Patent Description:
HHLS mice are generated by the transplantation of human hematopoietic stem and progenitor cells (HSPCs) and/or human fetal tissues into recipient mice deficient in the innate and adaptive arms of the immune response. The first models of HHLS mice were developed in the late <NUM> (<NPL>; <NPL>; <NPL>), and have been undergoing a series of improvements since then (<NPL>; <NPL>). The strains of mice currently used as recipients for human hematopoietic engraftment share three characteristics. First, they lack B and T cells due to the Scid mutation in the gene encoding the PRKDC protein (<NPL>; <NPL>), or due to deletion of one of the two Rag genes (<NPL>; <NPL>). Second, deletion or mutation of the Il2rg gene that encodes the common gamma chain (γc) of cytokine receptors abolishes IL-<NUM> signaling and results in the absence of NK cells (<NPL>; <NPL>). Third, the interaction between the SIRPA receptor expressed on mouse macrophages and the CD47 ligand on human cells provides an inhibitory signal to mouse macrophages and confers phagocytic tolerance for the human xenograft (<NPL>; <NPL>). Cross-species interaction between SIRPA expressed on mouse cells and human CD47 is achieved when using the NOD genetic background which contains a natural polymorphism in the Sirpa gene (<NPL>; <NPL>; <NPL>) or by BAC-transgenic expression of the human SIRPA gene (<NPL>). High levels of human hematopoietic cell engraftment, upon human HSPC transplantation, are achieved when using NOD Scid γc-/- (NOG (<NPL>) or NSG (<NPL>)) or hSIRPAtg RAG2-/- γc-/- (SRG (<NPL>)) mice as recipients.

Although human multi-lineage hematopoietic development is observed in these recipient strains, the terminal differentiation, homeostasis and/or effector function of most human cell types is sub-optimal. It has been hypothesized that this condition is due to reduced or absent cross-reactivity between cytokines secreted by mouse tissues and the human receptors expressed on hematopoietic cells (<NPL>; <NPL>). To circumvent this limitation, several strategies have been developed to deliver human cytokines in the mouse host. These methods include the injection of recombinant cytokines (<NPL>; <NPL>), lentiviral delivery of cytokine-encoding cDNA (<NPL>), hydrodynamic injection of plasmid DNA (<NPL>), transgenic expression of cDNA (<NPL>; <NPL>; <NPL>) or knock-in replacement of cytokine-encoding genes (<NPL>; <NPL>; <NPL>). The later method has the advantage of more physiological expression of the human gene. Furthermore, if the human cytokine is not fully cross-reactive on the mouse receptor, it can induce a defect in mouse cell populations and confer an additional competitive advantage to human cells. Using a knock-in gene replacement strategy, humanization of the gene encoding thrombopoietin (Tpo) resulted in better maintenance of functional human hematopoietic stem cells and increased engraftment in the bone marrow (<NPL>); replacement of the genes encoding interleukin-<NUM> and GM-CSF (Il3 and Csf2) induced the loss of mouse lung alveolar macrophages (AM) and the development of functional human AM (<NPL>); and substitution of the Csf1 gene, which encodes M-CSF, resulted in increased numbers of human monocytes in multiple tissues (<NPL>).

Human and mouse hemato-lymphoid systems differ in many aspects (<NPL>; <NPL>). One of the major differences between the two species lies in their white blood cell (WBC) differential. Human blood is rich in myeloid cells that represent <NUM>-<NUM>% of total WBCs. In contrast, mouse blood is dominated by lymphocytes and only <NUM>-<NUM>% of WBCs are of myeloid lineages. This species difference, whose functional and evolutionary significance is not understood, is not recapitulated in conventional HHLS mice such as NOG/NSG or SRG. Indeed, human myeloid development is particularly defective in these hosts, with myeloid cells representing only <NUM>-<NUM>% of human WBCs.

One application of mice with functional human immune systems is the development and testing of human vaccines. Historically, the induction of immune responses in vivo has been relatively inefficient (<NPL>; <NPL>; <NPL>; <NPL>; <NPL>). Several studies have reported successful pathogen-specific immune responses upon infection. Although it was reported that around <NUM>% of mice produced virus-specific IgM and IgG upon dengue virus infection (<NPL>), other studies reported frequencies below <NUM>% of mice producing antigen-specific IgM and IgG after HIV and EBV infection (<NPL>; <NPL>). Upon immunization with adjuvant and antigen, class switching of antigen-specific immunoglobulins is also historically inefficient with only a fraction of immunized animals showing antigen specific IgG responses (<NPL>; <NPL>; <NPL>; <NPL>; <NPL>; <NPL>). These studies included NSG and BALB/c RAG2-/- γc-/-mice and different adjuvant/antigen combinations. <NPL> discloses human hemato-lymphoid-system mice for modeling and studying human diseases in vivo, with a particular focus on improving the human hemato-lymphoid-system mice by human cytokine knock-in gene replacement.

