.
- a regulatory element is included in an expression cassette is a promoter in particular embodiments.
- the term "promoter” as used herein refers to a DNA sequence operably linked to a nucleic acid sequence to be transcribed such as a nucleic acid sequence encoding a desired molecule.
- a promoter is generally positioned upstream of a nucleic acid sequence to be transcribed and provides a site for specific binding by RNA polymerase and other transcription factors.
- a promoter is generally positioned upstream of the nucleic acid sequence transcribed to produce the desired molecule, and provides a site for specific binding by RNA polymerase and other transcription factors.
- An included promoter can be a constitutive promoter or can provide inducible expression; and can provide ubiquitous, tissue-specific or cell-type specific expression.
- Ubiquitous promoters that can be included in a human SCF expression construct include, but are not limited to, a 3-phosphoglycerate kinase (PGK-1) promoter, a beta-actin promoter, a ROSA26 promoter, a heat shock protein 70 (Hsp70) promoter, an EF-1 alpha gene encoding elongation factor 1 alpha (EF1) promoter, an eukaryotic initiation factor 4 A (eIF-4Al) promoter, a chloramphenicol acetyltransferase (CAT) promoter and a CMV (cytomegalovirus) promoter.
- PGK-1 3-phosphoglycerate kinase
- beta-actin beta-actin promoter
- ROSA26 promoter
- Hsp70 heat shock protein 70
- Hsp70 heat shock protein 70
- EF-1 alpha gene encoding elongation factor 1 alpha (EF1) promoter an eukaryotic initiation factor 4 A
- Tissue-specific promoters that can be included in a human SCF expression construct include, but are not limited to, a promoter of a gene expressed in the hematopoietic system, such as an SCF promoter, an IFN-beta promoter, a Wiskott-Aldrich syndrome protein (WASP) promoter, a CD45 (also called leukocyte common antigen) promoter, a Fit- 1 (fms-like tyrosine kinase, VEGF Receptor 1) promoter, an endoglin (CD105) promoter and an ICAM-2 (Intracellular Adhesion Molecule 2) promoter.
- a promoter of a gene expressed in the hematopoietic system such as an SCF promoter, an IFN-beta promoter, a Wiskott-Aldrich syndrome protein (WASP) promoter, a CD45 (also called leukocyte common antigen) promoter, a Fit- 1 (fms-like t
- one or more enhancer sequences may be included such as, but not limited to, cytomegalovirus (CMV) early enhancer element and an SV40 enhancer element.
- CMV cytomegalovirus
- Additional included sequences include an intron sequence such as the beta globin intron or a generic intron, a transcription termination sequence, and an mRNA polyadenylation (pA) sequence such as, but not limited to SV40-pA, beta-globin-pA and SCF-pA.
- intron sequence such as the beta globin intron or a generic intron
- pA mRNA polyadenylation
- An expression construct may include sequences necessary for amplification in bacterial cells, such as a selection marker (e.g. kanamycin or ampicillin resistance gene) and a replicon.
- a selection marker e.g. kanamycin or ampicillin resistance gene
- a replicon e.g. kanamycin or ampicillin resistance gene
- the expression construct is linearized before injection into non-human preimplantation embryos.
- the expression construct is injected into fertilized oocytes. Fertilized oocytes are collected from superovulated females the day after mating (0.5 dpc) and injected with the expression construct. The injected oocytes are either cultured overnight or transferred directly into oviducts of 0.5-day p.c. pseudopregnant females. Methods for superovulation, harvesting of oocytes, expression construct injection and embryo transfer are known in the art and described in Manipulating the Mouse Embryo: A Laboratory Manual, 3 rd edition, Cold Spring Harbor Laboratory Press; Dec. 15, 2002, ISBN-10: 0879695919.
- Offspring can be tested for the presence of the transgene by DNA analysis, such as PCR, Southern blot or sequencing. Mice which are carrying the transgene can be tested for protein expression by using such as ELISA or Western blot analysis.
- the expression construct may be transfected into stem cells (ES cells or iPS cells) using well-known methods, such as electroporation,
- the cells are screened for transgene integration by DNA analysis, such as PCR, Southern blot or sequencing. Cells with the correct integration can be tested for functional expression tested by protein analysis for SCF using for example ELISA or Western blot analysis.
- ES cells are grown in media optimized for the particular line. Typically ES media contains 15% fetal bovine serum (FBS) or synthetic or
- Selected cells incorporating the expression construct can be injected into preimplantation embryos.
- ES or iPS cell are rendered to single cells using a mixture of trypsin and EDTA, followed by resuspension in ES media.
- Groups of single cells are selected using a finely drawn-out glass needle (20-25 micrometer inside diameter) and introduced through the embryo's zona pellucida and into the blastocysts cavity (blastocoel) using an inverted microscope fitted with micromanipulators.
- stem cells can be injected into early stage embryos (e.g. 2-cell, 4-cell, 8-cell, premorula or morula). Injection may be assisted with a laser or piezo pulses drilled opening the zona pellucida.
- stem cells Approximately 9-10 selected stem cells (ES or iPS cells) are injected per blastocysts, or 8-cell stage embryo, 6-9 stem cells per 4-cell stage embryo, and about 6 stem cells per 2-cell stage embryo. Following stem cell introduction, embryos are allowed to recover for a few hours at 37 °C in 5% C0 2 , 5% 0 2 in nitrogen or cultured overnight before transfer into pseudopregnant recipient females. In a further alternative to stem cell injection, stem cells can be aggregated with morula stage embryos. All these methods are well established and can be used to produce stem cell chimeras. For a more detailed description see Manipulating the Mouse Embryo: A Laboratory Manual, 3 rd edition (A. Nagy, M. Gertsenstein, K.
- Pseudopregnant embryo recipients are prepared using methods known in the art. Briefly, fertile female mice between 6-8 weeks of age are mated with vasectomized or sterile males to induce a hormonal state conductive to supporting surgically introduced embryos. At 2.5 days post coitum (dpc) up to 15 of the stem cell containing blastocysts are introduced into the uterine horn very near to the uterus-oviduct junction. For early stage embryos and morula, such embryos are either cultured in vitro into blastocysts or implanted into 0.5 dpc or 1.5 dpc pseudopregnant females according to the embryo stage into the oviduct.
- dpc coitum
- Chimeric pups from the implanted embryos are born 16-20 days after the transfer depending on the embryo age at implantation. Chimeric males are selected for breeding.
- Offspring can be analyzed for transmission of the ES cell genome by coat color and genetic analysis, such as PCR, Southern blot or sequencing. Further the expression of human SCF can be analyzed by protein analysis (Western blot, ELISA) or other functional assays. Offspring expressing the transgene are intercrossed to create mice homozygous for the transgene. The transgenic mice are crossed to the
- the transgene is targeted into a specific locus of the stem cell genome which is known to result in reliable expression, such as mouse SCF, Hprt or the Rosa26 locus.
- Mouse SCF locus is preferable, since the expression of human SCF in the mouse, with expression pattern substantially same as that of mouse SCF in a mouse without transgene can be desired.
- a targeting construct is made using recombinant DNA techniques and includes 5' and 3' sequences which are homologous to the stem cell endogenous gene.
- the targeting construct further includes a selectable marker such as neomycin phosphotransferase, hygromycin or puromycin, a nucleic acid encoding human SCF and a polyadenylation signal.
- the nucleic acid encoding human SCF is either in frame with the endogenous gene locus, or a splice acceptor site and internal ribosome entry site (IRES) sequences are included.
- IRS internal ribosome entry site
- Such a targeting construct is transfected into stem cells and the stem cells are screened to detect the homologous recombination event using PCR, Southern blot or sequencing analysis. Cells with the correct homologous recombination event can be further analyzed for transgene expression by protein analysis, such as ELISA or Western blot analysis. If desired, the selectable marker can be removed by treating the stem cells with Cre recombinase.
- the cells are analyzed for the presence of the nucleic acid encoding xenogeneic SCF.
- Cells with the correct genomic event will be selected and injected into preimplantation embryos as described above. Chimeric males are selected for breeding.
- Offspring can be analyzed for transmission of the ES cell genome by coat color and genetic analysis, such as PCR, Southern blot or
- SCF protein expression such as by protein analysis (Western blot, ELISA) or other functional assays.
- Offspring expressing the by protein analysis (Western blot, ELISA) or other functional assays are intercrossed to create non-human animals homozygous for the transgene.
- the transgenic mice are crossed to the immunodeficient mice to create a congenic immunodeficient strain with the human SCF transgene.
- the transgenic immunodeficient mouse may comprise a human SCF transgene in substantially all of their cells. In further embodiment, the transgenic immunodeficient mouse may comprise a human SCF transgene in some, but not all their cells. One or multiple copies (such as concatamers) of the human SCF transgene may be integrated into the genome of the cells of the transgenic
- the present method uses a transgenic immunodeficient mouse having severe combined immunodeficiency or an IL2 receptor gamma chain deficiency in combination with severe combined immunodeficiency whose genome comprises a nucleic acid encoding human SCF operably linked to a promoter, wherein the mouse expresses the encoded human SCF.
- the present method uses a transgenic immunodeficient mouse having the scid mutation or an IL2 receptor gamma chain deficiency in
- the present method uses a transgenic NSG mouse expressing human SCF 248 , human SCF 220 and/or human sSCF.
- a transgenic immunodeficient mouse expressing human SCF , human SCF and/or human sSCF is generated by introduction of an expression cassette including a nucleic acid encoding an SCF protein operably linked to a promoter into cells to express the SCF protein in the transgenic mouse.
- An expression cassette can be introduced into the pronuclei of fertilized eggs of the desired immunodeficient mouse strain, such as NSG for example.
- microinjected eggs are either transferred the same day into oviducts of 0.5-day post coitus (p.c.) pseudopregnant females or cultured overnight and transferred the next day into oviducts of 0.5-day p.c. pseudopregnant females.
- the resulting pups are tested for presence of the transgene and expressed human SCF protein.
- the nucleic acid can be stably integrated into the chromosomal genome of a cell or maintained as a stable episome.
- an expression cassette can be introduced into the pronuclei of fertilized eggs of NOD-SCID (NOD.CB17-Prkdc scid /J).
- the microinjected eggs are either transferred the same day into oviducts of 0.5-day p.c. pseudopregnant females or cultured overnight and transferred the next day into oviducts of 0.5-day p.c. pseudopregnant female.
- the resulting pups are tested for presence of the transgene and those positive for the transgene can be crossed with NSG or NRG.
- expressing and “expresses” refer to transcription of a gene to produce a corresponding mRNA and/or translation of the mRNA to produce the corresponding protein.
- human HSC refers to multipotent stem cells expressing c-Kit receptor.
- multipotent stem cells expressing c-Kit receptor include, but are not limited to, haematopoietic stem cells, also known as hemocytoblasts.
- c-Kit receptor is well-known in the art, for example as described in Vandenbark G R et al., 1992, Cloning and structural analysis of the human c-kit gene, Oncogene 7(7): 1259-66; and Edling C E, Hallberg B, 2007, c-Kit-a hematopoietic cell essential receptor tyrosine kinase, Int. J. Biochem. Cell Biol. 39(11): 1995-8.
- immunodeficient mouse can be obtained from any tissue containing human HSC such as, but not limited to, human umbilical cord blood, bone marrow, GM-CSF-mobilized peripheral blood and fetal liver.
- tissue containing human HSC such as, but not limited to, human umbilical cord blood, bone marrow, GM-CSF-mobilized peripheral blood and fetal liver.
- Human HSC can be administered into newborn mouse by administration via various routes, such as, but not limited to, into the heart, liver and/or facial vein.
- Human HSC can be administered into adult mouse by various routes, such as, but not limited to, administration into the tail vein, into the femur bone marrow cavity or into the spleen.
- human HSC is administered into newborn mouse.
- Newborn mouse is preferably a mouse within two days of birth.
- the HSC as fetal liver can be engrafted under the renal capsule.
- Engraftment of human HSC can be assessed by any of various methods, such as, but not limited to, flow cytometric analysis of cells in the mouse to which the human HSC are administered at one or more time points following the administration of human HSC.
- the human HSC administered are isolated from an original source material to obtain a population of cells enriched in HSCs.
- the isolated human HSCs may or may not be pure.
- human HSCs are purified by selection for a cell marker, such as CD34.
- administered human HSCs are a population of cells in which CD34+ cells constitute about 1-100% of total cells, although a population of cells in which CD34+ cells constitute fewer than 1% of total cells can be used.
- administered human HSCs are T cell depleted cord blood cells in which CD34+ cells make up about 1-3% of total cells, lineage depleted cord blood cells in which CD34+ cells make up about 50% of total cells, or CD34+ positively selected cells in which CD34+ cells make up about 90% of total cells.
- administered human HSCs are lineage depleted cord blood cells in which 7AAD(-)lineage (hCD3/hCD4/hCD8/hCD19/hCD56)(-)CD34+CD38(-) cells make up about 50% of total cells, or lineage depleted and CD34+ positively selected cells in which 7AAD(-)lineage (hCD3/hCD4/hCD8/hCD19/hCD56)(-)CD34+CD38(-) cells make up about 90% of total cells. More preferably, administered human HSCs are lineage depleted and CD34+ positively selected cells in which 7AAD(-)lineage
- the number of human HSCs administered is not considered limiting with regard to generation of a human hematopoietic and immune system in an
- the number of administered HSCs is generally in the range of 3x10 to 1x10 CD34+ cells per mouse, although more or fewer can be used.
- the number of administered HSCs is in the range of 5xl0 2 to 5.3xl0 4 7AAD(-)lineage (hCD3/hCD4/hCD8/hCD19/hCD56)(-)CD34+CD38(-) cells.
- conditioning with either sub-lethal irradiation of the recipient mouse with high frequency electromagnetic radiation, generally using gamma radiation, or treatment with a radiomimetic drug such as busulfan or nitrogen mustard can be applied for the engraftment of human HSC in the
- immunodeficient mouse Conditioning is believed to reduce numbers of host hematopoietic cells, create appropriate microenvironmental factors for engraftment of xenogeneic HSC, and/or create microenvironmental niches for engraftment of human HSC. Standard methods for conditioning are known in the art, such as described herein and in J. Hayakawa et al, 2009, Stem Cells, 27(1):175-182.
- methods for producing an immune-system humanized mouse are provided according to the present invention which include delivery of human SCF to the human HSC in the immunodeficient mice, wherein the mice are irradiated prior to
- methods for producing an immune-system humanized mouse is provided according to the present invention which include delivery of human SCF to the human HSC in the immunodeficient mice, wherein the mice are administered with a radiomimetic drug, such as busulfan or nitrogen mustard, prior to administration of the HSC.
- methods for producing an immune-system humanized mouse is provided according to the present invention which include delivery of human SCF to the human HSC in the immunodeficient mice, without irradiating the mice prior to administration of the HSC.
- methods for producing an immune-system humanized mouse is provided according to the present invention which include delivery of human SCF to the human HSC in the immunodeficient mice, without irradiating the mice prior to administration of the HSC.
- immune- system humanized mouse is provided according to the present invention which include delivery of human SCF to the human HSC in the immunodeficient mice, without administering a radiomimetic drug, such as busulfan or nitrogen mustard, to the mouse prior to administration of the HSC.
- a radiomimetic drug such as busulfan or nitrogen mustard
- Engraftment is successful where human HSCs and cells differentiated from the human HSCs in the recipient animal are detected at a time when the majority of any administered non-HSC has degenerated. Detection of differentiated HSC cells can be achieved by detection of human DNA in the recipient mouse or detection of intact human HSCs and cells differentiated from the human HSCs, for example. Serial transfer of CD34+ cells into a secondary recipient and engraftment of a human hematopoietic system is a further test of HSC engraftment in the primary recipient. Engraftment can be detected by flow cytometry as 0.05% or greater human CD45+ cells in the blood, spleen or bone marrow at 10-12 weeks after administration of the HSC.
- the immune-system humanized mouse which can be obtained by the present method is characterized by greater numbers of differentiated human hematopoietic cells in the mouse in which human stem cell factor is delivered to the human hematopoietic stem cells compared to appropriate control mouse in which human stem cell factor is not delivered to the human hematopoietic stem cells.
- the immune system humanized mouse which can be produced by the present method is also provided according to embodiments of the present invention.
- immune-system humanized mouse refers to a mouse comprising human hematopoietic cells and human both acquired and innate immune cells, wherein the human hematopoietic cells and human both acquired and innate immune cells differenciated from the hematopoietic cells are surviving without being rejected from the host mouse, thereby human hematopoiesis and both acquired and innate immunity are reconstituted in the mouse.
- Aquired immune cells include T cells and B cells.
- Innate immune cells include macrophages, granulocytes (basophils, eosinophils, neutrophils), DCs, NK cells and mast cells.
- the immune-system humanized mouse of the present invention has
- a difference between the immune-system humanized mouse of the present inveniton and the control immune-system humanized mouse exists only in that the transgenic immunodeficient mouse used for prepareing the immune-system humanized mouse of the present inveniton comprises a nucleic acid encoding human Stem Cell Factor operably linked to a promoter in the genome and expresses the human Stem Cell Factor, whereas the transgenic immunodeficient mouse used for prepareing the control immune-system humanized mouse does not comprise a nucleic acid encoding human Stem Cell Factor operably linked to a promoter in the genome and does not express the human Stem Cell Factor.
- the engraftment level of human CD45+ cells in spleen of the immune-system humanized mouse of the present inveniton is generally 70% or more, preferably 80% or more, more preferably 85% or more, more preferably 90% or more.
- the engraftment level of human CD45+ cells in peripheral blood of the immune-system humanized mouse of the present inveniton is generally 60% or more, preferably 70% or more, more preferably 80% or more.
- the frequency of human CD33+ myeloid cells within the total human CD45+ population in the bone marrow of the immune-system humanized mouse of the present inveniton is generally 30% or more, preferably 40% or more, more preferably 45% or more.
- the frequency of human cKit+CD203c+ mast cells within the total human CD45+CD33+ myeloid cells in the spleen of the immune-system humanized mouse of the present inveniton is generally 65% or more, preferably 70% or more, more preferably 75% or more.
- the frequency of human c-kit+CD203c+ mast cells within the total human CD33+ population in the bone marrow of the immune-system humanized mouse of the present inveniton is greater than 15 %.
- Methods and immune-system humanized mouse provided by embodiments of the present invention have various utilities such as, but not limited to, as models of growth and differentiation of immune cells, in vivo study of immune response and for the testing of agents affecting hematopoietic and immune cell function.
- transgenic expression of human SCF results in the efficient development of human myeloid cells and human mast cells in hematopoietic organs and mucosal tissues (i.e. respiratory mucosa, gastric tissue) and achieves high chimerism of human hematopoietic cells in hematopoietic organs.
- embodiments of the present invention is useful as a model of growth and differentiation of human myeloid cells and/or human mast cells, in vivo study for testing of agents affecting human myeloid cells and/or human mast cells and agents for preventing or treating human immune/allergic/inflammatory disease relating myeloid cells and/or mast cells.
- Such disease includes type-I allergic disease (i.e. asthma, atopic dermatitis, allergic conjunctivitis, pollen allergy, food allergy).