There is a need in the art for humanized non-human animals able to support and sustain engraftment with human hematopoietic cells. The present invention addresses this unmet need in the art.

The invention relates to a method for testing the effect of an agent that modulates cancer cell growth or survival, or for the in vivo evaluation of a cancer treatment involving the agent, the method comprising: administering the agent to a genetically modified mouse, wherein the genetically modified mouse comprises in its genome: a replacement of a mouse M-CSF gene with a nucleic acid encoding a human M-CSF polypeptide operably linked to the mouse M-CSF promoter and regulatory elements at the mouse M-CSF gene locus, a replacement of a mouse IL-<NUM> gene with a nucleic acid encoding a human IL-<NUM> polypeptide operably linked to the mouse IL-<NUM> promoter and regulatory elements at the mouse IL-<NUM> gene locus, a replacement of a mouse GM-CSF gene with a nucleic acid encoding a human GM-CSF polypeptide operably linked to the mouse GM-CSF promoter and regulatory elements at the mouse GM-CSF gene locus, a replacement of a mouse TPO gene with a nucleic acid encoding a human TPO polypeptide operably linked to the mouse TPO promoter and regulatory elements at the mouse TPO gene locus, and an insertion of a nucleic acid encoding a human SIRPα polypeptide operably linked to a human SIRPA promoter and regulatory elements, wherein the genetically modified mouse expresses the human M-CSF polypeptide, the human IL-<NUM> polypeptide, the human GM-CSF polypeptide, the human SIRPα polypeptide, and the human TPO polypeptide, and wherein the genetically modified mouse is immunodeficient, engrafted with human hematopoietic cells, and engrafted with human cancer cells; and assessing effect of the agent on cancer cell growth or survival in the genetically modified mouse.

Genetically modified mice comprising a genome comprising a replacement of a mouse M-CSF gene with a nucleic acid encoding a human M-CSF polypeptide operably linked to the mouse M-CSF promoter and regulatory elements at the mouse M-CSF gene locus, a replacement of a mouse IL-<NUM> gene with a nucleic acid encoding a human IL-<NUM> polypeptide operably linked to the mouse IL-<NUM> promoter and regulatory elements at the mouse IL-<NUM> gene locus, a replacement of a mouse GM-CSF gene with a nucleic acid encoding a human GM-CSF polypeptide operably linked to the mouse GM-CSF promoter and regulatory elements at the mouse GM-CSF gene locus, a nucleic acid encoding human SIRPA operably linked to a human SIRPA promoter and regulatory elements and a nucleic acid encoding a human TPO polypeptide operably linked to the mouse TPO promoter and regulatory elements at the mouse TPO gene locus, and where the mouse expresses human M-CSF polypeptide, human IL-<NUM> polypeptide, human GM-CSF polypeptide, human SIRPA polypeptide and human TPO polypeptide wherein the genetically modified non-human animal is immunodeficient, engrafted with human hematopoietic cells, and engrafted with human cancer cells, are disclosed. In some embodiments, the genetically modified mouse does not express recombination activating gene <NUM> (Rag-<NUM>-/-). In some embodiments, the genetically modified mouse does not express IL2 receptor gamma chain (gamma chain-/-). In some embodiments, the genetically modified mouse does not express Rag-<NUM> and the genetically modified mouse does not express IL2 receptor gamma chain (Rag-<NUM>-/- gamma chain-/-). The genetically modified mouse also includes at least one human hematopoietic cell and at least one human cancer cell. In some embodiments, the human cancer cell is a leukemia cell or a melanoma cell.