- the present invention also provides a method of screening for a substance capable of preventing or treating immune/allergic/inflammatory disease relating myeloid cells and/or mast cells, comprising applying a test substance to the above-described immune-system humanized mouse provided by embodiments of the present invention or portion thereof affected with the immune/allergic/inflammatory disease relating myeloid cells and/or mast cells, and evaluating whether or not the test substance improves a condition or symptom of the immune/allergic/inflammatory disease.
- Portions of the immune-system humanized mouse includes isolated tissues comprising human myeloid cells and/or human mast cells (i.e. hematopoietic tissues, mucosal tissues (i.e. respiratory mucosa, gastric tissue)), isolated human cells (i.e. human myeloid cells, human mast cells), and the like.
- test substance subjected to the screening method of the present invention may be any commonly known compound or a novel compound; examples include nucleic acids, sugars, lipids, proteins, peptides, organic low molecular compounds, compound libraries prepared using combinatorial chemistry technology, random peptide libraries, or naturally occurring ingredients derived from microorganisms, animals, plants, marine organisms and the like, and the like.
- the test substance is administered to the immune-system humanized mouse provided by embodiments of the present invention or a portion thereof, and a condition or symptom of the immune/allergic/inflammatory disease relating myeloid cells and/or mast cells in the mouse or portion thereof is compared with the condition or symptom of a control mouse not administered with the test substance.
- Conditions or symptoms of the immune/allergic/inflammatory disease relating myeloid cells and/or mast cells includes, but are not limited to, release of chemical mediators (histamine and the like) or cytokines from myeloid cells and or mast cells, conditions or symptoms due to said chemical mediators or cytokine.
- condition or symptom in the control mouse or portion thereof not administered with the test substance may be a condition or symptom measured before or simultaneously with the measurement of the condition or symptom in the mouse or portion thereof administered with the test substance, it is preferable, from the viewpoint of experimental accuracy and reproducibility, that the former condition or symptom be a simultaneously measured.
- a substance that improves the condition or symptoms in the mouse or protion thereof, obtained as a result of the comparison is selected as a candidate substance for a drug for preventing or treating the immune/allergic/inflammatory disease.
- the human membrane bound SCF transgene driven by the human PGK promoter was backcrossed more than 10 generations from the original
- mice C3H/HeJ strain background onto the NSG strain. All the mice were bred and maintained at The Jackson Laboratory and animal facility at RIKEN RCAI under defined flora according to guidelines established by the Institutional Animal Committees at each respective institution.
- CB samples were first processed for isolation of MNCs using LSM lymphocyte separation medium (MP Biomedicals). CB MNCs were then enriched for human CD34+ cells by using anti -human CD34 microbeads (Miltenyi Biotec) and sorted for 7AAD(-)lineage (hCD3/hCD4/hCD8/hCD19/hCD56)(-)CD34+CD38(-) HSCs using FACSAria (BD Biosciences). To achieve high purity of donor HSCs, doublets were excluded by analysis of FSC-height/FSC-width and SSC-height/SSC-width.
- hSCF Tg and non-Tg NSG recipients received 150 cGy total body irradiation using a 137 Cs-source irradiator, followed by intravenous injection of 5xl 0 2 -5.3xl0 4 sorted HSCs via the facial vein.
- the recipient peripheral blood (PB) harvested from the retro-orbital plexus was evaluated for human hematopoietic engraftment every three to four weeks starting at four weeks post-transplantation.
- PB peripheral blood
- cells were stained with anti-hCD45, anti-msCD45, anti-hCD3, anti-hCD19, anti-hCD33, and anti-hCD56 to determine human hematopoietic chimerism and to analyze cell lineages engrafted in the recipients.
- the recipients were euthanized and single cell suspensions of BM and spleen were analyzed using flow cytometry.
- Antibodies used for flow cytometry are specified in Supplemental Methods.
- the labeled cells were analyzed using FACSCantoII or FACSAria (BD).
- Cytospin specimens of FACS-purified human myeloid cells were prepared with a Shandon Cytospin 4 cytocentrifuge (Thermo Electric) using standard procedures. To identify nuclear and cytoplasmic characteristics of each myeloid cell, cytospin specimens were stained with 100% May-Grunwald solution (Merck) for 3 minutes, followed by 50% May-Grunwald solution in phosphate buffer (Merck) for additional 5 minutes, and then with 5% Giemsa solution (Merck) in phosphate buffer for 15 minutes. All staining procedures were performed at room temperature. Light microscopy was performed with Zeiss Axiovert 200 (Carl Zeiss).
- FDR false discovery rate
- paraffin-embedded tissues were stained with H&E using standard procedures.
- IHC and immunofluorescence labeling were performed using standard procedures.
- Antibodies used for IHC and immunofluorescence labeling were mouse anti-human mast cell tryptase monocolonal antibody (Dako, clone AAl), mouse anti-human CD45 monoclonal antibody (Dako, clone 2B11+PD7/26), rabbit anti-human CD117 monoclonal antibody (Epitomics, clone YR145) and rabbit anti-human CD 14 polyclonal antibody (Atlas Antibodies). Light microscopy was performed using an Axiovert 200 (Carl Zeiss).
- hCDlc (BDCA-l-FITC) (Miltenyi Biotec, clone AD5-8E7)
- hCD3-V450 (BD Biosciences, clone UCHTl)
- hCDl lc-APC (BD Biosciences, clone B-ly6)
- hCD19-PE-Cy7 (BD Biosciences, clone SJ25C1)
- hCD14-Alexa700 BD Biosciences, clone M5E2
- hCD15-APC (BD Biosciences, clone HI98)
- hCD33-PE (BD Biosciences, clone WM53)
- hCD33-PE-Cy7 (BD Biosciences, clone P67.6)
- hCD45-APC (BD Biosciences, clone HI30)
- hCD45-V450 (BD Biosciences, clone HI30)
- the humanized mouse model system has served as a tool to investigate human hematopoiesis, immunity and diseases in vivo.
- the microenvironment supporting human hematopoiesis and immunity is primarily of mouse origin.
- the present inventors created a strain of NSG mice expressing membrane-bound human SCF (hSCF) to analyze the role of the BM microenvironment in human hematopoietic lineage
- c-Kit the receptor for SCF, is expressed at low levels in human CB
- Lin-CD34+CD38- HSCs were transplanted into newborn sublethally irradiated (1.5 Gy) hSCF Tg NSG mice and into non-Tg NSG controls (Table 1).
- Table 1 Summary of hSCF Tg MSG and non-Tg NSG recipients analyzed
- WBC white blood cell count
- RBC red blood cell count
- HGB hemoglobin concentration
- HCT hematocrit value
- PLT platelet count
- the present inventors performed flow cytometric analysis of peripheral blood every 3-4 weeks starting at 4 weeks post-transplantation. During long-term observation, all the 21 hSCF Tg NSG recipient mice became moribund at 8-35 weeks
- the present inventors examined the development of human myeloid subsets in the human membrane-bound SCF-expressing BM microenvironment.
- Flow cytometry scatter plots demonstrate the development of the side-scatter high granulocyte fraction in hSCF Tg NSG recipients which correlate with CD33+ HLA-DR(-) cells ( Figure 2A, B).
- the present inventors first identified CD33+c-Kit+CD203c+ mature human mast cells among human CD45+ cells.
- the present inventors identified
- APCs antigen presenting cells
- the present inventors then analyzed global transcriptional profiles of
- CYP51A1 cytochrome P450, family 51, subfemil A, polypeptide 1
- Gene Ontology terms functionally enriched among genes over-represented in immature granulocytes.
- Gene Ontology terms functionally enriched among genes under-represented in immature granulocytes.
- the present inventors next investigated the development of human mast cells in the membrane-bound hSCF expressing NSG mice.
- the frequency of cKit(+)CD203c+ cells in BM CD33+ cells was greater than 15% (Figure 2D, Table 1 ).
- Mast cell progenitors and mature mast cells reside in high frequencies in the spleen of normal immunocompetent mice 21 .
- the present inventors next examined the spleen of human HSC-engrafted hSCF Tg NSG recipients. Human
- CD33(high)c-Kit+CD203c+ mast cells accounted for the highest frequency among total hCD45+hCD33+ myeloid cells in the spleen of both hSCF Tg NSG and non-Tg NSG HSC-engrafted recipients ( Figures 4A,B).
- microenvironment enhances development of human mast cells from transplanted human HSCs within hematopoietic organs such as the BM and spleen, consistent with the activation of c-Kit signaling.
- the present inventors investigated whether the transgenic expression of hSCF results in the efficient development of mucosal tissue-type human mast cells in respiratory and gastrointestinal mucosal layers, as well as in hematopoietic organs.
- the present inventors performed IHC staining of human tryptase-expressing mast cells in the lung, stomach, small intestine, and large intestine in hSCF Tg NSG and control NSG recipients.
- human tryptase-positive mast cells were identified within cellular infiltrates (Figure 10).
- gastric tissue is one of the major sites of mast cell populations in man and mouse
- the present inventors quantified human mast cells in the gastric tissue of hSCF Tg and non-Tg NSG recipients transplanted with human HSCs.
- IHC staining for human mast cell tryptase followed by quantification of tryptase+ cells demonstrated the presence of human mast cells in gastric tissues of hSCF Tg NSG recipients (7.01+/- 0.63%, 3 sites per recipient analyzed in 3 mice) compared with non-Tg NSG recipients (2.53+/- 0.53%, 3 sites per recipient analyzed in 3 mice;
- a supportive microenvironment is essential for hematopoietic and immune system homeostasis. Critical roles played by various niches in the maintenance of cell cycle quiescence and self-renewal capacity of HSCs have been demonstrated, and the thymic microenvironment is critical for T cell education ' .
- the stromal microenvironment within the humanized mouse is predominately of mouse origin. While several key molecules such as SDF1 are cross reactive between man and mouse, a humanized microenvironment is required both to further improve human hematopoietic development in the recipients and to investigate in vivo the interactions between hematopoietic cells and their
- the present inventors created a novel NSG mouse strain that can support the engraftment of human HSCs and express hSCF in microenvironment.
- hSCF Tg NSG recipients transplanted with human HSCs the engraftment levels of human CD45+ cells were significantly higher compared with non-Tg NSG controls.
- the present inventors identified significant differences in human hematopoietic differentiation in hSCF Tg NSG recipients compared with non-Tg NSG recipients.
- human myeloid differentiation from HSCs there were substantially increased levels of human myeloid differentiation from HSCs in the hSCF Tg NSG mice, whereas human B cells accounted for the greatest population in the BM of non-Tg NSG mice.
- human SCF may be important in recapitulating human BM myelopoiesis in immunodeficient mice.
- membrane-bound human SCF may exert distinct effects on human myeloid development in the BM and in the spleen.
- the majority of human myeloid cells were c-Kit(-)CD203c(-)HLA-DR(-) granulocytes.
- myeloid cells at various levels of maturity were identified, with myelocytes and metamyelocytes predominating in the majority of hSCF Tg NSG recipients. Since immature cells were more prominent in hSCF Tg NSG recipients compared with non-Tg NSG recipients, the present inventors performed microarray analysis to identify transcriptional signature specific to the immature human granulocytes that developed in the hSCF Tg NSG mice. Approximately 300 genes were differentially transcribed in the immature granulocytes in hSCF Tg NSG recipients compared with the mature granulocytes in non-Tg NSG recipients. Some of the up-regulated genes were associated with cell cycle or metabolism.
- human mast cells comprised the greatest subfraction among engrafted human myeloid cells.
- human mast cells were present at the highest frequency among the myeloid lineage developed in the recipients. MGG staining revealed both mature and immature mast cells in hSCF Tg NSG recipient BM.
- Human mast cells were identified not only in hematopoietic organs but also in lung, gastric tissue, and intestinal tissues of hSCF Tg NSG recipients. Aberrant expression of CD30 and CD25 on mast cells is associated with systemic mastocytosis and other mast cell disorders ' . The present inventors did not find significantly upregulated expression of these antigens in the mast cells derived from BM or spleen of hSCF Tg NSG recipients.
- mice have been developed for supporting normal and malignant human hematopoietic cell engraftment and normal myeloid cell differentiation by using Il2rg nul1 immune-compromised mice (Table 4) 5>6>8>9>20>38 - 41 .
- TPO human thrombopoietin knock-in BALB/c x ⁇ 29(Rag2" u!1 Il2rg nul1 ) mice were reported to support both human hematopoietic engraftment and myeloid differentiation in the bone marrow. Both SCF and TPO exhibit
- the production method of the present invention and the immune-system humazized mouse produced by the method may serve as a novel platform for in vivo investigation of human mast cell development and allergic responses.
- Petzer AL Hogge DE, Landsdorp PM, Reid DS, Eaves CJ. Self-renewal of primitive human hematopoietic cells (long-term-culture-initiating cells) in vitro and their expansion in defined medium. Proc Natl Acad Sci U S A. 1996;93:1470-1474.
- IL2r gamma null mice a radioresistant model for human lymphohaematopoietic engraftment. Clin Exp Immunol. 2008;154:270-284.
- amino acid sequences of human SCF 220 , SCF 248 and soluble SCF are shown along with exemplary nucleic acid sequences encoding the proteins.
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Abstract
The present invention provides a method for producing an immune-system humanized mouse, comprising administering human hematopoietic stem cells to a first transgenic immunodeficient mouse whose genome comprises a nucleic acid encoding human Stem Cell Factor operably linked to a promoter, and an immune-system humanized mouse produced by the method.
Description
DESCRIPTION
METHOD FOR PRODUCING IMMUNE-SYSTEM HUMANIZED MOUSE CROSS-REFERENCE TO RELATED APPLICATIONS
[0001]
This application is based on a U.S. provisional patent application No. 61/551,209 (filing date: October 25, 2011), the contents of which are incorporated in full herein. FIELD OF THE INVENTION
[0002]
The present invention relates generally to methods for producing a
immune-system humanized mouse, and the immune-system humanized mouse capable of being produced by the methods.
BACKGROUND OF THE INVENTION
[0003]
The humanized mouse system, a xenogeneic transplantation and engraftment model for human hematopoietic stem cells (HSCs) and peripheral blood mononuclear cells (MNCs), facilitates the investigation of human hematopoietic and immune systems in vivo1'2. Since the pioneering work using SCID-hu3 and Hu-PBL-SCID models4, investigators have attempted to better recapitulate human biology in mice across xenogeneic immunological barriers. Recently, the introduction of targeted null mutations of immune-related genes such as Ragl, Rag2, Il2rg, or Prfl in recipient mice has improved engraftment levels of human CD45+ leukocytes ' " . However, limitations remain in the ability of the host mouse hematopoietic microenvironment to support human hematopoiesis. The impaired development of human T-lymphoid and myeloid lineage cells compared with human B-lymphoid lineage cells in NOD/SCID and other immune-compromised mice may be due to the lack of appropriate
microenvironmental support. The recently created HLA class I expressing
immune-compromised NOD/SCID/IL2r gamma null (NSG) mice partially addresses this issue for human T cell development. Human CD8+ T cells developing within these recipients of transplanted human HSCs exhibited cytokine production and cytotoxicity in an HLA-restricted manner10"12.
SUMMARY OF THE INVENTION
[0004]
In order to create a hematopoietic microenvironment more suitable for human myeloid development, the present inventors developed a new immune-compromised mouse strain that expresses human membrane bound stem cell factor (SCF) under the control of the phosphoglycerate kinase (PGK) promoter (hSCF Tg NSG). By using hSCF Tg NSG mice as recipients of human HSC, the present inventors aimed to clarify the role of membrane-bound form of SCF in supporting the engraftment of human hematopoietic cells and influencing the differentiation of the human myeloid lineage in the recipient mouse bone marrow (BM), spleen and other organs. Here the present inventors show nearly complete human hematopoietic chimerism in the BM of hSCF Tg NSG recipients. In the BM of these recipients, human granulocytes accounted for the majority of engrafted human cells reflecting the physiological human BM status. In addition to the development of immature and mature granulocytes including
metamyelocytes and neutrophils, c-Kit+ human mast cells differentiated efficiently in BM, spleen, and mucosal tissues. The hSCF Tg NSG mice, by supporting efficient human myeloid development including mast cells, may serve as a novel platform for in vivo investigation of human mast cell development and allergic responses.
[0005]
Accordingly, the present invention is the folio wings:
(1) A method for producing a first immune-system humanized mouse, comprising administering human hematopoietic stem cells to a first transgenic immunodeficient mouse whose genome comprises a nucleic acid encoding human Stem Cell Factor operably linked to a promoter,
wherein the first transgenic immunodeficient mouse expresses the human Stem Cell Factor, and
wherein the immune-system humanized mouse has
greater human CD45+ leukocyte population in bone marrow, spleen and peripheral blood,
greater human CD33+ myeloid cell population in bone marrow human CD45+ leukocytes, and
- greater human CD203c+c-kit+ mast cell population in splenic human
CD45+CD33+ myeloid cells
as compared to a second immune-system humanized mouse prepared by administering the human hematopoietic stem cells to a second transgenic immunodeficient mouse which does not express the human Stem Cell Factor.
(2) The method of (1), wherein the first transgenic immunodeficient mouse has NOD background, the severe combined immune deficiency (scid) mutation, and a complete knockout of the interleukin-2 receptor gamma chain.
(3) The method of (2), wherein the first transgenic mouse is a NOD/LtSZ-scid IL2Ry null (NSG) mouse.
(4) The method of (1), wherein the human Stem Cell Factor is a human
membrane-associated stem cell factor.
(5) A method for producing an immune-system humanized mouse, comprising administering human hematopoietic stem cells to a transgenic immunodeficient mouse whose genome comprises a nucleic acid encoding human Stem Cell Factor operably linked to a promoter,
wherein the transgenic immunodeficient mouse expresses the human Stem Cell Factor, and
wherein engraftment level of human CD45+ cells in the bone marrow of the
immune-system humanized mouse is 70% or more,
the frequency of human CD33+ myeloid cells within the total human CD45+ population in the bone marrow of the immune-system humanized mouse is 30% or more, and the frequency of human c-kit+CD203c+ mast cells within the total human CD45+CD33+ myeloid cells in the spleen of the immune-system humanized mouse is 65% or more. (6). The method of (5), wherein the engraftment level of human CD45+ cells in the bone marrow of the immune-system humanized mouse is 95% or more.
(7) The method of (5), wherein the frequency of human CD33+ myeloid cells within the total human CD45+ population in the bone marrow of the immune-system humanized mouse is 45% or more.
(8) The method of (5), wherein the frequency of human c-kit+CD203c+ mast cells within the total human CD45+CD33+ myeloid cells in the spleen of the immune-system humanized mouse is 75% or more.
(9) A transgenic immunodeficient mouse, comprising engrafted human hematopoietic cells and human both acquired and innate immune cells differenciated from the hematopoietic cells surviving without being rejected from the mouse;
wherein engraftment level of human CD45+ cells in the bone marrow of the
immune-system humanized mouse is 70% or more,
the frequency of human CD33+ myeloid cells within the total human CD45+ population in the bone marrow of the immune-system humanized mouse is 30% or more, and
the frequency of human c-kit+CD203c+ mast cells within the total human CD45+CD33+ myeloid cells in the spleen of the immune-system humanized mouse is 65% or more; wherein the the transgenic immunodeficient mouse comprises a nucleic acid encoding human Stem Cell Factor operably linked to a promoter in the genome, and expresses the human Stem Cell Factor.