The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

The disclosure relates generally to a genetically modified mouse expressing human M-CSF, human IL-<NUM>, human GM-CSF, human SIRPA and human TPO. The disclosure also relates to methods of generating and methods of using the genetically modified non-human animals described herein. In some examples, the genetically modified mouse described herein is engrafted with human hematopoietic cells. In various examples, the human hematopoietic cell engrafted, genetically modified non-human animals of the disclosure are useful for the in vivo evaluation of the growth and differentiation of hematopoietic and immune cells, for the in vivo evaluation of human hematopoiesis, for the in vivo evaluation of cancer cells, for the in vivo assessment of an immune response, for the in vivo evaluation of vaccines and vaccination regimens, for the use in testing the effect of agents that modulate cancer cell growth or survival, for the in vivo evaluation of a treatment of cancer, for the in vivo production and collection of immune mediators, including human antibodies, and for use in testing the effect of agents that modulate hematopoietic and immune cell function.

Such terms are found defined and used in context in various standard references illustratively including<NPL>; <NPL>; <NPL>;<NPL>; and <NPL>. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

As used herein, each of the following terms has the meaning associated with it in this section.

"About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±<NUM>% or ±<NUM>%, more preferably ±<NUM>%, even more preferably ±<NUM>%, and still more preferably ±<NUM>% from the specified value, as such variations are appropriate to perform the disclosed methods.

The term "abnormal" when used in the context of organisms, tissues, cells or components thereof, refers to those organisms, tissues, cells or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from those organisms, tissues, cells or components thereof that display the "normal" (expected) respective characteristic. Characteristics which are normal or expected for one cell or tissue type, might be abnormal for a different cell or tissue type.

The term "antibody," as used herein, refers to an immunoglobulin molecule which is able to specifically bind to a specific epitope on an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. The antibodies in the present disclosure may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies ("intrabodies"), Fv, Fab and F(ab)<NUM>, as well as single chain antibodies (scFv), heavy chain antibodies, such as camelid antibodies, and humanized antibodies (<NPL>; <NPL>; <NPL>; <NPL>).

The term "cancer" as used herein is defined as disease characterized by the uncontrolled proliferation and/or growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Cancer as here herein includes both solid tumors and hematopoietic malignancies. Examples of various cancers amenable to the invention include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, bone cancer, brain cancer, lymphoma, leukemia, lung cancer, myeloidysplastic syndromes, myeloproliferative disorders and the like.

"Constitutive" expression is a state in which a gene product is produced in a living cell under most or all physiological conditions of the cell.

A "coding region" of a gene consists of the nucleotide residues of the coding strand of the gene and the nucleotides of the non-coding strand of the gene which are homologous with or complementary to, respectively, the coding region of an mRNA molecule which is produced by transcription of the gene.

A "coding region" of a mRNA molecule also consists of the nucleotide residues of the mRNA molecule which are matched with an anti-codon region of a transfer RNA molecule during translation of the mRNA molecule or which encode a stop codon. The coding region may thus include nucleotide residues comprising codons for amino acid residues which are not present in the mature protein encoded by the mRNA molecule (e.g., amino acid residues in a protein export signal sequence).

A "disease" is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.

In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.

A disease or disorder is "alleviated" if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by a patient, or both, is reduced.

An "effective amount" or "therapeutically effective amount" of a compound is that amount of compound which is sufficient to provide a beneficial effect to the subject to which the compound is administered. An "effective amount" of a delivery vehicle is that amount sufficient to effectively bind or deliver a compound.

"Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

As used herein "endogenous" refers to any material from or produced inside an organism, cell, tissue or system.

As used herein, the term "exogenous" refers to any material introduced from or produced outside an organism, cell, tissue or system.

The terms "expression construct" and "expression cassette" are used herein to refer to a double-stranded recombinant DNA molecule containing a desired nucleic acid human coding sequence and containing one or more regulatory elements necessary or desirable for the expression of the operably linked coding sequence.

As used herein, the term "fragment," as applied to a nucleic acid or polypeptide, refers to a subsequence of a larger nucleic acid or polypeptide. A "fragment" of a nucleic acid can be at least about <NUM> nucleotides in length; for example, at least about <NUM> nucleotides to about <NUM> nucleotides; at least about <NUM> to about <NUM> nucleotides, at least about <NUM> to about <NUM> nucleotides, at least about <NUM> nucleotides to about <NUM> nucleotides; or about <NUM> nucleotides to about <NUM> nucleotides; or about <NUM> nucleotides (and any integer value in between). A "fragment" of a polypeptide can be at least about <NUM> nucleotides in length; for example, at least about <NUM> amino acids to about <NUM> amino acids; at least about <NUM> to about <NUM> amino acids, at least about <NUM> to about <NUM> amino acids, at least about <NUM> amino acids to about <NUM> amino acids; or about <NUM> amino acids to about <NUM> amino acids; or about <NUM> amino acids (and any integer value in between).