(10) The transgenic immunodeficient mouse of (9), wherein the engraftment level of human CD45+ cells in the bone marrow of the immune-system humanized mouse is 95% or more.
(11) The transgenic immunodeficient mouse of (9), wherein the frequency of human CD33+ myeloid cells within the total human CD45+ population in the bone marrow of the immune-system humanized mouse is 45% or more.
(12) The transgenic immunodeficient mouse of (9), wherein the frequency of human c-kit+CD203c+ mast cells within the total human CD45+CD33+ myeloid cells in the spleen of the immune-system humanized mouse is 75% or more.
(13) A method of screening for a substance capable of preventing or treating
immune/allergic/inflammatory disease relating myeloid cells and/or mast cells, comprising applying a test substance to the transgenic immunodeficient mouse of (9) or portion thereof affected with the immune/allergic/inflammatory disease relating myeloid cells and/or mast cells, and evaluating whether or not the test substance improves a condition or symptom of the immune/allergic/inflammatory disease.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006]
Figures 1 A- IE show that human hematopoietic engraftment is enhanced in hSCF Tg NSG recipients. (A) hSCF Tg NSG recipients developed progressive anemia as evidenced by reduced hemoglobin concentration compared with non-Tg NSG mice transplanted with human HSCs from the same donor source. (B) Human CD45+ chimerism was analyzed over time in PB of hSCF Tg and non-Tg NSG recipients. (C) Representative flow cytometry contour plots demonstrating the presence of human CD45+ cells, CD19+ B cells, CD33+ myeloid cells, CD3+ T cells and CD56+CD3- NK cells in recipient BM. (D) At the time of sacrifice, engraftment levels of human CD45+ cells in the BM, spleen, and PB of hSCF Tg NSG recipients were significantly higher compared with non-Tg NSG controls (BM: hSCF Tg n=21, non-Tg n=13, pO.0001; spleen: hSCF Tg n=21, non-Tg n=13, p=0.0065; PB: hSCF Tg n=21, non-Tg n=13, pO.0001). (E) In hSCF Tg NSG recipient BM, significantly greater human CD33+
myeloid lineage development was observed (hSCF Tg n=21, non-Tg n=13, p=0.0002).
[0007]
Figures 2A-2G show that HLA-DR-negative human myeloid cells predominate in hSCF Tg NSG recipient BM. (A) Flow cytometry contour plots demonstrating forward- and side-scatter characteristics of 6 hSCF Tg NSG recipient BM (Sl-1, SI -2, S9-1, S4-1, SI 2-2, S2-1) and 3 non-Tg NSG recipient BM (Nl-1, Nl-2, N9-1) are shown. Polymorphonuclear myeloid cells (red asterisks) are present at high frequencies in hSCF Tg NSG recipient BM. (B) Flow cytometry contour plots demonstrating hCD33 and HLA-DR expression in the same recipients as shown in (A). Consistent with their FSC and SSC characteristics, hSCF Tg NSG recipient BM contained a prominent
CD33+HLA-DR(-) granulocyte population (red asterisks). (Nl-1 : sacrificed at 21 weeks; Nl-2: sacrificed at 16 weeks; N9-1 : sacrificed at 20 weeks; Sl-1 : sacrificed at 23 weeks; Sl-2: sacrificed at 20 weeks; S9-1 : sacrificed at 16 weeks; S4-1 : sacrificed at 13 weeks; SI 2-2: sacrificed at 8 weeks; S2-1 : sacrificed at 16 weeks) (C) Representative flow cytometry scatter plots of hSCF Tg NSG recipient BM demonstrating the
identification of human c-Kit+CD203c+ mast cells within the hCD33+ fraction and HLA-DR(-)SSC high granulocytes and HLA-DR+SSC low APCs within the
c-Kit(-)CD203c(-) fraction. (S4-1 : sacrificed at 13 weeks; S2-1 : sacrificed at 16 weeks) (D) Frequencies of human c-Kit+CD203c+ mast cells, CD33+HLA-DR(-) granulocytes, and CD33+HLA-DR+ APCs within the total hCD45+hCD33+ myeloid cell population in the BM of hSCF Tg and non-Tg NSG recipients are shown. Numbers of cells in the granulocyte/neutrophil fraction was significantly higher in hSCF Tg NSG recipient BM (hSCF Tg n=20, non-Tg n=12, p=0.0001). (E) CD33+HLA-DR(-) cells from hSCF Tg and non-Tg NSG recipient BM were FACS-purified and examined by MGG staining. In eight of 13 hSCF Tg recipients (S4-1 and SI 2-2 shown as
representative), immature myeloid cells comprised the majority of cells in this fraction. In four of 13 hSCF Tg recipients (S2-1 shown as representative) and four of five non-Tg NSG recipients (N12-1 shown as representative), mature neutrophils (band and
segmented forms) were observed. (N12-1 : sacrificed at 8 weeks; S2-1: sacrificed at 16 weeks; S4-1 : sacrificed at 13 weeks; S12-2: sacrificed at 8 weeks) (F, G) Global transcriptional profiles of FACS-purified CD33+cKit(-)CD203(-)HLA-DR(-)
granulocytes and CD33+c-Kit(-)CD203c(-)HLA-DR+CD14+ monocytes derived from hSCF Tg NSG and non-Tg NSG recipient BM as well as human CD 16+ neutrophils and CD 14+ monocytes were compared. (F) Unsupervised clustering for each group is shown. (G) The expression heatmap demonstrates genes that are significantly under-
and over- represented in each population.
[0008]
Figures 3A-3C show human mast cell development in hSCF Tg NSG recipient BM. (A) Representative flow cytometry scatter plot and histogram demonstrating the identification of human CD45+CD33+CD117+ mast cells. (B) FACS-sorted hCD45+CD33+CD117+CD203c+ human mast cells from a representative non-Tg NSG recipient BM (Nl-1: 0.9% human mast cells within hCD45+CD33+ population) and hSCF Tg NSG recipient BM (Sl-3: 14.6%, S12-3: 8.8%, and S3-2: 7.3% human mast cells within the hCD45+CD33+ population) were examined by MGG staining. (Nl-1: sacrificed at 21 weeks; Sl-3: sacrificed at 21 weeks; SI 2-3: sacrificed at 13 weeks; S3-2: sacrificed at 15 weeks) (C) H&E- and anti-mast cell tryptase antibody- stained bone sections demonstrate hypercellular BM with high frequency of tryptase+ human mast cells in hSCF Tg NSG recipients. Non-Tg NSG recipient - Nll-1: 70.7% hCD45+. hSCF Tg NSG recipients -SI 1-1: 98.0% and S9-1: 99.9% hCD45+. (Nll-1: sacrificed at 20 weeks; SI 1-1: sacrificed at 10 weeks; S9-1:
sacrificed at 16 weeks)
[0009]
Figure 4 (A) Human mast cell development is enhanced in hSCF Tg NSG recipient spleens (S4-1: sacrificed at 13 weeks; S2-1: sacrificed at 16 weeks).
(B) Frequencies of human c-Kit+CD203c+ mast cells, CD33+HLA-DR(-) granulocyte population, and CD33+HLA-DR+ antigen-presenting cells (APCs) within total hCD45+hCD33+ myeloid cells in the spleens of hSCF Tg and non-Tg NSG recipients are shown. Human mast cell development in the spleen was significantly greater in the hSCF Tg NSG recipients (hSCF Tg: n=20, non-Tg NSG: n=12, p=0.0304). (C) FACS-sorted hCD45+CD33+CDl 17+CD203c+ human mast cells from a representative non-Tg NSG recipient spleen (Nl-1: 59.3% human mast cells within hCD45+CD33+ population) and hSCF Tg NSG recipient spleen (Sl-2: 85.7%, Sl-3: 77.7%, and S12-3: 56.1% human mast cells within hCD45+CD33+ population) were examined by MGG staining (Nl-1: sacrificed at 21 weeks; Sl-2: sacrificed at 20 weeks; Sl-3: sacrificed at 21 weeks; SI 2-3: sacrificed at 13 weeks). (D) H&E- and anti-mast cell tryptase antibody-stained spleen sections demonstrating the presence of human mast cells in non-Tg NSG recipients and hSCF Tg NSG recipients. Non-Tg NSG recipient -Nll-1: 94.0% hCD45+. hSCF Tg NSG recipients - Sll-1: 95.3% and S9-1: 97.0% hCD45+ (Nll-1: sacrificed at 20 weeks; Sll-1: sacrificed at 10 weeks; S9-1: sacrificed at 16 weeks).
[0010]
Figures 5A-5D show human mast cell development in hSCF Tg NSG recipient stomach, small and large intestine. H&E- and anti-mast cell tryptase antibody-stained sections of (A) non-Tg NSG recipient stomach (NSG control: Nl-3), small intestine (N5-1) and large intestine (N9-1) and (B) hSCF Tg NSG recipient stomach (Sl-3), small intestine (SI 2-3) and large intestine (SI 2-3) demonstrating the presence of human mast cells (Nl-3: sacrificed at 24 weeks; N5-1 : sacrificed at 35 weeks; N9-1 : sacrificed at 20 weeks; Sl-3: sacrificed at 21 weeks; SI 2-3: sacrificed at 13 weeks). (C) Confocal immunofluorescence images of hSCF Tg stomach (SI -9) demonstrate human CD45+ (green) and human CD117+ (red) mast cells. (D) Frequencies of tryptase+ cells were quantified by sampling three areas each from hSCF Tg (n=3) and non-Tg (n=3) NSG recipients. hSCF Tg NSG recipients: 7.0+/- 0.6%. Non-Tg NSG recipients: 2.5+/- 0.5%. pO.0001 by two tailed t test.
[0011]
Figures 6A-6B show PB hematological parameters in human HSC-engrafted hSCF Tg NSG and non-Tg NSG recipients and non-irradiated, non-human
HSC-engrafted hSCF Tg NSG mice. (A) Mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration
(MCHC) measurements in the PB of hSCF Tg NSG and non-Tg NSG recipients did not show significant differences. (B) While human HSC-engrafted hSCF Tg NSG
recipients showed anemia (n=7), non-irradiated non-transplanted hSCF Tg NSG mice showed normal hemoglobin levels at 11-12 weeks of age (n=5).
[0012]
Figure 7 shows human erythroid cell chimerism in BM. Human erythroid chimerism was determined by calculating the frequency of human glycophorin A
(GlyA)(+)mouse Terll9(-) cells within hGlyA(+)mouse Terl l9(-) and hGlyA(-)mouse
Terl l9(+) cells combined.
[0013]
Figures 8A-8B shows Colony-forming cell (CFC) assay with HSCs and hematopoietic progenitor cells (HPC) derived from hSCF Tg and non-Tg NSG recipients. (A) CFC assay with hCD45+CD34+CD38- HSC-enriched fraction derived from hSCF Tg and non-Tg NSG recipients. (B) CFC assay with hCD45+CD34+CD38+
HPC-enriched fraction derived from hSCF Tg and non-Tg NSG recipients.
[0014]
Figure 9 shows that human mast cell tryptase+ cells in HSC-engrafted hSCF Tg
NSG recipient BM do not co-label with antibodies to human CD 14. Confocal immunofluorescence images of BM from hSCF Tg recipient S9-1 demonstrate human mast cell tryptase+ (green) BM cells do not express human CD 14 (red). Low and high magnification images are shown. Immunohistochemical labeling of human mast cell tryptase+ cells using the same recipient BM is shown in Figure 3C.
[0015]
Figure 10 shows human mast cell development in the lung of hSCF Tg and non-Tg NSG recipients. H&E- and anti-mast cell tryptase antibody-stained sections of lung (N5-1, SI 2-3, SI -2) demonstrates the presence of human mast cells.
DETAILED DESCRIPTION OF THE INVENTION
[0016]
Methods for producing an immue-system humanized mouse are provided according to embodiments of the present invention which include administration of human hematopoitetic stem cells (HSC) to a transgenic immunodeficient mouse whose genome comprises a nucleic acid encoding human Stem Cell Factor operably linked to a promoter, wherein the transgenic immunodeficient mouse expresses the human Stem Cell Factor,
[0017]
Production of the immune-system humanized mouse can be achieved by the engraftment of human hematopoietic stem cells in a human stem cell factor-expressing transgenic immunodeficient mouse whose genome comprises a nucleic acid encoding human Stem Cell Factor operably linked to a promoter, which is characterized by presence of differentiated human hematopoietic cells in the transgenic immunodeficient mouse in which human stem cell factor expressed by the transgenic immunodeficient mouse is delivered to the human hematopoietic stem cells.
[0018]
Scientific and technical terms used herein are intended to have the meanings commonly understood by those of ordinary skill in the art. Such terms are found defined and used in context in various standard references illustratively including J. Sambrook and D. W. Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rd Ed., 2001; F. M. Ausubel, Ed., Short Protocols in
Molecular Biology, Current Protocols; 5th Ed., 2002; B. Alberts et al., Molecular
Biology of the Cell, 4th Ed., Garland, 2002; D. L. Nelson and M. M. Cox, Lehninger Principles of Biochemistry, 4th Ed., W.H. Freeman & Company, 2004; and Herdewijn, P.
(Ed.), Oligonucleotide Synthesis: Methods and Applications, Methods in Molecular
Biology, Humana Press, 2004.
[0019]
Stem Cell factor (SCF)
The terms "stem cell factor" and "SCF" are used interchangeably herein to refer to a well-known cytokine that binds to the c-Kit receptor (CD117). SCF is also known as kit ligand, SF, Kitl, KL- 1 and other names. Various isoforms of SCF are known including transmembrane (membrane-associated) and soluble isoforms generated by alternative splicing. Particular isoforms include human membrane-associated stem cell factor (i.e. human membrane-associated stem cell factor 248 (SCF248), human
000
membrane-associated stem cell factor 220 (SCF )) and human soluble stem cell factor (SCF), see Anderson, D. M. et al., 1990, Cell 63, 235; Flannagan, J. G. et al., 1991, Cell 64, 1025; Anderson, D. M. et al., 1991 , Cell Growth Differ. 2, 373; Martin, F. H. et al., Cell, 63 :203, 1990; Huang E. J. et al., Mol. Biol. Cell, 3:349, 1992; and Huang E. et al., Cell, 63 :225, 1990. Amino acid sequences of human soluble SCF, human SCF220 and
248
human SCF along with exemplary nucleic acid sequences encoding human soluble SCF, human SCF220 or human SCF248 are shown herein. It will be appreciated by those of ordinary skill in the art that, due to the degenerate nature of the genetic code, alternate nucleic acid sequences encode human soluble SCF, human SCF220 and human SCF248 and variants thereof and that such alternate nucleic acids may be used in compositions and methods described herein.
[0020]
In addition to these isolated naturally occurring human SCF amino acid
LH O C
sequences, such as human SCF , human SCF and human sSCF, the term human SCF encompasses variants of human SCF , human SCF and human sSCF which may be delivered to an immunodeficient mouse according to embodiments of methods of the present invention. As used herein, the term "variant" defines either an isolated naturally occurring genetic mutant of a human SCF or a recombinantly prepared variation of a human SCF, each of which contain one or more mutations in its genome compared to the corresponding wild-type human SCF. For example, such mutations can be one or more amino acid substitutions, additions, and/or deletions. The term
"variant" further refers to non-human SCF orthologues.
[0021]
The term "wild-type" refers to a naturally occurring or unmutated organism, protein or nucleic acid.
[0022]
In particular embodiments, a variant SCF protein delivered to an
immunodeficient animal according to embodiments of the present invention has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to human SCF248, human SCF220 or human sSCF.
[0023]
To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical overlapping positions/total number of positions XI 00%). In one embodiment, the two sequences are the same length.
[0024]
The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. A preferred, non limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, PNAS 87:2264 2268, modified as in Karlin and Altschul, 1993, PNAS. 90:5873 5877. Such an algorithm is incorporated into the NBLAST and BLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches are performed with the NBLAST nucleotide program parameters set, e.g., for score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the present invention. BLAST protein searches are performed with the XBLAST program parameters set, e.g., to score 50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST are utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389 3402. Alternatively, PSI BLAST is used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) are used (see, e.g., the NCBI website). Another preferred, non limiting example of a mathematical
algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11 17. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 is used.
[0025]
The percent identity between two sequences is determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.
[0026]
Mutations can be introduced using standard molecular biology techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. One of skill in the art will recognize that one or more amino acid mutations can be introduced without altering the functional properties of human SCF proteins.
[0027]
Assays for assessment of functional properties of SCF and variants are known in the art as exemplified in Blume-Jensen, P. et al, J. Biol. Chem., 269(34):21793-21802, 1994.
[0028]
Conservative amino acid substitutions can be made in human SCF proteins to produce human SCF protein variants. Conservative amino acid substitutions are art recognized substitutions of one amino acid for another amino acid having similar characteristics. For example, each amino acid may be described as having one or more of the following characteristics: electropositive, electronegative, aliphatic, aromatic, polar, hydrophobic and hydrophilic. A conservative substitution is a substitution of one amino acid having a specified structural or functional characteristic for another amino acid having the same characteristic. Acidic amino acids include aspartate, glutamate; basic amino acids include histidine, lysine, arginine; aliphatic amino acids include isoleucine, leucine and valine; aromatic amino acids include phenylalanine, glycine, tyrosine and tryptophan; polar amino acids include aspartate, glutamate, histidine, lysine, asparagine, glutamine, arginine, serine, threonine and tyrosine; and hydrophobic amino acids include alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, proline, valine and tryptophan; and conservative substitutions include substitution among amino acids within each group. Amino acids may also be described in terms of relative size, alanine, cysteine, aspartate, glycine, asparagine, proline, threonine, serine,
valine, all typically considered to be small.
[0029]
Human SCF variants can include synthetic amino acid analogs, amino acid derivatives and/or non-standard amino acids, illustratively including, without limitation, alpha-aminobutyric acid, citrulline, canavanine, cyanoalanine, diaminobutyric acid, diaminopimelic acid, dihydroxy-phenylalanine, djenkolic acid, homoarginine, hydroxyproline, norleucine, norvaline, 3-phosphoserine, homoserine,
5-hydroxytryptophan, 1 -methylhistidine, methylhistidine, and ornithine.
[0030]
Human SCF variants are encoded by nucleic acids having a high degree of identity with a nucleic acid encoding a wild-type human SCF. The complement of a nucleic acid encoding a human SCF variant specifically hybridizes with a nucleic acid encoding a wild-type human SCF under high stringency conditions.
[0031]
The term "nucleic acid" refers to R A or DNA molecules having more than one nucleotide in any form including single-stranded, double-stranded, oligonucleotide or polynucleotide. The term "nucleotide sequence" refers to the ordering of nucleotides in an oligonucleotide or polynucleotide in a single- stranded form of nucleic acid.
[0032]
The term "complementary" refers to Watson-Crick base pairing between nucleotides and specifically refers to nucleotides hydrogen bonded to one another with thymine or uracil residues linked to adenine residues by two hydrogen bonds and cytosine and guanine residues linked by three hydrogen bonds. In general, a nucleic acid includes a nucleotide sequence described as having a "percent complementarity" to a specified second nucleotide sequence. For example, a nucleotide sequence may have 80%, 90%, or 100% complementarity to a specified second nucleotide sequence, indicating that 8 of 10, 9 of 10 or 10 of 10 nucleotides of a sequence are complementary to the specified second nucleotide sequence. For instance, the nucleotide sequence 3'-TCGA-5' is 100% complementary to the nucleotide sequence 5'-AGCT-3'. Further, the nucleotide sequence 3'-TCGA-5' is 100% complementary to a region of the nucleotide sequence 5'-TTAGCTGG-3'.
[0033]
The terms "hybridization" and "hybridizes" refer to pairing and binding of complementary nucleic acids. Hybridization occurs to varying extents between two nucleic acids depending on factors such as the degree of complementarity of the nucleic
acids, the melting temperature, Tm, of the nucleic acids and the stringency of
hybridization conditions, as is well known in the art. The term "stringency of hybridization conditions" refers to conditions of temperature, ionic strength, and composition of a hybridization medium with respect to particular common additives such as formamide and Denhardt's solution. Determination of particular hybridization conditions relating to a specified nucleic acid is routine and is well known in the art, for instance, as described in J. Sambrook and D. W. Russell, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rd Ed., 2001; and F. M.
Ausubel, Ed., Short Protocols in Molecular Biology, Current Protocols; 5th Ed., 2002. High stringency hybridization conditions are those which only allow hybridization of substantially complementary nucleic acids. Typically, nucleic acids having about 85-100% complementarity are considered highly complementary and hybridize under high stringency conditions. Intermediate stringency conditions are exemplified by conditions under which nucleic acids having intermediate complementarity, about 50-84% complementarity, as well as those having a high degree of complementarity, hybridize. In contrast, low stringency hybridization conditions are those in which nucleic acids having a low degree of complementarity hybridize.
[0034]
The terms "specific hybridization" and "specifically hybridizes" refer to hybridization of a particular nucleic acid to a target nucleic acid without substantial hybridization to nucleic acids other than the target nucleic acid in a sample.
[0035]
Stringency of hybridization and washing conditions depends on several factors, including the Tm of the probe and target and ionic strength of the hybridization and wash conditions, as is well-known to the skilled artisan. Hybridization and conditions to achieve a desired hybridization stringency are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001; and Ausubel, F. et al., (Eds.), Short Protocols in Molecular Biology, Wiley, 2002.
[0036]
An example of high stringency hybridization conditions is hybridization of nucleic acids over about 100 nucleotides in length in a solution containing 6*SSC, 5*Denhardt's solution, 30% formamide, and 100 micrograms/ml denatured salmon sperm DNA at 37 °C overnight followed by washing in a solution of 0.1 * SSC and 0.1% SDS at 60 °C for 15 minutes. SSC is 0.15M NaCl/0.015M Na citrate. Denhardt's solution is 0.02% bovine serum albumin/0.02% FICOLL/0.02% polyvinylpyrrolidone.
[0037]
Nucleic acids encoding human SCF or an human SCF variant can be isolated or generated recombinantly or synthetically using well-known methodology.
[0038]
Immunodeficient mouse
Kinds of immunodeficient mouse used in the present method are not particulary limited as long as the immune-system humanized mouse can be produced by the present method.
[0039]
In one embodiment, the immunodeficient mouse used in the present invention is a mouse having severe combined immune deficiency. The term "severe combined immune deficiency (SCID)" refers to a condition characterized by absence of T cells and lack of B cell function. Common forms of SCID include: X-linked SCID which is characterized by gamma chain gene mutations in the IL2RG gene and the lymphocyte phenotype T(-) B(+) NK(-); and autosomal recessive SCID characterized by Jak3 gene mutations and the lymphocyte phenotype T(-) B(+) NK(-), ADA gene mutations and the lymphocyte phenotype T(-) B(-) NK(-), IL-7R alpha-chain mutations and the lymphocyte phenotype T(-) B(+) NK(+), CD3 delta or epsilon mutations and the lymphocyte phenotype T(-) B(+) NK(+), RAG1/RAG2 mutations and the lymphocyte phenotype T(-) B(-) NK(+), Artemis gene mutations and the lymphocyte phenotype T(-) B(-) NK(+), CD45 gene mutations and the lymphocyte phenotype T(-) B(+) NK(+).
[0040]
In one embodiment, the immunodeficient mouse used in the present invention is a mouse having the severe combined immunodeficiency mutation (Prkdcscid), commonly referred to as the scid mutation. The scid mutation is well-known and located on mouse chromosome 16 as described in Bosma, et al., Immunogenetics 29:54-56, 1989. Mice homozygous for the scid mutation are characterized by an absence of functional T cells and B cells, lymphopenia, hypoglobulinemia and a normal hematopoetic microenvironment. The scid mutation can be detected, for example, by detection of markers for the scid mutation using well-known methods.
[0041]
In one embodiment, the immunodeficient mouse used in the present invention is a mouse having an IL2 receptor gamma chain deficiency in combination with the severe combined immunodeficiency (scid) mutation. The term "IL2 receptor gamma chain deficiency" refers to decreased IL2 receptor gamma chain. Decreased IL2
receptor gamma chain can be due to gene deletion or mutation. Decreased IL2 receptor gamma chain can be detected, for example, by detection of IL2 receptor gamma chain gene deletion or mutation and/or detection of decreased IL2 receptor gamma chain expression using well-known methods.
[0042]
In one embodiment, the immunodeficient mouse used in the present invention is a mouse having a NOD background. NOD background is a well-known background of a diabetes susceptible nonobese diabetic (NOD) mouse (Diabetes. 1993
Jan;42(l):44-55.). NOD background includes NOD/ShiLtJ background and NOD/Shi background (http://jaxmice.jax.org/strain/001976.html). NOD background is preferably NOD/ShiLtJ background.
[0043]
In a preferred embodiment, the immunodeficient mouse used in the present invention is a mouse having the NOD background, the severe combined immune deficiency (scid) mutation, and a complete knockout of the interleukin-2 receptor gamma chain. Such mouse include NOD/ShiLtJ- Prkdcscid-I12rg null (NSG) (The Journal of Immunology, 1995, 154: 180-191 ; Nature Reviews, 2007, 7: 118-130) and
NOD/Shi-SCID-yc null (NOG) (US7145055). NSG mouse is preferable. NSG mice is also known as NOO.Cg-Prkdcscid Il2rgtml Wjl ISzi mice, NOD/LtSz-sc d Il2rg-I- mice and other names. NOG mice is also known as
Il2rgtm,Sug/iic. NSG mice combine multiple immune deficits from the NOD/ShiLtJ background, the severe combined immune deficiency (scid) mutation, and a complete knockout of the interleukin-2 receptor gamma chain. As a result, NSG mice lack mature T, B and NK cells, and are deficient in cytokine signaling. NSG mice are characterized by lack of IL2R/y (gamma c) expression, no detectable serum immunoglobulin, no hemolytic complement, no mature T lymphocytes, and no mature natural killer cells.
[0044]
In another embodiment, the immunodeficient mouse used in the present invention is Rag2 and I12rg double knockout mice such as BALB/c-Rag2-/-fl2rg-/- (C.Cg-Rag2'mdFwa Il2rgtmlSugIYic), md-Rag2-AIl2rg-/- (Stock
( d)-Rag2tmlFwaIl2rglmlKrf/B ) and other mice strain have the same immunodeficient phenotype like NSG.
[0045]
In the present method, a transgenic immunodeficient mouse whose genome . comprises a nucleic acid encoding human SCF operably linked to a promoter, wherein
the mouse expresses the encoded human SCF, are used.
[0046]
In one embodiment, the genome of the transgenic immunodeficient mouse comprises an expression cassette including a nucleic acid encoding human SCF, wherein the nucleic acid is operably linked to a promoter and a polyadenylation signal and further contains an intron, and the mouse expresses the encoded human SCF.
[0047]
Any of various methods can be used to introduce a human SCF transgene into an immunodeficient mouse to produce a transgenic immunodeficient mouse expressing human SCF. Such techniques are well-known in the art and include, but are not limited to, pronuclear microinjection and transformation of embryonic stem cells. Methods for generating transgenic mouse that can be used include, but are not limited to, those described in J. P. Sundberg and T. Ichiki, Eds., Genetically Engineered Mice Handbook, CRC Press; 2006; M. H. Hofker and J. van Deursen, Eds., Transgenic Mouse Methods and Protocols, Humana Press, 2002; A. L. Joyner, Gene Targeting: A Practical Approach, Oxford University Press, 2000; Manipulating the Mouse Embryo: A Laboratory Manual, 3<rd >edition, Cold Spring Harbor Laboratory Press; Dec. 15, 2002, ISBN- 10:
0879695919; Kursad Turksen (Ed.), Embryonic stem cells: methods and protocols in Methods Mol Biol. 2002; 185, Humana Press; Current Protocols in Stem Cell Biology, ISBN: 978047015180; Meyer et al. PNAS USA, vol. 107 (34), 15022-15026.
[0048]
Generation of a transgenic mouse expressing human SCF can be achieved by methods such as DNA injection of an expression construct into a preimplantation embryo or by use of stem cells, such as embryonic stem (ES) cells or induced pluripotent stem (iPS) cells.
[0049]
The terms "expression construct" and "expression cassette" are used herein to refer to a double-stranded recombinant DNA molecule containing a desired nucleic acid coding for human SCF sequence and containing one or more regulatory elements necessary or desirable for the expression of the operably linked coding sequence. In one embodiment, the expression construct is an expression vector comprising a nucleic acid encoding human SCF operably linked to a promoter, wherein the human SCF can be expressed in a mouse when transferred into a mouse cell. In one embodiment, the expression construct is an artificial chromosome comprising human genome fragment encoding human SCF, wherein the human SCF can be expressed when the artificial
chromosome is integrated in mouse genome. The term "regulatory element" as used herein refers to a nucleotide sequence which controls some aspect of the expression of nucleic acid sequences. Exemplary regulatory elements illustratively include an enhancer, an internal ribosome entry site (IRES), an intron; an origin of replication, a polyadenylation signal (pA), a promoter, a transcription termination sequence, and an upstream regulatory domain, which contribute to the replication, transcription, post-transcriptional processing of a nucleic acid sequence. Those of ordinary skill in the art are capable of selecting and using these and other regulatory elements in an expression construct with no more than routine experimentation. Expression constructs can be generated recombinantly or synthetically using well-known methodology.
[0050]
The term "operably linked" as used herein refers to a nucleic acid in functional relationship with a second nucleic acid.
[0051]
A regulatory element is included in an expression cassette is a promoter in particular embodiments. The term "promoter" as used herein refers to a DNA sequence operably linked to a nucleic acid sequence to be transcribed such as a nucleic acid sequence encoding a desired molecule. A promoter is generally positioned upstream of a nucleic acid sequence to be transcribed and provides a site for specific binding by RNA polymerase and other transcription factors. In specific embodiments, a promoter is generally positioned upstream of the nucleic acid sequence transcribed to produce the desired molecule, and provides a site for specific binding by RNA polymerase and other transcription factors. An included promoter can be a constitutive promoter or can provide inducible expression; and can provide ubiquitous, tissue-specific or cell-type specific expression.
[0052]
Ubiquitous promoters that can be included in a human SCF expression construct include, but are not limited to, a 3-phosphoglycerate kinase (PGK-1) promoter, a beta-actin promoter, a ROSA26 promoter, a heat shock protein 70 (Hsp70) promoter, an EF-1 alpha gene encoding elongation factor 1 alpha (EF1) promoter, an eukaryotic initiation factor 4 A (eIF-4Al) promoter, a chloramphenicol acetyltransferase (CAT) promoter and a CMV (cytomegalovirus) promoter.
[0053]
Tissue-specific promoters that can be included in a human SCF expression construct include, but are not limited to, a promoter of a gene expressed in the
hematopoietic system, such as an SCF promoter, an IFN-beta promoter, a Wiskott-Aldrich syndrome protein (WASP) promoter, a CD45 (also called leukocyte common antigen) promoter, a Fit- 1 (fms-like tyrosine kinase, VEGF Receptor 1) promoter, an endoglin (CD105) promoter and an ICAM-2 (Intracellular Adhesion Molecule 2) promoter.
[0054]
These and other promoters are known in the art as exemplified in Abboud, S. L. et al, J. Histochem & Cytochem., 51(7) .941-949, 2003; Schorpp et al, Nucl. Acids Res., 24(9): 1787-1788, 19%; McBurney, M. W. et al, Devel. Dynamics, 200:278-293, 1994; and Majumder, M. et al, Blood, 87(8):3203-3211, 1996.
[0055]
In addition to a promoter, one or more enhancer sequences may be included such as, but not limited to, cytomegalovirus (CMV) early enhancer element and an SV40 enhancer element.
[0056]
Additional included sequences include an intron sequence such as the beta globin intron or a generic intron, a transcription termination sequence, and an mRNA polyadenylation (pA) sequence such as, but not limited to SV40-pA, beta-globin-pA and SCF-pA.
[0057]
An expression construct may include sequences necessary for amplification in bacterial cells, such as a selection marker (e.g. kanamycin or ampicillin resistance gene) and a replicon.
[0058]
For methods of DNA injection of an expression construct into a
preimplantation embryo, the expression construct is linearized before injection into non-human preimplantation embryos. Preferably the expression construct is injected into fertilized oocytes. Fertilized oocytes are collected from superovulated females the day after mating (0.5 dpc) and injected with the expression construct. The injected oocytes are either cultured overnight or transferred directly into oviducts of 0.5-day p.c. pseudopregnant females. Methods for superovulation, harvesting of oocytes, expression construct injection and embryo transfer are known in the art and described in Manipulating the Mouse Embryo: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press; Dec. 15, 2002, ISBN-10: 0879695919. Offspring can be tested for the presence of the transgene by DNA analysis, such as PCR, Southern blot or
sequencing. Mice which are carrying the transgene can be tested for protein expression by using such as ELISA or Western blot analysis.
[0059]
Alternatively the expression construct may be transfected into stem cells (ES cells or iPS cells) using well-known methods, such as electroporation,
calcium-phosphate precipitation and lipofection. The cells are screened for transgene integration by DNA analysis, such as PCR, Southern blot or sequencing. Cells with the correct integration can be tested for functional expression tested by protein analysis for SCF using for example ELISA or Western blot analysis.
[0060]
Mouse ES cells are grown in media optimized for the particular line. Typically ES media contains 15% fetal bovine serum (FBS) or synthetic or
semi-synthetic equivalents, 2 mM glutamine, 1 mM Na Pyruvate, 0.1 mM non-essential amino acids, 50 U/ml penicillin and streptomycin, 0.1 mM 2-mercaptoethanol and 1000 U/ml LIF (plus, for some cell lines chemical inhibitors of differentiation) in Dulbecco's Modified Eagle Media (DMEM). A detailed description is known in the art (Tremml et al., 2008, Current Protocols in Stem Cell Biology, Chapter l :Unit 1C.4. For review of inhibitors of ES cell differentiation, see Buehr, M., et al. (2003). Genesis of embryonic stem cells. Philosophical Transactions of the Royal Society B: Biological Sciences 358, 1397-1402.
[0061]
Selected cells incorporating the expression construct can be injected into preimplantation embryos. For microinjection, ES or iPS cell are rendered to single cells using a mixture of trypsin and EDTA, followed by resuspension in ES media.
Groups of single cells are selected using a finely drawn-out glass needle (20-25 micrometer inside diameter) and introduced through the embryo's zona pellucida and into the blastocysts cavity (blastocoel) using an inverted microscope fitted with micromanipulators. Alternatively to blastocyst injection, stem cells can be injected into early stage embryos (e.g. 2-cell, 4-cell, 8-cell, premorula or morula). Injection may be assisted with a laser or piezo pulses drilled opening the zona pellucida. Approximately 9-10 selected stem cells (ES or iPS cells) are injected per blastocysts, or 8-cell stage embryo, 6-9 stem cells per 4-cell stage embryo, and about 6 stem cells per 2-cell stage embryo. Following stem cell introduction, embryos are allowed to recover for a few hours at 37 °C in 5% C02, 5% 02 in nitrogen or cultured overnight before transfer into pseudopregnant recipient females. In a further alternative to stem cell injection, stem
cells can be aggregated with morula stage embryos. All these methods are well established and can be used to produce stem cell chimeras. For a more detailed description see Manipulating the Mouse Embryo: A Laboratory Manual, 3rd edition (A. Nagy, M. Gertsenstein, K. Vintersten, R. Behringer, Cold Spring Harbor Laboratory Press; Dec. 15, 2002, ISBN- 10: 0879695919, Nagy et al., 1990, Development 110, 815-821; U.S. Pat. No. 7,576,259: Method for making genetic modifications, U.S. Pat. No. 7,659,442, U.S. Pat. No. 7,294,754, Kraus et al. 2010, Genesis 48, 394-399).
[0062]
Pseudopregnant embryo recipients are prepared using methods known in the art. Briefly, fertile female mice between 6-8 weeks of age are mated with vasectomized or sterile males to induce a hormonal state conductive to supporting surgically introduced embryos. At 2.5 days post coitum (dpc) up to 15 of the stem cell containing blastocysts are introduced into the uterine horn very near to the uterus-oviduct junction. For early stage embryos and morula, such embryos are either cultured in vitro into blastocysts or implanted into 0.5 dpc or 1.5 dpc pseudopregnant females according to the embryo stage into the oviduct. Chimeric pups from the implanted embryos are born 16-20 days after the transfer depending on the embryo age at implantation. Chimeric males are selected for breeding. Offspring can be analyzed for transmission of the ES cell genome by coat color and genetic analysis, such as PCR, Southern blot or sequencing. Further the expression of human SCF can be analyzed by protein analysis (Western blot, ELISA) or other functional assays. Offspring expressing the transgene are intercrossed to create mice homozygous for the transgene. The transgenic mice are crossed to the
immunodeficient mice to create a congenic immunodeficient strain with the human SCF transgene.
[0063]
Alternatively the transgene is targeted into a specific locus of the stem cell genome which is known to result in reliable expression, such as mouse SCF, Hprt or the Rosa26 locus. Mouse SCF locus is preferable, since the expression of human SCF in the mouse, with expression pattern substantially same as that of mouse SCF in a mouse without transgene can be desired. For targeted transgenics, a targeting construct is made using recombinant DNA techniques and includes 5' and 3' sequences which are homologous to the stem cell endogenous gene. The targeting construct further includes a selectable marker such as neomycin phosphotransferase, hygromycin or puromycin, a nucleic acid encoding human SCF and a polyadenylation signal. To insure correct transcription and translation of the nucleic acid encoding human SCF, the nucleic acid
encoding human SCF is either in frame with the endogenous gene locus, or a splice acceptor site and internal ribosome entry site (IRES) sequences are included. Such a targeting construct is transfected into stem cells and the stem cells are screened to detect the homologous recombination event using PCR, Southern blot or sequencing analysis. Cells with the correct homologous recombination event can be further analyzed for transgene expression by protein analysis, such as ELISA or Western blot analysis. If desired, the selectable marker can be removed by treating the stem cells with Cre recombinase. After Cre recombinase treatment the cells are analyzed for the presence of the nucleic acid encoding xenogeneic SCF. Cells with the correct genomic event will be selected and injected into preimplantation embryos as described above. Chimeric males are selected for breeding. Offspring can be analyzed for transmission of the ES cell genome by coat color and genetic analysis, such as PCR, Southern blot or
sequencing and can be tested for SCF protein expression such as by protein analysis (Western blot, ELISA) or other functional assays. Offspring expressing the by protein analysis (Western blot, ELISA) or other functional assays are intercrossed to create non-human animals homozygous for the transgene. The transgenic mice are crossed to the immunodeficient mice to create a congenic immunodeficient strain with the human SCF transgene.