As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame encoding a polypeptide. Such natural allelic variations can typically result in <NUM>-<NUM>% variance in the nucleotide sequence of a given gene. Alternative alleles can be identified by sequencing the gene of interest in a number of different individuals. This can be readily carried out by using hybridization probes to identify the same genetic locus in a variety of individuals. Any and all such nucleotide variations and resulting amino acid polymorphisms or variations that are the result of natural allelic variation and that do not alter the functional activity are intended to be within the scope of the disclosure.

"Homologous" as used herein, refers to the subunit sequence similarity between two polymeric molecules, e.g. between two nucleic acid molecules, e.g., two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions, e.g. if half (e.g., five positions in a polymer ten subunits in length) of the positions in two compound sequences are homologous then the two sequences are <NUM>% homologous, if <NUM>% of the positions, e.g. <NUM> of <NUM>, are matched or homologous, the two sequences share <NUM>% homology. By way of example, the DNA sequences <NUM>'-ATTGCC-<NUM>' and <NUM>'-TATGGC-<NUM>' share <NUM>% homology.

The terms "human hematopoietic stem and progenitor cells" and "human HSPC" as used herein, refer to human self-renewing multipotent hematopoietic stem cells and hematopoietic progenitor cells.

"Inducible" expression is a state in which a gene product is produced in a living cell in response to the presence of a signal in the cell.

As used herein, an "instructional material" includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of a compound, composition, vector, or delivery system of the disclosure in the kit for effecting alleviation of the various diseases or disorders recited herein. Optionally, or alternately, the instructional material can describe one or more methods of alleviating the diseases or disorders in a cell or a tissue of a mammal. The instructional material of the kit of the disclosure can, for example, be affixed to a container which contains the identified compound, composition, vector, or delivery system of the disclosure or be shipped together with a container which contains the identified compound, composition, vector, or delivery system. Alternatively, the instructional material can be shipped separately from the container with the intention that the instructional material and the compound be used cooperatively by the recipient.

The term "operably linked" as used herein refers to a polynucleotide in functional relationship with a second polynucleotide. By describing two polynucleotides as "operably linked" is meant that a single-stranded or double-stranded nucleic acid moiety comprises the two polynucleotides arranged within the nucleic acid moiety in such a manner that at least one of the two polynucleotides is able to exert a physiological effect by which it is characterized, upon the other. By way of example, a promoter operably linked to the coding region of a gene is able to promote transcription of the coding region. Preferably, when the nucleic acid encoding the desired protein further comprises a promoter/regulatory sequence, the promoter/regulatory sequence is positioned at the <NUM>' end of the desired protein coding sequence such that it drives expression of the desired protein in a cell. Together, the nucleic acid encoding the desired protein and its promoter/regulatory sequence comprise a "transgene.

The term "polynucleotide" as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric "nucleotides. " The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.

As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof. The term "peptide" typically refers to short polypeptides. The term "protein" typically refers to large polypeptides.

The term "progeny" as used herein refers to a descendent or offspring and includes the differentiated or undifferentiated decedent cell derived from a parent cell. In one usage, the term progeny refers to a descendent cell which is genetically identical to the parent. In another use, the term progeny refers to a descendent cell which is genetically and phenotypically identical to the parent. In yet another usage, the term progeny refers to a descendent cell that has differentiated from the parent cell.

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.

Ranges: throughout this disclosure, various aspects can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claims. For example, description of a range such as from <NUM> to <NUM> should be considered to have specifically disclosed subranges such as from <NUM> to <NUM>, from <NUM> to <NUM>, from <NUM> to <NUM>, from <NUM> to <NUM>, from <NUM> to <NUM>, from <NUM> to <NUM> etc., as well as individual numbers within that range, for example, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>.

A "recombinant polypeptide" is one, which is produced upon expression of a recombinant polynucleotide.

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, an enhancer, 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.

By the term "specifically binds," as used herein with respect to an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific.

In some instances, the terms "specific binding" or "specifically binding", can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope "A", the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled "A" and the antibody, will reduce the amount of labeled A bound to the antibody.

By the term "synthetic antibody" as used herein, is meant an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.

"Variant" as the term is used herein, is a nucleic acid sequence or a peptide sequence that differs in sequence from a reference nucleic acid sequence or peptide sequence respectively, but retains essential biological properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions and truncations. Changes in the sequence of peptide variants are typically limited or conservative, so that the sequences of the reference peptide and the variant are closely similar overall and, in many regions, identical. A variant and reference peptide can differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A variant of a nucleic acid or peptide can be a naturally occurring such as an allelic variant, or can be a variant that is not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides may be made by mutagenesis techniques or by direct synthesis.