[0064]
In one embodiment, the transgenic immunodeficient mouse may comprise a human SCF transgene in substantially all of their cells. In further embodiment, the transgenic immunodeficient mouse may comprise a human SCF transgene in some, but not all their cells. One or multiple copies (such as concatamers) of the human SCF transgene may be integrated into the genome of the cells of the transgenic
immunodeficient mouse.
[0065]
In one embodiment, the present method uses a transgenic immunodeficient mouse having severe combined immunodeficiency or an IL2 receptor gamma chain deficiency in combination with severe combined immunodeficiency whose genome comprises a nucleic acid encoding human SCF operably linked to a promoter, wherein the mouse expresses the encoded human SCF.
[0066]
In one embodiment, the present method uses a transgenic immunodeficient mouse having the scid mutation or an IL2 receptor gamma chain deficiency in
combination with the scid mutation are provided according to embodiments of the
present invention whose genome comprises a nucleic acid encoding human SCF operably linked to a promoter, wherein the mouse expresses the encoded human SCF.
[0067]
In one embodiment, the present method uses a transgenic NSG mouse expressing human SCF248, human SCF220 and/or human sSCF.
[0068]
A transgenic immunodeficient mouse expressing human SCF , human SCF and/or human sSCF is generated by introduction of an expression cassette including a nucleic acid encoding an SCF protein operably linked to a promoter into cells to express the SCF protein in the transgenic mouse.
[0069]
An expression cassette can be introduced into the pronuclei of fertilized eggs of the desired immunodeficient mouse strain, such as NSG for example. The
microinjected eggs are either transferred the same day into oviducts of 0.5-day post coitus (p.c.) pseudopregnant females or cultured overnight and transferred the next day into oviducts of 0.5-day p.c. pseudopregnant females. The resulting pups are tested for presence of the transgene and expressed human SCF protein. The nucleic acid can be stably integrated into the chromosomal genome of a cell or maintained as a stable episome.
[0070]
In a further embodiment, an expression cassette can be introduced into the pronuclei of fertilized eggs of NOD-SCID (NOD.CB17-Prkdcscid/J). The microinjected eggs are either transferred the same day into oviducts of 0.5-day p.c. pseudopregnant females or cultured overnight and transferred the next day into oviducts of 0.5-day p.c. pseudopregnant female. The resulting pups are tested for presence of the transgene and those positive for the transgene can be crossed with NSG or NRG.
[0071]
The terms "expressing" and "expresses" refer to transcription of a gene to produce a corresponding mRNA and/or translation of the mRNA to produce the corresponding protein.
[0072]
Human HSC and administration of human HSC into transgenic immunodeficient mouse
The term "human HSC" as used herein refers to multipotent stem cells expressing c-Kit receptor. Examples of multipotent stem cells expressing c-Kit receptor include, but are not limited to, haematopoietic stem cells, also known as
hemocytoblasts. c-Kit receptor is well-known in the art, for example as described in Vandenbark G R et al., 1992, Cloning and structural analysis of the human c-kit gene, Oncogene 7(7): 1259-66; and Edling C E, Hallberg B, 2007, c-Kit-a hematopoietic cell essential receptor tyrosine kinase, Int. J. Biochem. Cell Biol. 39(11): 1995-8.
[0073]
Isolation of human HSC, administration of the human HSC to a host mouse and methods for assessing engraftment thereof are well-known in the art.
[0074]
Human hematopoietic stem cells for administration to a transgenic
immunodeficient mouse can be obtained from any tissue containing human HSC such as, but not limited to, human umbilical cord blood, bone marrow, GM-CSF-mobilized peripheral blood and fetal liver.
[0075]
Human HSC can be administered into newborn mouse by administration via various routes, such as, but not limited to, into the heart, liver and/or facial vein.
Human HSC can be administered into adult mouse by various routes, such as, but not limited to, administration into the tail vein, into the femur bone marrow cavity or into the spleen. Preferably, human HSC is administered into newborn mouse. Newborn mouse is preferably a mouse within two days of birth. In a further example, the HSC as fetal liver can be engrafted under the renal capsule.
[0076]
Engraftment of human HSC can be assessed by any of various methods, such as, but not limited to, flow cytometric analysis of cells in the mouse to which the human HSC are administered at one or more time points following the administration of human HSC.
[0077]
Exemplary methods for isolation of human HSC, administration of the human HSC to a host mouse and methods for assessing engraftment thereof are described herein and in T. Pearson et al., Curr. Protoc. Immunol. 81 :15.21.1-15.21.21, 2008; Ito, M. et al, Blood 100: 3175-3182; Traggiai, E. et al, Science 304: 104-107; Ishikawa, F. et al, Blood 106: 1565-1573; Shultz, L. D. et al, J. Immunol. 174: 6477-6489; Holyoake T L et al, Exp Hematol., 1999, 27(9):1418-27.
[0078]
The human HSC administered are isolated from an original source material to obtain a population of cells enriched in HSCs. The isolated human HSCs may or may
not be pure. According to embodiments, human HSCs are purified by selection for a cell marker, such as CD34. According to embodiments, administered human HSCs are a population of cells in which CD34+ cells constitute about 1-100% of total cells, although a population of cells in which CD34+ cells constitute fewer than 1% of total cells can be used. According to embodiments, administered human HSCs are T cell depleted cord blood cells in which CD34+ cells make up about 1-3% of total cells, lineage depleted cord blood cells in which CD34+ cells make up about 50% of total cells, or CD34+ positively selected cells in which CD34+ cells make up about 90% of total cells.
Preferably, administered human HSCs are lineage depleted cord blood cells in which 7AAD(-)lineage (hCD3/hCD4/hCD8/hCD19/hCD56)(-)CD34+CD38(-) cells make up about 50% of total cells, or lineage depleted and CD34+ positively selected cells in which 7AAD(-)lineage (hCD3/hCD4/hCD8/hCD19/hCD56)(-)CD34+CD38(-) cells make up about 90% of total cells. More preferably, administered human HSCs are lineage depleted and CD34+ positively selected cells in which 7AAD(-)lineage
(hCD3/hCD4/hCD8/hCDl 9/hCD56)(-)CD34+CD38(-) cells make up about 95% of total cells.
[0079]
The number of human HSCs administered is not considered limiting with regard to generation of a human hematopoietic and immune system in an
immunodeficient mouse expressing human SCF. A single HSC can generate a hematopoietic and immune system. Thus, the number of administered HSCs is generally in the range of 3x10 to 1x10 CD34+ cells per mouse, although more or fewer can be used. Preferably, the number of administered HSCs is in the range of 5xl02 to 5.3xl04 7AAD(-)lineage (hCD3/hCD4/hCD8/hCD19/hCD56)(-)CD34+CD38(-) cells.
[0080]
Prior to administration of the human HSC, conditioning with either sub-lethal irradiation of the recipient mouse with high frequency electromagnetic radiation, generally using gamma radiation, or treatment with a radiomimetic drug such as busulfan or nitrogen mustard can be applied for the engraftment of human HSC in the
immunodeficient mouse. Conditioning is believed to reduce numbers of host hematopoietic cells, create appropriate microenvironmental factors for engraftment of xenogeneic HSC, and/or create microenvironmental niches for engraftment of human HSC. Standard methods for conditioning are known in the art, such as described herein and in J. Hayakawa et al, 2009, Stem Cells, 27(1):175-182. In an embodiment of the invention, methods for producing an immune-system humanized mouse are provided
according to the present invention which include delivery of human SCF to the human HSC in the immunodeficient mice, wherein the mice are irradiated prior to
administration of the HSC. In an embodiment of the invention, methods for producing an immune-system humanized mouse is provided according to the present invention which include delivery of human SCF to the human HSC in the immunodeficient mice, wherein the mice are administered with a radiomimetic drug, such as busulfan or nitrogen mustard, prior to administration of the HSC. In an embodiment of the invention, methods for producing an immune-system humanized mouse is provided according to the present invention which include delivery of human SCF to the human HSC in the immunodeficient mice, without irradiating the mice prior to administration of the HSC. In an embodiment of the invention, methods for producing an
immune- system humanized mouse is provided according to the present invention which include delivery of human SCF to the human HSC in the immunodeficient mice, without administering a radiomimetic drug, such as busulfan or nitrogen mustard, to the mouse prior to administration of the HSC.
[0081]
Engraftment is successful where human HSCs and cells differentiated from the human HSCs in the recipient animal are detected at a time when the majority of any administered non-HSC has degenerated. Detection of differentiated HSC cells can be achieved by detection of human DNA in the recipient mouse or detection of intact human HSCs and cells differentiated from the human HSCs, for example. Serial transfer of CD34+ cells into a secondary recipient and engraftment of a human hematopoietic system is a further test of HSC engraftment in the primary recipient. Engraftment can be detected by flow cytometry as 0.05% or greater human CD45+ cells in the blood, spleen or bone marrow at 10-12 weeks after administration of the HSC.
[0082]
In particular embodiments, the immune-system humanized mouse which can be obtained by the present method is characterized by greater numbers of differentiated human hematopoietic cells in the mouse in which human stem cell factor is delivered to the human hematopoietic stem cells compared to appropriate control mouse in which human stem cell factor is not delivered to the human hematopoietic stem cells. The immune system humanized mouse which can be produced by the present method is also provided according to embodiments of the present invention.
[0083]
The term "immune-system humanized mouse" refers to a mouse comprising
human hematopoietic cells and human both acquired and innate immune cells, wherein the human hematopoietic cells and human both acquired and innate immune cells differenciated from the hematopoietic cells are surviving without being rejected from the host mouse, thereby human hematopoiesis and both acquired and innate immunity are reconstituted in the mouse. Aquired immune cells include T cells and B cells. Innate immune cells include macrophages, granulocytes (basophils, eosinophils, neutrophils), DCs, NK cells and mast cells.
[0084]
In particular embodiments, the immune-system humanized mouse of the present invention has
greater human CD45+ leukocyte population in bone marrow, spleen and peripheral blood,
greater human CD33+ myeloid cell population in bone marrow human CD45+ leukocytes, and
- greater human CD203c+c-kit+ mast cell population in splenic human
CD45+CD33+ myeloid cells
as compared to a control immune-system humanized mouse prepared by administering the human hematopoietic stem cells to a transgenic immunodeficient mouse which does not express the human Stem Cell Factor. The comparison is performed at 12 weeks after administration of the HSC.
[0085]
Preferably, a difference between the immune-system humanized mouse of the present inveniton and the control immune-system humanized mouse exists only in that the transgenic immunodeficient mouse used for prepareing the immune-system humanized mouse of the present inveniton comprises a nucleic acid encoding human Stem Cell Factor operably linked to a promoter in the genome and expresses the human Stem Cell Factor, whereas the transgenic immunodeficient mouse used for prepareing the control immune-system humanized mouse does not comprise a nucleic acid encoding human Stem Cell Factor operably linked to a promoter in the genome and does not express the human Stem Cell Factor.
[0086]
In particular embodiments, engraftment level of human CD45+ cells
(calculated as % hCD45+ cells relative to total numbers of mouse and human CD45+ cells in the nucleated cell, gate) in the bone marrow of the immune-system humanized mouse of the present inveniton is generally 70% or more, preferably 80% or more, more
preferably 90% or more, particularly preferably 95% or more. The engraftment level of human CD45+ cells in spleen of the immune-system humanized mouse of the present inveniton is generally 70% or more, preferably 80% or more, more preferably 85% or more, more preferably 90% or more. The engraftment level of human CD45+ cells in peripheral blood of the immune-system humanized mouse of the present inveniton is generally 60% or more, preferably 70% or more, more preferably 80% or more.
[0087]
In particular embodiments, the frequency of human CD33+ myeloid cells within the total human CD45+ population in the bone marrow of the immune-system humanized mouse of the present inveniton is generally 30% or more, preferably 40% or more, more preferably 45% or more.
[0088]
In particular embodiments, the frequency of human cKit+CD203c+ mast cells within the total human CD45+CD33+ myeloid cells in the spleen of the immune-system humanized mouse of the present inveniton is generally 65% or more, preferably 70% or more, more preferably 75% or more.
[0089]
In particular embodiments, the frequency of human c-kit+CD203c+ mast cells within the total human CD33+ population in the bone marrow of the immune-system humanized mouse of the present inveniton is greater than 15 %.
[0090]
Methods and immune-system humanized mouse provided by embodiments of the present invention have various utilities such as, but not limited to, as models of growth and differentiation of immune cells, in vivo study of immune response and for the testing of agents affecting hematopoietic and immune cell function. Particularly, in the immune-system humanized mouse of the present invention transgenic expression of human SCF results in the efficient development of human myeloid cells and human mast cells in hematopoietic organs and mucosal tissues (i.e. respiratory mucosa, gastric tissue) and achieves high chimerism of human hematopoietic cells in hematopoietic organs. Accordingly, the method and immune-system humanized mouse provided by
embodiments of the present invention is useful as a model of growth and differentiation of human myeloid cells and/or human mast cells, in vivo study for testing of agents affecting human myeloid cells and/or human mast cells and agents for preventing or treating human immune/allergic/inflammatory disease relating myeloid cells and/or mast cells. Such disease includes type-I allergic disease (i.e. asthma, atopic dermatitis,
allergic conjunctivitis, pollen allergy, food allergy).
[0091]
Accordingly, the present invention also provides a method of screening for a substance capable of preventing or treating immune/allergic/inflammatory disease relating myeloid cells and/or mast cells, comprising applying a test substance to the above-described immune-system humanized mouse provided by embodiments of the present invention or portion thereof affected with the immune/allergic/inflammatory disease relating myeloid cells and/or mast cells, and evaluating whether or not the test substance improves a condition or symptom of the immune/allergic/inflammatory disease. Portions of the immune-system humanized mouse includes isolated tissues comprising human myeloid cells and/or human mast cells (i.e. hematopoietic tissues, mucosal tissues (i.e. respiratory mucosa, gastric tissue)), isolated human cells (i.e. human myeloid cells, human mast cells), and the like.
[0092]
The test substance subjected to the screening method of the present invention may be any commonly known compound or a novel compound; examples include nucleic acids, sugars, lipids, proteins, peptides, organic low molecular compounds, compound libraries prepared using combinatorial chemistry technology, random peptide libraries, or naturally occurring ingredients derived from microorganisms, animals, plants, marine organisms and the like, and the like.
[0093]
In a embodiment, the test substance is administered to the immune-system humanized mouse provided by embodiments of the present invention or a portion thereof, and a condition or symptom of the immune/allergic/inflammatory disease relating myeloid cells and/or mast cells in the mouse or portion thereof is compared with the condition or symptom of a control mouse not administered with the test substance. Conditions or symptoms of the immune/allergic/inflammatory disease relating myeloid cells and/or mast cells includes, but are not limited to, release of chemical mediators (histamine and the like) or cytokines from myeloid cells and or mast cells, conditions or symptoms due to said chemical mediators or cytokine.
[0094]
The comparison of the conditions or symptoms can be made preferably on the basis of the presence or absence of a significant difference. Although the condition or symptom in the control mouse or portion thereof not administered with the test substance may be a condition or symptom measured before or simultaneously with the
measurement of the condition or symptom in the mouse or portion thereof administered with the test substance, it is preferable, from the viewpoint of experimental accuracy and reproducibility, that the former condition or symptom be a simultaneously measured.
[0095]
Then, a substance that improves the condition or symptoms in the mouse or protion thereof, obtained as a result of the comparison, is selected as a candidate substance for a drug for preventing or treating the immune/allergic/inflammatory disease.
[0096]
Any patents or publications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication is specifically and individually indicated to be incorporated by reference.
[0097]
Embodiments of the invention are illustrated in the following examples. These examples are provided for illustrative purposes and are not considered limitations on the scope of invention.
EXAMPLES
[0098]
Methods
Mice
NOO.Cg-Prkdcscid IL2rgtmlWjl (NSG) mice and NOO.Cg-Prkdcscid IL2rgtmlWjl Tg(PGKl-KITLG*220)441Daw/J, abbreviated as hSCF Tg NSG mice, were generated at The Jackson Laboratory. The human membrane bound SCF transgene driven by the human PGK promoter was backcrossed more than 10 generations from the original
13
C3H/HeJ strain background onto the NSG strain. All the mice were bred and maintained at The Jackson Laboratory and animal facility at RIKEN RCAI under defined flora according to guidelines established by the Institutional Animal Committees at each respective institution.
[0099]
Purification and transplantation of human HSCs
All experiments were performed with authorization from the Institutional Review Board for Human Research at RIKEN RCAI. Cord blood (CB) samples were first processed for isolation of MNCs using LSM lymphocyte separation medium (MP Biomedicals). CB MNCs were then enriched for human CD34+ cells by using anti -human CD34 microbeads (Miltenyi Biotec) and sorted for 7AAD(-)lineage
(hCD3/hCD4/hCD8/hCD19/hCD56)(-)CD34+CD38(-) HSCs using FACSAria (BD Biosciences). To achieve high purity of donor HSCs, doublets were excluded by analysis of FSC-height/FSC-width and SSC-height/SSC-width. Purity of each sorted sample was higher than 95%. Newborn (within two days of birth) hSCF Tg and non-Tg NSG recipients received 150 cGy total body irradiation using a 137Cs-source irradiator, followed by intravenous injection of 5xl 02-5.3xl04 sorted HSCs via the facial vein.
[0100]
Analysis of human cell engraftment by flow cytometry
The recipient peripheral blood (PB) harvested from the retro-orbital plexus was evaluated for human hematopoietic engraftment every three to four weeks starting at four weeks post-transplantation. After lysis of erythrocytes, cells were stained with anti-hCD45, anti-msCD45, anti-hCD3, anti-hCD19, anti-hCD33, and anti-hCD56 to determine human hematopoietic chimerism and to analyze cell lineages engrafted in the recipients. At 8-35 weeks post-transplantation, the recipients were euthanized and single cell suspensions of BM and spleen were analyzed using flow cytometry.
Antibodies used for flow cytometry are specified in Supplemental Methods. The labeled cells were analyzed using FACSCantoII or FACSAria (BD).
[0101]
Morphological analysis of cytospin specimens
Cytospin specimens of FACS-purified human myeloid cells were prepared with a Shandon Cytospin 4 cytocentrifuge (Thermo Electric) using standard procedures. To identify nuclear and cytoplasmic characteristics of each myeloid cell, cytospin specimens were stained with 100% May-Grunwald solution (Merck) for 3 minutes, followed by 50% May-Grunwald solution in phosphate buffer (Merck) for additional 5 minutes, and then with 5% Giemsa solution (Merck) in phosphate buffer for 15 minutes. All staining procedures were performed at room temperature. Light microscopy was performed with Zeiss Axiovert 200 (Carl Zeiss).
[0102]
Microarray analysis
Purified hCD45+CD33+c-Kit(-)CD203c(-)HLA-DR(-) granulocytes and hCD45+CD33+ c-Kit(-)CD203c(-)HLA-DR+CD14+ monocytes from BM of four hSCF Tg NSG recipients and three non-Tg NSG recipients as well as neutrophils and monocytes from two healthy individuals were evaluated using Human Genome U133 plus 2.0 GeneChips (Affymetrix, USA). Total RNA was extracted with TRIzol
(Invitrogen, USA) from > 104 sorted cells, and amplified to cDNA using the Ovation
Pico WTA System (Nugen, USA). Biotinylated cDNA was synthesized with Two-Cycle Target Labeling Kit (Affymetrix). Microarray data were analyzed using the
Bioconductor package (Bioconductor, http://www.bioconductor.org/). The signal intensities of the probe sets were normalized using the GC-RMA program (Bioconductor, http://www.bioconductor.org/). The RankProd program was used to select differentially expressed genes with a cutoff p-value of <0.01 and an estimated false-positive rate of <0.0514. Gene annotation was obtained from Ingenuity Pathway Analysis and Gene Ontology Annotation databases (Ingenuity systems, http://www.ingenuity.com; Gene Ontology Annotation, http://www.ebi.ac.up/GOA/). For differentially transcribed genes, GO term enrichment analysis was performed according to a method described by
Draghici et a/.15 with a correction of multiple testing using false discovery rate (FDR)16. Eventually, GO terms with the FDR corrected p-value <0.05 were selected as
functionally enriched terms. Raw data for microarray analysis is in the process of being deposited into a public database and series accession numbers will be available by the time of publication. Differences in expression levels were considered significant if p<0.05 using either Kruskal-Wallis, Wilcoxon-Mann- Whitney or student's t-test in KaleidaGraph (Synergy Software, USA).
[0103]
Immunohistochemistry (IHC) and immunofluorescence imaging
Thin (~5um) sections prepared from paraformaldehyde-fixed
paraffin-embedded tissues were stained with H&E using standard procedures. IHC and immunofluorescence labeling were performed using standard procedures. Antibodies used for IHC and immunofluorescence labeling were mouse anti-human mast cell tryptase monocolonal antibody (Dako, clone AAl), mouse anti-human CD45 monoclonal antibody (Dako, clone 2B11+PD7/26), rabbit anti-human CD117 monoclonal antibody (Epitomics, clone YR145) and rabbit anti-human CD 14 polyclonal antibody (Atlas Antibodies). Light microscopy was performed using an Axiovert 200 (Carl Zeiss).
For quantification of tryptase+ cell frequency, three high-power fields from three different recipients were examined using AutoMeasure module of Axio Vision software (Release 4, Carl Zeiss). Confocal microscopy analysis was performed using a LSM710 (Carl Zeiss).
[0104]
Antibodies used for flow cytometry
The following monoclonal antibodies were used for the identification of human hematopoietic lineages and myeloid subsets: hCDlc (BDCA-l-FITC) (Miltenyi Biotec,
clone AD5-8E7), hCD3-V450 (BD Biosciences, clone UCHTl), hCDl lc-APC (BD Biosciences, clone B-ly6), hCD19-PE-Cy7 (BD Biosciences, clone SJ25C1), hCD14-Alexa700 (BD Biosciences, clone M5E2), hCD15-APC (BD Biosciences, clone HI98), hCD33-PE (BD Biosciences, clone WM53), hCD33-PE-Cy7 (BD Biosciences, clone P67.6), hCD45-APC (BD Biosciences, clone HI30), hCD45-V450 (BD Biosciences, clone HI30), hCD56-FITC (BD Biosciences, clone NCAM16.2), hCD117-PE (BD Biosciences, clone YB5.B8), hCD117-PerCP-Cy5.5 (BD Biosciences, clone 104D2), hCD123-PerCP-Cy5.5 (BD Biosciences, clone 7G3), hCD141 (BDCA-3-PE) (Miltenyi Biotec, clone AD5-14H12) , hCD203c-PE (BioLegend, clone NP4D6), hHLA-DR-APC-H7 (BD Biosciences, clone L243(G46-6)), mCD45-APC-Cy7 (BD Biosciences, clone 30-F11).
[0105]
Results
Human hematopoietic repopulation is enhanced in hSCF Tg NSG recipients
The humanized mouse model system has served as a tool to investigate human hematopoiesis, immunity and diseases in vivo. However, one of the major limitations in the system is that the microenvironment supporting human hematopoiesis and immunity is primarily of mouse origin. In the present study, the present inventors created a strain of NSG mice expressing membrane-bound human SCF (hSCF) to analyze the role of the BM microenvironment in human hematopoietic lineage
determination and development.
[0106]
c-Kit, the receptor for SCF, is expressed at low levels in human CB
Lin-CD34+CD38- early HSCs and at high levels in mast cells17"19. For reconstitution of human myeloid and lymphoid cells, 5xl02-5.3xl04 FACS-purified CB
Lin-CD34+CD38- HSCs were transplanted into newborn sublethally irradiated (1.5 Gy) hSCF Tg NSG mice and into non-Tg NSG controls (Table 1).
Table 1 Summary of hSCF Tg MSG and non-Tg NSG recipients analyzed
[0107]
Table 1 Summary of hSCF Tg NSG and non-Tg NSG recipients analyzed.
A total of 21 human HSC-engrafted hSCF Tg NSG (S) recipients and 15 human HSC-engrafted non-Tg NSG (N) recipients were created. WBC: white blood cell count; RBC: red blood cell count; HGB: hemoglobin concentration; HCT: hematocrit value; PLT: platelet count
[0108]
To determine the kinetics of human hematopoietic chimerism in the recipient circulation, the present inventors performed flow cytometric analysis of peripheral blood every 3-4 weeks starting at 4 weeks post-transplantation. During long-term observation, all the 21 hSCF Tg NSG recipient mice became moribund at 8-35 weeks
post-transplantation. Complete blood count analysis demonstrated reduced erythrocyte hemoglobin concentration in the peripheral blood of hSCF Tg NSG recipients compared with non-Tg NSG recipients. Anemia in hSCF Tg NSG recipients was not associated with abnormalities in MCV, MCH or MCHC (Figure 6). The suppression of host erythropoiesis in the hSCF Tg NSG recipients was related to the irradiation and engraftment of the human HSCs, since unmanipulated non-transplanted hSCF Tg NSG mice did not develop anemia (Figure 6). Impaired mouse erythropoiesis in these engrafted hSCF Tg NSG mice was associated with rapid expansion of hCD45+
hematopoietic cells compared with non-Tg NSG recipients (Figure IB).
[0109]
At 8-35 weeks post-transplantation, individual hSCF Tg NSG recipients were analyzed to determine levels of reconstitution of human hematopoiesis and immunity in the BM, spleen and PB. At the time of necropsy, the present inventors did not observe any gross macroscopic abnormalities in these recipients. The present inventors performed flow cytometric analysis to evaluate the engraftment levels of human CD45+ cells (calculated as %hCD45+ cells relative to total numbers of mouse and human
CD45+ cells in the nucleated cell gate). Engraftment levels of human CD45+
leukocytes in the BM, spleen and PB were significantly higher in hSCF Tg NSG recipients (mean value +/- standard error; 97.1 +/- 1.1%, 94.5 +/- 1.6%, 83.1 +/- 3.9%, respectively; n=21) compared with engrafted non-Tg NSG recipients (63.1 +/- 7.3%, 77.3 +/- 6.9%, 49.5 +/- 6.5%, respectively; n=13) (pO.0001, p=0.0065, pO.0001 by two-tailed t test, respectively) (Figure 1C, D). Compared with enhanced engraftment of human leukocytes in the recipient BM, development of human erythroid precursors was not significantly different in hSCF Tg NSG recipients compared with non-Tg NSG
recipients (Figure 7). The present inventors next analyzed the development of human lymphoid and myeloid cells in the engrafted human CD45+ hematopoietic cell populations by flow cytometry using monoclonal antibodies against hCD3, hCD19, hCD56, and hCD33 along with anti-human and anti-mouse CD45 antibodies (Figure 1C, E). While there was recipient-to-recipient variability, the frequency of human CD33+ myeloid cells within the total human CD45+ population was significantly higher in the hSCF Tg NSG recipient BM than in the non-Tg NSG recipient BM and constituted the majority of human hematopoietic cells (hSCF Tg: 49.7 +/- 4.0%; n=21 and non-Tg NSG controls: 26.2 +/- 2.9%; n=13) (p=0.0002 by two-tailed t test). In contrast, in non-Tg NSG recipients, the majority of human hematopoietic cells in the BM were B cells (53.3 +/- 4.5%), consistent with previous reports5'9'20. These findings demonstrate that the expression of membrane-bound hSCF in BM microenvironment results in significantly more efficient engraftment of human HSCs as well as enhanced development of human CD33+ myeloid cells from the engrafted human HSCs.
[0110]
Human myeloid lineage development in hSCF Tg NSG recipients
Next, the present inventors examined the development of human myeloid subsets in the human membrane-bound SCF-expressing BM microenvironment. Flow cytometry scatter plots demonstrate the development of the side-scatter high granulocyte fraction in hSCF Tg NSG recipients which correlate with CD33+ HLA-DR(-) cells (Figure 2A, B). To quantify the frequencies of different myeloid subsets in these recipients, the present inventors first identified CD33+c-Kit+CD203c+ mature human mast cells among human CD45+ cells. Next, the present inventors identified
CD33+HLA-DR(-) human granulocyte lineage cells and human CD33+HLA-DR+ antigen presenting cells (APCs) among human CD45+ cells excluding mature mast cells (Figure 2C).
[0111]
In the hSCF Tg NSG recipient BM, there were increased percentages of CD33+HLA-DR(-) granulocytes and decreased percentages of CD33+HLA-DR+ APCs compared with non-Tg NSG controls (hSCF Tg: 46.7 +/- 5.9% and 38.3 +/- 4.0%, respectively; n=20 and non-Tg NSG controls: 25.8 +/- 5.0% and 65.5 +/- 4.1%, respectively; n=12) (p=0.0204 and p=0.0001 by two-tailed t test, respectively) (Figure 2D). In the BM of 11 out of 20 hSCF Tg NSG recipients examined,
c-Kit(-)CD203c(-)HLA-DR(-)side scatter (high) granulocytes accounted for the highest frequency of total human myeloid cells (Figure 2D, Table 1). To examine the
morphological features of the human granulocytes developing in the hSCF Tg recipients, the present inventors carried out May-Grunwald Giemsa (MGG) staining using cytospin specimens of FACS-purified CD33+c-Kit(-)CD203c(-)HLA-DR(-) cells from BM of hSCF Tg and non-Tg NSG recipients. In nine out of 13 hSCF Tg recipient BM cells examined, the majority of myeloid cells showed the morphology of immature
granulocytes with large nuclear-to-cytoplasmic ratio and nuclei with few lobulations, largely consisting of myelocytes and metamyelocytes (S4-1 and SI 2-2 shown as representative in Figure 2E). In four out of 13 hSCF Tg recipients examined and in four out of five non-Tg NSG recipients, mature segmented neutrophils were present in the sorted CD33+c-Kit(-)CD203c(-)HLA-DR(-) granulocyte population (N12-1 and S2-1 shown as representative in Figure 2E). These findings indicate that both by quantitative and morphological examinations, human granulocytic cells with various degrees of maturity predominate among the CD33+ myeloid cells developing within the hSCF Tg NSG recipients. To examine the myeloid differentiation capacity of hematopoietic stem and progenitor populations in a functional manner, the present inventors performed a colony-forming cell (CFC) assay using CD34+CD38- and CD34+CD38+ cells derived from BM of hSCF Tg NSG recipients and non-Tg NSG recipients. In both cell populations, myeloid and erythroid colony formation were similar between hSCF Tg NSG and non-Tg NSG recipient BM (Figure 8).
[0112]
The present inventors then analyzed global transcriptional profiles of
CD33+HLA-DR(-)c-Kit(-)CD203c(-) granulocytes from hSCF Tg NSG recipient BM (n=4), non-Tg NSG recipient BM (n=3) and primary human BM (n=2). Additional control samples included BM monocytes from hSCF Tg recipients (n=3), non-Tg NSG recipient BM monocytes (n=3), and primary human BM monocytes (n=2).
Unsupervised clustering demonstrated a clear segregation of transcriptional profiles between granulocytes and monocytes regardless of the source. This suggests that human granulocytes and monocytes in humanized mouse undergo distinct differentiation process similar to their counterparts in human BM (Figure 2F). The present inventors next examined whether there were any differences in gene expression within three distinct granulocyte sources (hSCF Tg recipient BM, non-Tg NSG recipient BM and primary human BM neutrophils) (Figure 2G). As seen in the heat-map representation, the present inventors found clusters of genes differentially transcribed in the distinct sources of granulocytes (Figure 2G). Multiple genes associated with transcriptional regulation were included in the genes up-regulated in human immature granulocytes
derived from the BM of hSCF Tg NSG mice, suggesting that these cells are more actively cycling and proliferating compared with mature granulocytes from the BM of hSCF Tg NSG and non-Tg NSG mice and primary human BM neutrophils (Figure 2G, Tables 2 and 3).
Table 2-1 Genes over-represented in immature granulocytes
probelD symbol i Description
218395 at ACTR6 ARP6 a *n-related protein 6 homolog (yeast)
205209 at ACVR1B activin A receptor, type IB
218568 at AGK acylglycerol kinase
20 952 at ALCAM activated leukocyte cell adhesion molecule
218093~s at A KRD10 ankyiin repeat domain 10
205423 at AP1B1 adaptor-related protein complex 1, beta 1 subunit
241701 at ARHGAP21 Rho GTPase activating protein 21
226871 s at ATG4D ATG4 autophagy related 4 homolog D (S. cerevisiae)
223452 s at ATL3 atiastin GTPase 3
213366 x at ATP5C1 ATP synthase, H+ transporting, mitochondrial F1 complex, gamma polypeptide 1
226463 at ATP6V1C1 ATPase, H+ transporting, lysosomal 42kDa, V1 subunit C1
224729 s at ATPAF1 ATP synthase mitochondrial F1 complex assembly factor 1
224677 x at C 1orf31 chromosome 11 open reading frame 31
219526 at C14orf169 chromosome 14 open reading frame 169
203173 s at C16orf62 chromosome 16 open reading frame 62
225749 at C16orf91 chromosome 16 open reading frame 91
223401 at C17orf48 chromosome 1 open reading frame 48
224574 at C17orf49 chromosome 1 open reading frame 49
212055 at C18orf10 chromosome 18 open reading frame 10
225841 at C1orf59 chromosome 1 open reading frame 59
200042 at C22orf28 chromosome 22 open reading frame 28
219176 at C2orf47 chromosome 2 open reading frame 47
224604 at C4orf3 chromosome 4 open reading frame 3
226385 s at C7ori30 c romosome 7 open reading frame 30
218992 at C9orf46 chromosome 9 open reading frame 46
218545 at CCDC91 coiled-coil domain containing 91
200812 at CCT7 chaperonin containing TCP1, subunit 7 (eta)
202256 at CD2BP2 CD2 (cytoplasmic taiQ binding protein 2
217880 at CDC27 cell division cycle 27 homolog (S. cerevisiae)
218062 x_at CDC42EP4 CDC42 effector protein (Rho GTPase binding) 4
220935 s at CDK5RAP2 CDK5 regulatory subunit associated protein 2
204203 at CE8PG CCAAT/enhancer binding protein (C EBP), gamma
219472 at CENPO centromere protein O
226449 at CEP120 cerrtrosomal protein 1 0kQa
218572 at CH P4A chromatin modifying protein 4A
218597 s at CISD1 CDGSH iron sulfur domain 1
214252~s_at CLN5 ceroid-lipofuscinosis, neuronal 5
202799 at CLPP ClpP caseinolytic peptidase, ATP-dependerrt, proteolytic subunit homolog (E. coli)
226702 at CMPK2 cytidine monophosphate (UMP-CMP) kinase 2, mitochondrial
226017 at C TM7 CKLF-like MARVEL transmembrane domain containing 7
201913 S_at COASY CoA synthase
222637 at COM D10 COMM domain containing 10
201405 s at COPS6 C0P9 constitutive photomorphogenic homotog subunit 6 (Anabidopsis)
202646_S_at ICSDE1 fcold shock domain containing E1, RMA-btnding
201633 S at CYB5B j cytochrome b5 type B (outer mitochondrial membrane)
202314 aT CYP51A1 (cytochrome P450, family 51, subfemil A, polypeptide 1
201095 at DAP death-associated protein
209389 x_at DBl Idiazepam binding inhibitor (GABA receptor modulator, acyi-CoA binding protein) 201082 s at DCTN1 idynactin 1
203261_at DCTN6 jdynactin 6
208896 at DDX18 iDEAD (Asp-Glu-AIa-Asp) box polypeptide 18
202577_js_at DDX19A iDEAD (Asp-Glu-AIa-As) box polypeptide 19A
202534_x_at DHFR dihydrotblate reductase
207831 x_at DHPS deoxyhypusine synthase
218409_s_at DNAJC1 DnaJ (Hsp40) homolog, subfamily C, member 1
212911_at DNAJC16 DnaJ (Hsp40) homolog, subfamily C, member 16
202416 at DMAJC7 DnaJ (Hsp40) homolog, subfamily C, member 7
223446_s at DT BP1 dystrobrevin binding protein 1
200703_at" DYNLL1 dynein, light chain, LC8-type 1
201 43 s_at EIF2S1 eukaryotic translation initiation factor 2, subunit 1 alpha, 35kDa
208756~at E1F3I eukaryotic translation initiation factor 3, subunit I
201303_at E1F4A3 eukaryotic translation initiation factor 4A3
208625~S_at E1F4G1 eukaryotic translation initiation factor 4 gamma, 1
210213_s_at E1F6 eukaryotic translation initiation factor 6
202012_s_at EXT2 exostosin 2
208623_s_at EZR ezrin
203262_s_at FAM50A family with sequence similarity 50, member A
20 275 at FDPS famesyl diphosphate synthase
231846 at FOX ED2 FAD-dependent oxidoreductase domain containing 2
200959 at FUS j fused in sarcoma
204618_s_at GABPB1 ;GA binding protein transcription factor, beta subunit 1
207574 s_at GADD45B I growth arrest and D A-damage-inducibte, beta
201816_s_at GBAS glioblastoma amplified sequence
232296_s_at GFM1 G elongation factor, mitochondrial 1
201576 s_at GLB1 galactosidase, beta 1
202678_at GTF2A2 general transcription factor ilA, 2, 12kDa
200075 s at GU 1 guanylate kinase 1
20l145lat HAX1 HCLS1 associated protein X-1
209314_s_at HBS1L HBS1-like (S. cerevisiae)
223908 at HDAC8 histone deacetylase 8