As used herein, the term "genetically modified" means an animal, the germ cells of which comprise an exogenous human nucleic acid or human nucleic acid sequence. By way of non-limiting examples a genetically modified animal can be a transgenic animal or a knock-in animal, so long as the animal comprises a human nucleic acid sequence.

As used herein, "knock-in" is meant a genetic modification that replaces the genetic information encoded at a chromosomal locus in a non-human animal with a different DNA sequence.

The disclosure relates to a genetically modified non-human animal expressing human M-CSF, human IL-<NUM>/GM-CSF, human SIRPA and human TPO (herein referred to as MIST). The disclosure also relates to methods of generating and methods of using the genetically modified non-human animals described herein. Within the context of the invention, the genetically modified non-human animal is a mouse. In some examples, the genetically modified non-human animal is an immunodeficient mouse. In a particular example, the immunodeficient mouse is a RAG2-/- γc-/- mouse. In another particular example, the genetically modified non-human animal of the disclosure expresses human M-CSF, human IL-<NUM>/GM-CSF, and human TPO and does not express RAG2 or γc (referred to herein as MITRG). In another particular example, the genetically modified non-human animal of the disclosure expresses human M-CSF, human IL-<NUM>/GM-CSF, human SIRPA and human TPO and does not express RAG2 or γc (referred to herein as MISTRG). In some examples, the genetically modified non-human animals described herein are engrafted with a human hematopoietic cell.

In various examples, the human hematopoietic cell engrafted, genetically modified non-human animals of the disclosure are useful for the in vivo evaluation of the growth and differentiation of hematopoietic and immune cells, for the in vivo evaluation of human hematopoiesis, for the in vivo evaluation of cancer cells, for the in vivo assessment of an immune response, for the in vivo evaluation of vaccines and vaccination regimens, for the use in testing the effect of agents that modulate cancer cell growth or survival, for the in vivo evaluation of a treatment of cancer, for the in vivo production and collection of immune mediators, including human antibodies, and for use in testing the effect of agents that modulate hematopoietic and immune cell function.

The disclosure includes a genetically modified non-human animal that expresses at least one of human M-CSF, human IL-<NUM>/GM-CSF, human SIRPA, human TPO, and any combination thereof. Within the context of the invention, the genetically modified animal is a mouse. In some examples, the genetically modified non-human animal that expresses a human nucleic acid also expresses the corresponding non-human animal nucleic acid. In other examples, the genetically modified non-human animal that expresses a human nucleic acid does not also express the corresponding non-human animal nucleic acid. In some examples, the genetically modified animal is an animal having one or more genes knocked out to render the animal an immunodeficient animal, as elsewhere described herein. To create a genetically modified non-human animal, a nucleic acid encoding a human protein can be incorporated into a recombinant expression vector in a form suitable for expression of the human protein in a non-human host cell. In various examples, the recombinant expression vector includes one or more regulatory sequences operatively linked to the nucleic acid encoding the human protein in a manner which allows for transcription of the nucleic acid into mRNA and translation of the mRNA into the human protein. The term "regulatory sequence" is art-recognized and intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are known to those skilled in the art and are described in <NPL>. It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transfected and/or the amount of human protein to be expressed.

A genetically modified non-human animal can be created, for example, by introducing a nucleic acid encoding the human protein (typically linked to appropriate regulatory elements, such as a constitutive or tissue-specific enhancer) into an oocyte, e.g., by microinjection, and allowing the oocyte to develop in a female foster animal. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. Methods for generating genetically modified animals, particularly animals such as mice, have become conventional in the art and are described, for example, in <CIT> and <CIT> and <NPL>. A genetically modified founder animal can be used to breed additional animals carrying the transgene. Genetically modified animals carrying a transgene encoding the human protein of the disclosure can further be bred to other genetically modified animals carrying other transgenes, or be bred to knockout animals, e.g., a knockout animal that does not express one or more of its genes.