221896 s at HIGD1A HIG1 hypoxia inducible domain family, member A
201277_s_at HNRNPAB heterogeneous nuclear ribonucleoprotein PJB
202557_at HSPA13 heat shock protein 70kDa femily, member 13
226887 at HSPA1 heat shock 70kDa protein 14
204868_at ICT1 immature colon carcinoma transcript 1
208881 x_ at 1D11 isopentenyl-diphosphate delta isomerase 1
226450~at INSR insulin receptor
2 713 x_at KIAA0101 ΪΚΙΑΑ0101
209654 at KIAA0947 jKIAA0947
228334_x_at KIAA1712 ΪΚΙΑΑ1712
221219_S_at LHDC4 kelch domain containing 4
200914 X_at ΚΓΝ1 kinectin 1 (kinesin receptor)
210732_s at LGALS8 lectin, galactoside-birtding, soluble, 8
212S58 at" L.HFPL2 lipoma H GIC fusion partner-like 2
222099_S 3t LS 14A LSM14A, SCD6 homolog A (S. cerevisiae)
225469_aT LYR 5 LYR motif containing 5
212567_s_at MAP4 microtubule-assodated protein 4
203218_at APK9 imitogen-activatsd protein kinase 9
200712_s_at IMAPRE1 microtubule-associated protein, RP EB family, member 1
203496_s_at mediator complex subunit 1
222567_s_at EX3C mex-3 homolog C (C. elegans)
222997_s at RPS21 mitochondrial ribosomal protein SZ1
226257_x_at MRPS22 mitochondrial ribosomal protein S22
2135 1_s_3t MTMR1 myotubularin related protein 1
2041 3_at YL6B myosin, light chain 6B, alkali, smooth muscle and non-muscle
225344_at NCOA7 nudear receptor coactivator 7
217860_at NDUFA10 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 2kDa
218563_at NDUFA3 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 3, 9kDa
203478_at NDUFC1 NADH dehydrogenase (ubiquinone) 1, subcomplex unknown, 1, 6kDa
203039 s_at NDUFS1 NADH dehydrogenase (ubiquinone) Fe-S protein 1 , 75kDa (NADH-coenzyme Q reductase)
218860_at NOC4L nucleolar complex associated 4 homolog (S. cerevisiae)
229353 s_at NUCKS1 nuclear casein kinase and cyclin-dependerrt kinase substrate 1
210574_s_at NUDC nuclear distribution gene C homolog (A. nidulans)
202153 s_at NUP62 nucleoporin 62kDa
229624_at OPA3 optic atrophy 3 (autosomal recessive, with chorea and spastic paraplegia)
210283_x_at PAIP1 poly(A) binding protein interacting protein
200006_at PARK? Parkinson disease (autosomal recessive, early onset) 7
210023_s_at PCGF1 polycomb group ring finger 1
204025 s_at PDCD2 programmed cell death 2
223037_at PD2D11 PDZ domain containing 11
2035 8 s_at PER2 period homolog 2 (Drosophila)
213239_at PIBF1 progesterone immunomodulatory binding factor 1
217806_s at POLDIP2 polymerase (DNA-directed), detta interacting protein 2
203664~s~at POLR2D polymerase (RNA) II (DNA directed) polypeptide D
202868~s~at POP4 processing of precursor 4, rioonuetease P MRP subunit (S. cerevisiae)
219875_s at PPPDE1 PPPDE peptidase domain containing 1
39729_at PRDX2 peroxiredoxin 2
202408_s_at PRPF31 PRP31 pre-mRNA processing factor 31 homolog (S. cerevisiae)
213729_at PRPF40A PRP40 pre-mRNA processing factor 0 homolog A (S. cerevisiae)
201576 x at PSMA1 proteasome (prosome, macropain) subunit, alpha type, 1
208805_aF PSMA6 proteasome (prosome, macropain) subunit, alpha type, 6
208827 at PSMB6 proteasome (prosome, macropain) subunit, beta type, 6
209503_s at PS C5 proteasome (prosome, macropain) 26S subunit, ATPase, 5
200830 aT PSMD2 proteasome (prosome, macropain) 26S subunit, non-ATPase, 2
201388_at PSMD3 proteasome {prosome, macropain) 26S subunit, non-ATPase, 3
201052 s_at PSMF1 proteasome (prosome, macropain) inhibitor subunit 1 (P131)
202716_at PTPN1 protein tyrosine phosphatase, non-receptor type 1
213136 at FTPN2 I protein tyrosine phosphatase, non-receptor type 2
209899 s at PUF60 jpoly-U binding splicing factor 60 Da
217597 x at RAB40B IRAB40B, member RAS oncogene family
201039 s at AD23A i AD23 homolog A (S. cerevisiae)
205740 s at BM42 I RNA binding motif protein 42
213404 s at RHE B fRas homolog enriched in brain
204208 at RNGTT RNA guanytyltransferase and 5'-phosphatase
206050 s at RNH1 ribonudease/angiogenin inhibitor 1
21829 at R0BLD3 roadblock domain containing 3
221593 s_at RPL31 ribosoma! protein L31
209360 s at RUNX1 rurrt-related transcription factor 1
1554482 a at SAR1B SAR1 homolog B (S. cerevisiae)
202361 at SEC24C SEC24 family, member C (S. cerevisiae)
214298 x at SEPT6 septin 6
212465 at SETD3 SET domain containing 3
214197 s at SETDB1 SETT domain, bifurcated 1
226639 at SFT2D3 SFT2 domain containing 3
225619~at SLAIN1 SLAIN motif family, member 1
223222 at SLC25A19 solute carrier family 25 (mitochondrial thiamine pyrophosphate earner), member 19
224626 at SLC35A4 solute carrier family 35, member A4
205398 s at SMAD3 S AD family member 3
227607 at STAMBPL1 STAM binding protein-like 1
215004 s at SUGP1 SURP and G patch domain containing 1
204295 at SURF1 surfeit 1
222978 at SURF4 surfeit 4
218996 at TFPT TCF3 (E2A) fusion partner (in childhood Leukemia)
218118 s at TI M23 Iranslocase of inner mitochondrial membrane 23 homolog (yeast)
228619 x at TIPRL TIP41, TOR signaling pathway regulator-like (S. cerevisiae)
218930 s at TMEM106B transmembrane protein 106B
223335 at TMEM69 transmembrane protein 69
223324 s at TRPM7 transient receptor potential cation channel, subfamily M, member 7
244189 at TTC28AS TTC28 arrb'sense RNA (non-protein coding)
201113 at TUF Tu translation elongation factor, mitochondria!
223017~at TXNDC12 thioredoxin domain containing 12 (endoplasmic reticulum)
213535 s at UBE21 ubiquitin-conjugating enzyme E2l (UBC9 homolog, yeast)
213128 s at UBE3A ubiquitin protein ligase E3A
208998 at UCP2 uncoupling protein 2 (mitochondrial, proton earner)
212600 s at UQCRC2 ubiquinol-cytochrome c reductase core protein II
212399 s at VGLL4 vestigial like 4 (Drosophila)
212880 at WDR7 WD repeat domain 7
221193 s at ZCCHC10 zinc finger, CCHC domain containing 10
201857 at ZFR zinc finger RNA binding protein
215239 x at ZNF273 zinc finger protein 273
23851 oTat Z F720 Izinc finger protein 720
218916 at ZNF768 izinc finger protein 768
Table 2-2 Genes under-represented in immature granulocytes
probelD symbol Description
224455 s at ADPG ADP-dependent glucokinase
1555536 at ANTXR2 anthrax toxin receptor 2
224451 x at ARHGAP9 Rho GTPase activating protein 9
210828 s at ARNT aryl hydrocarbon receptor nuclear translocates
243829_at BRAF v-raf murine sarcoma viral oncogene homolog B1
231860 at BRWD1 bromodomain and WD repeat domain containing 1
229514 at C14orf118 chromosome 14 open reading frame 118
1553338 at C1orf55 chromosome 1 open reading frame 55
1560558_at C9orf80 chromosome 9 open reading frame 80
203357 s at CAPN7 ca!pain 7
203450 aT CBY1 chibby homolog 1 (Drosophila) .
208783 s at CD46 CD46 molecule, complement regulatory protein
235226 aT CDK19 cyclin-dependent kinase 19
236023 at CDK9 cyclin-dependent kinase 9
1554052 at CNOT1 CCR4-NOT transcription complex, subunit 1
1554339 a at COG3 component of oligomeric golgi complex 3
208719 s at DDX17 DEAD (Asp-Glu-Ala-Asp) box polypeptide 17
212514 x at DDX3X DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, X-linked
242970 aT DIP2B DIP2 disco-interacting protein 2 homolog B (Drosophila)
220572 at DKFZp547G183 hypothetical LOC55525
1554534 at DPYD dihydropyrim'tdine dehydrogenase
229017 s at DSTYK dual serine threonine and tyrosine protein kinase
208706 s at BF5 eukaryotic translation initiation factor 5
1556357 s at ER!CHI glutamate-rich 1
218297 at FAM188A family with sequence similarity 188, member A
204072 s at FRY furry homolog (Drosophila)
231918 s at GF 2 G elongation factor, mitochondrial 2
222109 aT GNL3L guanine nucleotide binding protein-like 3 (nudeolar)-like
220265 at GPR107 G protein-coupled receptor 107
226840 at H2AFY H2A histone family, member Y
212291 at HIPK1 homeodomain interacting protein kinase 1
225107 at HNRNPA2B1 heterogeneous nuclear ribonucleoprotein A2/B1
203204 s at KD 4A lysine (K)-specific demethylase 4A
2 2359 s at K1AA0913 K1AA091
215936 s at K1AA 033 KIAA1033
1553955 at KLRAQ1 KLRAQ motif containing 1
202058 s at KPNA1 karyopherin alpha 1 (importin alpha 5)
242364 x at LOC100131096 hypothetical LOC100131096
1563571 at LOC285463 hypothetical protein LOC285463
214107 x at LOC440434 hypothetical protein FU11822
1555860 x at LOC440944 hypothetical LOC440944
227383 at LOC727820 hypothetical protein LOC727820
1558111 at MBNL1 muscleblind-HKe (Drosophila)
222636 at ME028 mediator complex subunit 28
211801 x at MFN1 mitofusin 1
200898 s at MGEA5 meningioma expressed antigen 5 (hyaluronidase)
1557158 s at LL3 myeloid/lymphoid or mixed-lineage leukemia 3
218926 at MYNN myoneurin
22l899_at N4BP2L2 NEDD4 binding protein 2-li! e 2
1S6837 at COA6 nuclear receptor coactivator 6
200856 x at NCOR1 nuclear receptor corepressor 1
1558515 at NCRNA00182 non-protein coding RNA 182
210786 s at NOTCH2 notch 2
218295 s at NUP50 nucleoporin 50kDa
212307 s at OGT O-linked N-acetylglucosamine (GlcNAc) transferase (UDP-N-acety1gIuoosamine:polypeptide- -acetylglucosaminyl transferase)
210028 s at 0RC3L origin recognition complex, subunit 3-like (yeast)
202876 s at PBX2 pre-B-oell leukemia homeobo
213173~af PCNX pecanex homolog (Drosophila)
208591 s at PDE3B phosphodiesterase 3B, cGMP-inhibited
206792 x~at PDE4C phosphodiesterase 4C, cA P-specific
209700 x at PDE4DIP phosphodiesterase 4D interacting protein
1552621 at POLR2J2 polymerase (RNA) II (DNA directed) polypeptide J2
2050S6 at P0M121 POM121 membrane glycoprotein
229027 at PPM1A protein phosphatase, Mg2+ n2+ dependent, 1A
203057 a at PRDM2 PR domain containing 2, with ZNF domain
1567443 at PSEN1 presenilin 1
222796 at"" PTCD1 pentatricopeptide repeat domain 1
209895 at PTPN11 protein tyrosine phosphatase, non-receptor type 11
209694 at PTS 6-pyruvoyltetrahydropterin synthase
219681 S at RAB11RP1 RAB1 family interacting protein 1 (class I)
208731 at RAB2A RAB2A, member RAS oncogene family
204478 S at RA8IF RAB interacting factor
232500~at RALGAPA2 Ral GTPase activating protein, alpha subunit 2 (catalytic)
239438 at RAPGEF6 Rap guanine nucleotide exchange factor (GEF) 6
202677 at RASA1 RAS p21 protein activator (GTPase activating protein) 1
227223 at RBM39 RNA binding motif protein 39
209936~at RBM5 RNA binding motif protein 5
1552617 a at RFWD2 ring finger and WD repeat domain 2
2095 0 at RNF139 ring finger protein 139
225931 S at RNF213 ring finger protein 213
226975 rt RNPC3 RNA-bind'mg region (RNP1 , RRM) containing 3
203528 at SEMA4D sema domain, immunoglobulin domain (Ig), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 4D
210172 at SF1 splicing factor 1
33322 i at SFN stratifin
220123 at SLC35F5 solute carrier family 35, member F5
223160 s at SMEK1 S EK homolog 1, suppressor of mekl (Dictyostelium)
235324 at SRSF3 serine/argirtine-rich splicing factor 3
223053 x_at SSU72 SSU72 RNA polymerase II CTO phosphatase homolog (S. cerevisiae)
212572 at STK38L serine/threonine kinase 38 like
223746 at STK4 serine/threonine kinase 4
[0113]
Table 2 Genes over- and under-represented in immature granulocytes of hSCF Tg NSG mice.
(1) Genes over-represented in immature granulocytes. (2) Genes under-represented in immature granulocytes.
Table 3-1 Gene Ontology terms functionally enriched among genes over-represented in immature granulocytes
Table 3-2 Gene Ontology terms functionally enriched among genes under-represented in immature granulocytes
Q
3
a
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3
o
a
o
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o
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&.
o
3
OQ
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3
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o
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under-represented in immature granulocytes of hSCF Tg NSG mice.
(1) Gene Ontology terms functionally enriched among genes over-represented in immature granulocytes. (2) Gene Ontology terms functionally enriched among genes under-represented in immature granulocytes.
[0115]
Development of human mast cells in hSCF Tg NSG recipients
The present inventors next investigated the development of human mast cells in the membrane-bound hSCF expressing NSG mice. Overall, the frequencies of cKit(+)CD203c+ cells within total BM CD33+ myeloid cells were similar between hSCF Tg NSG and non-Tg NSG recipients when excluding two non-Tg NSG recipients observed for more than 8 months (p=0.1439 by two-tailed t test) (Figure 2D, Table 1). In seven of 20 hSCF Tg NSG recipients, compared with one of ten non-Tg NSG recipients, the frequency of cKit(+)CD203c+ cells in BM CD33+ cells was greater than 15% (Figure 2D, Table 1 ). When these cKit+CD203c+ cells were FACS-purified and examined by MGG staining, their morphology was consistent with mast cells with varying degrees of cytoplasmic granulation (Figure 3A,B). Histological examination of HE-stained bone sections showed increased cellularity in hSCF Tg NSG recipients compared with non-Tg NSG recipients (Figure 3C). The present inventors then performed IHC staining for mast cell tryptase to identify human mast cells in the BM. Consistent with the quantitative analysis by flow cytometry, tryptase+ cells were abundantly observed in the hSCF Tg NSG recipients compared with non-Tg NSG recipients (Figure 3C). This does not reflect an increase in mouse mast cells since nearly all nucleated hematopoietic cells in the hSCF Tg NSG recipients are of human origin (Figure ID). The same sections were further subjected to the
immunofluorescence staining followed by confocal imaging demonstrating that these are mast cells and not CD 14+ monocytes (Figure 9).
[0116]
Mast cell progenitors and mature mast cells reside in high frequencies in the spleen of normal immunocompetent mice21. The present inventors next examined the spleen of human HSC-engrafted hSCF Tg NSG recipients. Human
CD33(high)c-Kit+CD203c+ mast cells accounted for the highest frequency among total hCD45+hCD33+ myeloid cells in the spleen of both hSCF Tg NSG and non-Tg NSG HSC-engrafted recipients (Figures 4A,B). However, the frequencies of human mast cells in the myeloid cell population were significantly higher in hSCF Tg NSG recipients than in non-Tg NSG controls (hSCF Tg: 77.4 +/- 4.5%; n=20 and non-Tg NSG controls:
62.5 +/- 3.9%; n=12) (p=0.0304 by two-tailed t test) (Figure 4B). These human cells with surface expression phenotype of mast cells also showed morphological features of mature mast cells (Figure 4C). Mast cell tryptase IHC staining confirmed the presence of human mast cells within the recipient spleen (Figure 4D). These findings indicate that the expression of membrane-bound human SCF in the recipient mouse
microenvironment enhances development of human mast cells from transplanted human HSCs within hematopoietic organs such as the BM and spleen, consistent with the activation of c-Kit signaling.
[0117]
Next, the present inventors investigated whether the transgenic expression of hSCF results in the efficient development of mucosal tissue-type human mast cells in respiratory and gastrointestinal mucosal layers, as well as in hematopoietic organs. For this purpose, the present inventors performed IHC staining of human tryptase-expressing mast cells in the lung, stomach, small intestine, and large intestine in hSCF Tg NSG and control NSG recipients. In the hSCF Tg NSG recipient lungs, human tryptase-positive mast cells were identified within cellular infiltrates (Figure 10). In tissue sections from the stomach, small intestine and large intestine, human tryptase-positive mast cells were present in both hSCF Tg NSG and non-Tg NSG recipients (Figures 5A, B). The mast cell tryptase+ cells in gastrointestinal tissues of hSCF Tg mice were further examined by immunofluorescence microscopy using anti-human CD45 and anti-human-c-Kit antibodies. The present inventors found the presence of hCD45+c-Kit+ cells in the gastric tissues of the hSCF Tg recipients consistent with IHC staining for mast cell tryptase (Figure 5C). Since gastric tissue is one of the major sites of mast cell populations in man and mouse, the present inventors quantified human mast cells in the gastric tissue of hSCF Tg and non-Tg NSG recipients transplanted with human HSCs. IHC staining for human mast cell tryptase followed by quantification of tryptase+ cells demonstrated the presence of human mast cells in gastric tissues of hSCF Tg NSG recipients (7.01+/- 0.63%, 3 sites per recipient analyzed in 3 mice) compared with non-Tg NSG recipients (2.53+/- 0.53%, 3 sites per recipient analyzed in 3 mice;
pO.0001 by two tailed t test) (Figure 5D). Collectively, transgenic expression of human membrane-bound SCF influences human myeloid development and mast cell development in hematopoietic organs and mucosal tissues along with the achievement of high chimerism of human hematopoietic cells in hematopoietic organs.
[0118]
Discussion
A supportive microenvironment is essential for hematopoietic and immune system homeostasis. Critical roles played by various niches in the maintenance of cell cycle quiescence and self-renewal capacity of HSCs have been demonstrated, and the thymic microenvironment is critical for T cell education ' . However, despite significant progress over the last decade, the stromal microenvironment within the humanized mouse is predominately of mouse origin. While several key molecules such as SDF1 are cross reactive between man and mouse, a humanized microenvironment is required both to further improve human hematopoietic development in the recipients and to investigate in vivo the interactions between hematopoietic cells and their
microenvironment.
[0119]
In the present study, the present inventors humanized membrane-bound stem cell factor [SCF=Kit ligand (KL)] using the construct and mouse strain created by Williams and colleagues . Toksoz et al. reported that human membrane-bound SCF expressed by mouse stromal cells efficiently supports long-term human hematopoiesis in vitro24. In human hematopoiesis, SCF-cKit signaling is critical for the maintenance of stem and progenitor cell activities . Human SCF/KL has been shown to drive cell cycle entry by primitive hematopoietic cells in vitro . Both long-term colony-initiation and colony-forming capacities are expanded ex vivo by cytokine supplementation that includes SCF/KL " . Therefore, to elucidate the role of membrane-bound human SCF in differentiation, proliferation, and maturation of human hematopoiesis in vivo, the present inventors created a novel NSG mouse strain that can support the engraftment of human HSCs and express hSCF in microenvironment. In hSCF Tg NSG recipients transplanted with human HSCs, the engraftment levels of human CD45+ cells were significantly higher compared with non-Tg NSG controls. In addition to the greatly increased levels of human hematopoietic repopulation, the present inventors identified significant differences in human hematopoietic differentiation in hSCF Tg NSG recipients compared with non-Tg NSG recipients. Namely, there were substantially increased levels of human myeloid differentiation from HSCs in the hSCF Tg NSG mice, whereas human B cells accounted for the greatest population in the BM of non-Tg NSG mice. Since normal human BM contains myeloid cells at a relatively high frequency (36.2-62.2%) , human SCF may be important in recapitulating human BM myelopoiesis in immunodeficient mice. In addition, membrane-bound human SCF may exert distinct effects on human myeloid development in the BM and in the spleen. In the BM of hSCF Tg recipients, the majority of human myeloid cells were
c-Kit(-)CD203c(-)HLA-DR(-) granulocytes. Among these granulocytes, myeloid cells at various levels of maturity were identified, with myelocytes and metamyelocytes predominating in the majority of hSCF Tg NSG recipients. Since immature cells were more prominent in hSCF Tg NSG recipients compared with non-Tg NSG recipients, the present inventors performed microarray analysis to identify transcriptional signature specific to the immature human granulocytes that developed in the hSCF Tg NSG mice. Approximately 300 genes were differentially transcribed in the immature granulocytes in hSCF Tg NSG recipients compared with the mature granulocytes in non-Tg NSG recipients. Some of the up-regulated genes were associated with cell cycle or metabolism.
[0120]
In several hSCF Tg NSG recipients, human mast cells comprised the greatest subfraction among engrafted human myeloid cells. In the spleens of hSCF Tg NSG engrafted mice, human mast cells were present at the highest frequency among the myeloid lineage developed in the recipients. MGG staining revealed both mature and immature mast cells in hSCF Tg NSG recipient BM. Human mast cells were identified not only in hematopoietic organs but also in lung, gastric tissue, and intestinal tissues of hSCF Tg NSG recipients. Aberrant expression of CD30 and CD25 on mast cells is associated with systemic mastocytosis and other mast cell disorders ' . The present inventors did not find significantly upregulated expression of these antigens in the mast cells derived from BM or spleen of hSCF Tg NSG recipients.
[0121]
To date, several mouse strains have been developed for supporting normal and malignant human hematopoietic cell engraftment and normal myeloid cell differentiation by using Il2rgnul1 immune-compromised mice (Table 4)5>6>8>9>20>38-41.
U2ya'
[0122]
Table 4 Summary of immune-compromised mouse strains using 112 ygnu".
[0123]
Among these, human thrombopoietin (TPO) knock-in BALB/c x \29(Rag2"u!1 Il2rgnul1) mice were reported to support both human hematopoietic engraftment and myeloid differentiation in the bone marrow. Both SCF and TPO exhibit
species-specificity between man and mouse in supporting HSCs and myeloid cells in both species. These approaches focusing on the two distinct molecules based on the two immune-compromised mouse backgrounds will allow us to investigate human hematopoiesis and immunity from stem cells to myeloid progenitors to mature myeloid effector cells in vivo. Altogether, the newly created hSCF Tg NSG mouse model engrafted with purified human HSCs will facilitate the in vivo understanding of human hematopoietic hierarchy and mast cell biology. INDUSTRIAL APPLICABILITY
[0124]
The production method of the present invention and the immune-system humazized mouse produced by the method may serve as a novel platform for in vivo investigation of human mast cell development and allergic responses.
[0125]
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[0126]
SEQUENCES
The amino acid sequences of human SCF220, SCF248 and soluble SCF are shown along with exemplary nucleic acid sequences encoding the proteins.
[0127]
Human SCF220 (245 aa) (SEQ ID NO: 2)
MKKTQTWILTCIYLQLLLFNPLVKTEGICRNRVTNNVKDVTKLVANL
PKDYMITLKYVPGMDVLPSHCWISEMVVQLSDSLTDLLDKFSNISEG
LSNYSIIDKLVNIVDDLVECVKENSSKDLKKSFKSPEPRLFTPEEFF
RIFNRSIDAFKDFVVASETSDCVVSSTLSPEKGKAKNPPGDSSLHWA
AMALPALFSLIIGFAFGALYWKKRQPSLTRAVENIQINEEDNEISML QEKEREFQEV
[0128]
Nucleotide Sequence Encoding Human SCF220 (SEQ ID NO: 1)
ATGAAGAAGACACAAACTTGGATTCTCACTTGCATTTATCTTCAGCT
GCTCCTATTTAATCCTCTCGTCAAAACTGAAGGGATCTGCAGGAATC
GTGTGACTAATAATGTAAAAGACGTCACTAAATTGGTGGCAAATCTT
CCAAAAGACTACATGATAACCCTCAAATATGTCCCCGGGATGGATGT
TTTGCCAAGTCATTGTTGGATAAGCGAGATGGTAGTACAATTGTCAG
ACAGCTTGACTGATCTTCTGGACAAGTTTTCAAATATTTCTGAAGGC
TTGAGTAATTATTCCATCATAGACAAACTTGTGAATATAGTGGATGA
CCTTGTGGAGTGCGTGAAAGAAAACTCATCTAAGGATCTAAAAAAAT
CATTCAAGAGCCCAGAACCCAGGCTCTTTACTCCTGAAGAATTCTTT
AGAATTTTTAATAGATCCATTGATGCCTTCAAGGACTTTGTAGTGGC
ATCTGAAACTAGTGATTGTGTGGTTTCTTCAACATTAAGTCCTGAGA
AAGGGAAGGCCAAAAATCCCCCTGGAGACTCCAGCCTACACTGGGCA
GCCATGGCATTGCCAGCATTGTTTTCTCTTATAATTGGCTTTGCTTT
TGGAGCCTTATACTGGAAGAAGAGACAGCCAAGTCTTACAAGGGCAG
TTGAAAATATACAAATTAATGAAGAGGATAATGAGATAAGTATGTTG
CAAGAGAAAGAGAGAGAGTTTCAAGAAGTGTAA
[0129]
Human SCF248 (273 aa) (SEQ ID NO: 4)
MKKTQTWILTCIYLQLLLFNPLVKTEGICRNRVTNNVKDVTKLVANL PKDYMITLKYVPGMDVLPSHCWISEMVVQLSDSLTDLLDKFSNISEG LSNYSIIDKLVNIVDDLVECVKENSSKDLKKSFKSPEPRLFTPEEFF RIFNRSIDAFKDFVVASETSDCVVSSTLSPEKDSRVSVTKPFMLPPV
AAS S LRND S S S SNRKAKNPPGD S S LH WAAM ALPALF S LIIGFAFG AL
YWKKRQPSLTRAVENIQINEEDNEISMLQEKEREFQEV
[0130]
Coding Sequence for Human SCF248 (SEQ ID NO: 3)
ATGAAGAAGACACAAACTTGGATTCTCACTTGCATTTATCTTCAGCT GCTCCTATTTAATCCTCTCGTCAAAACTGAAGGGATCTGCAGGAATC GTGTGACTAATAATGTAAAAGACGTCACTAAATTGGTGGCAAATCTT CCAAAAGACTACATGATAACCCTCAAATATGTCCCCGGGATGGATGT TTTGCCAAGTCATTGTTGGATAAGCGAGATGGTAGTACAATTGTCAG ACAGCTTGACTGATCTTCTGGACAAGTTTTCAAATATTTCTGAAGGC TTGAGTAATTATTCCATCATAGACAAACTTGTGAATATAGTGGATGA CCTTGTGGAGTGCGTGAAAGAAAACTCATCTAAGGATCTAAAAAAAT CATTCAAGAGCCCAGAACCCAGGCTCTTTACTCCTGAAGAATTCTTT AGAATTTTTAATAGATCCATTGATGCCTTCAAGGACTTTGTAGTGGC ATCTGAAACTAGTGATTGTGTGGTTTCTTCAACATTAAGTCCTGAGA AAGATTCCAGAGTCAGTGTCACAAAACCATTTATGTTACCCCCTGTT GCAGCCAGCTCCCTTAGGAATGACAGCAGTAGCAGTAATAGGAAGGC CAAAAATCCCCCTGGAGACTCCAGCCTACACTGGGCAGCCATGGCAT TGCCAGCATTGTTTTCTCTTATAATTGGCTTTGCTTTTGGAGCCTTA TACTGGAAGAAGAGACAGCCAAGTCTTACAAGGGCAGTTGAAAATAT ACAAATTAATGAAGAGGATAATGAGATAAGTATGTTGCAAGAGAAAG AGAGAGAGTTTCAAGAAGTGTAA
[0131]
Nucleotide Sequence Encoding Human SCF248 Including 5' and 3' Non-Coding Sequences (SEQ ID NO: 5)
CCGCCTCGCGCCGAGACTAGAAGCGCTGCGGGAAGCAGGGACAGTGG AGAGGGCGCTGCGCTCGGGCTACCCAATGCGTGGACTATCTGCCGCC GCTGTTCGTGCAATATGCTGGAGCTCCAGAACAGCTAAACGGAGTCG CCACACCACTGTTTGTGCTGGATCGCAGCGCTGCCTTTCCTTATGAA GAAGACACAAACTTGGATTCTCACTTGCATTTATCTTCAGCTGCTCC TATTTAATCCTCTCGTCAAAACTGAAGGGATCTGCAGGAATCGTGTG ACTAATAATGTAAAAGACGTCACTAAATTGGTGGCAAATCTTCCAAA AGACTACATGATAACCCTCAAATATGTCCCCGGGATGGATGTTTTGC CAAGTCATTGTTGGATAAGCGAGATGGTAGTACAATTGTCAGACAGC TTGACTGATCTTCTGGACAAGTTTTCAAATATTTCTGAAGGCTTGAG
TAATTATTCCATCATAGACAAACTTGTGAATATAGTGGATGACCTTG
TGGAGTGCGTGAAAGAAAACTCATCTAAGGATCTAAAAAAATCATTC
AAGAGCCCAGAACCCAGGCTCTTTACTCCTGAAGAATTCTTTAGAAT
TTTTAATAGATCCATTGATGCCTTCAAGGACTTTGTAGTGGCATCTG
AAACTAGTGATTGTGTGGTTTCTTCAACATTAAGTCCTGAGAAAGAT
TCCAGAGTCAGTGTCACAAAACCATTTATGTTACCCCCTGTTGCAGC
CAGCTCCCTTAGGAATGACAGCAGTAGCAGTAATAGGAAGGCCAAAA
ATCCCCCTGGAGACTCCAGCCTACACTGGGCAGCCATGGCATTGCCA
GCATTGTTTTCTCTTATAATTGGCTTTGCTTTTGGAGCCTTATACTG
GAAGAAGAGACAGCCAAGTCTTACAAGGGCAGTTGAAAATATACAAA
TTAATGAAGAGGATAATGAGATAAGTATGTTGCAAGAGAAAGAGAGA
GAGTTTCAAGAAGTGTAAATTGTGGCTTGTATCAACACTGTTACTTT
CGTACATTGGCTGGTAACAGTTCATGTTTGCTTCATAAATGAAGCAG
CTTTAAACAAATTCATATTCTGTCTGGAGTGACAGACCACATCTTTA
TCTGTTCTTGCTACCCATGACTTTATATGGATGATTCAGAAATTGGA
ACAGAATGTTTTACTGTGAAACTGGCACTGAATTAATCATCTATAAA
GAAGAACTTGCATGGAGCAGGACTCTATTTTAAGGACTGCGGGACTT
GGGTC TC ATTTAG A ACTTGC AGC TG ATGTTGG A AG AG A A AGC AC GTG
TCTCAGACTGCATGTACCATTTGCATGGCTCCAGAAATGTCTAAATG
CTGAAAAAACACCTAGCTTTATTCTTCAGATACAAACTGCAG
[0132]
Human Soluble SCF (164 aa and 25 aa N-Terminal Signal Peptide) (SEQ ID NO: 7)
MKKTQTWILTCIYLQLLLFNPLVKTEGICRNRVTNNVKDVTKLVANL
PKDYMITLKYVPGMDVLPSHCWISEMVVQLSDSLTDLLDKFSNISEG
LSNYSIIDKLVNIVDDLVECVKENSSKDLKKSFKSPEPRLFTPEEFF
RIFNRSIDAFKDFVVASETSDCVVSSTLSPEKDSRVSVTKPFMLPPV A
[0133]
Nucleotide Sequence Encoding Human Soluble SCF (164 aa and 25 aa N-Terminal Signal Peptide) (SEQ ID NO: 6)
ATGAAGAAGACACAAACTTGGATTCTCACTTGCATTTATCTTCAGCT GCTCCTATTTAATCCTCTCGTCAAAACTGAAGGGATCTGCAGGAATC GTGTGACTAATAATGTAAAAGACGTCACTAAATTGGTGGCAAATCTT CCAAAAGACTACATGATAACCCTCAAATATGTCCCCGGGATGGATGT TTTGCCAAGTCATTGTTGGATAAGCGAGATGGTAGTACAATTGTCAG ACAGCTTGACTGATCTTCTGGACAAGTTTTCAAATATTTCTGAAGGC
TTGAGTAATTATTCCATCATAGACAAACTTGTGAATATAGTGGATGA CCTTGTGGAGTGCGTGAAAGAAAACTCATCTAAGGATCTAAAAAAAT CATTCAAGAGCCCAGAACCCAGGCTCTTTACTCCTGAAGAATTCTTT AGAATTTTTAATAGATCCATTGATGCCTTCAAGGACTTTGTAGTGGC ATCTGAAACTAGTGATTGTGTGGTTTCTTCAACATTAAGTCCTGAGA
AAGATTCCAGAGTCAGTGTCACAAAACCATTTATGTTACCCCCTGTT GCA
Claims
- 1. A method for producing a first immune-system humanized mouse, comprising administering human hematopoietic stem cells to a first transgenic immunodeficient mouse whose genome comprises a nucleic acid encoding human Stem Cell Factor operably linked to a promoter,
wherein the first transgenic immunodeficient mouse expresses the human Stem Cell Factor, and
wherein the immune-system humanized mouse has
- greater human CD45+ leukocyte population in bone marrow, spleen and peripheral blood,
greater human CD33+ myeloid cell population in bone marrow human CD45+ leukocytes, and
greater human CD203c+c-kit+ mast cell population in splenic human CD45+CD33+ myeloid cells
as compared to a second immune-system humanized mouse prepared by administering the human hematopoietic stem cells to a second transgenic immunodeficient mouse which does not express the human Stem Cell Factor.
2. The method of claim 1, wherein the first transgenic immunodeficient mouse has NOD background, the severe combined immune deficiency (scid) mutation, and a complete knockout of the interleukin-2 receptor gamma chain.
3. The method of claim 2, wherein the first transgenic mouse is a NOD/LtSZ-scid IL2Ry null (NSG) mouse.
4. The method of claim 1, wherein the human Stem Cell Factor is a human
membrane-associated stem cell factor.
5. A method for producing an immune-system humanized mouse, comprising
administering human hematopoietic stem cells to a transgenic immunodeficient mouse whose genome comprises a nucleic acid encoding human Stem Cell Factor operably linked to a promoter,
wherein the transgenic immunodeficient mouse expresses the human Stem Cell Factor, and wherein engraftment level of human CD45+ cells in the bone marrow of the
immune-system humanized mouse is 70% or more,
the frequency of human CD33+ myeloid cells within the total human CD45+ population in the bone marrow of the immune-system humanized mouse is 30% or more, and the frequency of human c-kit+CD203c+ mast cells within the total human CD45+CD33+ myeloid cells in the spleen of the immune-system humanized mouse is 65% or more.
6. The method of claim 5, wherein the engraftment level of human CD45+ cells in the bone marrow of the immune-system humanized mouse is 95% or more.
7. The method of claim 5, wherein the frequency of human CD33+ myeloid cells within the total human CD45+ population in the bone marrow of the immune-system humanized mouse is 45% or more.
8. The method of claim 5, wherein the frequency of human c-kit+CD203c+ mast cells within the total human CD45+CD33+ myeloid cells in the spleen of the immune-system humanized mouse is 75% or more.
9. A transgenic immunodeficient mouse, comprising engrafted human hematopoietic cells and human both acquired and innate immune cells differenciated from the hematopoietic cells surviving without being rejected from the mouse;
wherein engraftment level of human CD45+ cells in the bone marrow of the
immune-system humanized mouse is 70% or more,
the frequency of human CD33+ myeloid cells within the total human CD45+ population in the bone marrow of the immune-system humanized mouse is 30% or more, and the frequency of human c-kit+CD203c+ mast cells within the total human CD45+CD33+ myeloid cells in the spleen of the immune-system humanized mouse is 65% or more; wherein the the transgenic immunodeficient mouse comprises a nucleic acid encoding human Stem Cell Factor operably linked to a promoter in the genome, and expresses the human Stem Cell Factor.
10. The transgenic immunodeficient mouse of claim 9, wherein the engraftment level of human CD45+ cells in the bone marrow of the immune-system humanized mouse is 95% or more.
11. The transgenic immunodeficient mouse of claim 9, wherein the frequency of human CD33+ myeloid cells within the total human CD45+ population in the bone marrow of the immune-system humanized mouse is 45% or more.
12. The transgenic immunodeficient mouse of claim 9, wherein the frequency of human c-kit+CD203c+ mast cells within the total human CD45+CD33+ myeloid cells in the spleen of the immune-system humanized mouse is 75% or more.
13. A method of screening for a substance capable of preventing or treating immune/allergic/inflammatory disease relating myeloid cells and/or mast cells, comprising applying a test substance to the transgenic immunodeficient mouse of claim 9 or portion thereof affected with the immune/allergic/inflammatory disease relating myeloid cells and/or mast cells, and evaluating whether or not the test substance improves a condition or symptom of the immune/allergic/inflammatory disease.
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Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2017066561A2 (en) | 2015-10-16 | 2017-04-20 | President And Fellows Of Harvard College | Regulatory t cell pd-1 modulation for regulating t cell effector immune responses |
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WO2017066561A2 (en) | 2015-10-16 | 2017-04-20 | President And Fellows Of Harvard College | Regulatory t cell pd-1 modulation for regulating t cell effector immune responses |
WO2017165412A2 (en) | 2016-03-21 | 2017-09-28 | Dana-Farber Cancer Institute, Inc. | T-cell exhaustion state-specific gene expression regulators and uses thereof |
WO2019199799A1 (en) * | 2018-04-09 | 2019-10-17 | The Wistar Institute | Humanized mouse model |
WO2020008066A1 (en) * | 2018-07-06 | 2020-01-09 | Institut Pasteur | Human immune system mouse model |
WO2020033791A1 (en) | 2018-08-09 | 2020-02-13 | Verseau Therapeutics, Inc. | Oligonucleotide compositions for targeting ccr2 and csf1r and uses thereof |
WO2022216942A1 (en) | 2021-04-07 | 2022-10-13 | Dana-Farber Cancer Institute, Inc. | Compositions and methods for the treatment of cancer |
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