In some examples, the genetically modified non-human animal of the disclosure expresses one or more human nucleic acids from the non-human animal's native promoter and native regulatory elements. In other examples, the genetically modified non-human animal of the disclosure expresses a human nucleic acid from the native human promoter and native regulatory elements. The skilled artisan will understand that the genetically modified non-human animal of the disclosure includes genetically modified non-human animals that express at least one human nucleic acid from any promoter. Examples of promoters useful in the disclosure include, but are not limited to, DNA pol II promoter, PGK promoter, ubiquitin promoter, albumin promoter, globin promoter, ovalbumin promoter, SV40 early promoter, the Rous sarcoma virus (RSV) promoter, retroviral LTR and lentiviral LTR. Promoter and enhancer expression systems useful in the disclosure also include inducible and/or tissue-specific expression systems.

In some examples, the disclosure includes genetically modified immunodeficient non-human animals having a genome that includes a nucleic acid encoding a human polypeptide operably linked to a promoter, wherein the animal expresses the encoded human polypeptide. In various examples, the disclosure includes genetically modified immunodeficient non-human animals having a genome that comprises an expression cassette that includes a nucleic acid encoding at least one human polypeptide, wherein the nucleic acid is operably linked to a promoter and a polyadenylation signal and further contains an intron, and wherein the animal expresses the encoded human polypeptide.

In various examples, various methods are used to introduce a human nucleic acid sequence into an immunodeficient non-human animal to produce a genetically modified immunodeficient animal that expresses a human gene. Such techniques are well-known in the art and include, but are not limited to, pronuclear microinjection, transformation of embryonic stem cells, homologous recombination and knock-in techniques. Methods for generating genetically modified animals that can be used include, but are not limited to, those described in <NPL>), <NPL>), <NPL>), <NPL>), <NPL>), and <NPL>), <CIT>, <NPL>), <NPL>), <NPL>) and <NPL>). The methods disclosed herein comprise genetically modified immunodeficient non-human animals deficient in B cell and/or T cell number and/or function, alone, or in combination with a deficiency in NK cell number and/or function (for example, due to an IL2 receptor gamma chain deficiency (i.e., γc-/-)), and having a genome that comprises a human nucleic acid operably linked to a promoter, wherein the animal expresses the encoded human polypeptide. The generation of the genetically modified non-human animal of the disclosure 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.

In one example, the human nucleic acid is expressed by the native regulatory elements of the human gene. In other examples, the human nucleic acid is expressed by the native regulatory elements of the non-human animal. In other examples, human nucleic acid is expressed from a ubiquitous promoter. Nonlimiting examples of ubiquitous promoters useful in the expression construct of the compositions and methods of the invention include, a <NUM>-phosphoglycerate kinase (PGK-<NUM>) promoter, a beta-actin promoter, a ROSA26 promoter, a heat shock protein <NUM> (Hsp70) promoter, an EF-<NUM> alpha gene encoding elongation factor <NUM> alpha (EF1) promoter, an eukaryotic initiation factor 4A (eIF-4A1) promoter, a chloramphenicol acetyltransferase (CAT) promoter and a CMV (cytomegalovirus) promoter.

The human nucleic acid is expressed from a tissue-specific promoter. Nonlimiting examples of tissue-specific promoters useful in the genetically modified mice of the methods of the invention include a promoter of a gene expressed in the hematopoietic system, such as a M-CSF promoter, an IL-<NUM> promoter, a GM-CSF promoter, a SIRPA promoter, a TPO promoter, an IFN-β promoter, a Wiskott-Aldrich syndrome protein (WASP) promoter, a CD45 (also called leukocyte common antigen) promoter, a Flt-<NUM> promoter, an endoglin (CD105) promoter and an ICAM-<NUM> (Intracellular Adhesion Molecule <NUM>) promoter. These and other promoters useful in the compositions and methods of the disclosure are known in the art as exemplified in <NPL>), <NPL>), <NPL>) and <NPL>). Further to comprising a promoter, one or more additional regulatory elements, such as an enhancer element or intron sequence, is included in the genetically modified mice used in methods of the invention. Examples of enhancers useful in the compositions and methods of the invention include, but are not limited to, a cytomegalovirus (CMV) early enhancer element and an SV40 enhancer element. Examples of intron sequences useful in the compositions and methods of the invention include, but are not limited to, the beta globin intron or a generic intron. Other additional regulatory elements useful in some embodiments of the invention include, but are not limited to, a transcription termination sequence and an mRNA polyadenylation (pA) sequence.

In some examples, the methods of introduction of the human nucleic acid expression construct into a preimplantation non-human embryo include linearization of the expression construct before it is injected into a preimplantation non-human embryo. In preferred examples, the expression construct is injected into fertilized non-human oocytes. Fertilized oocytes can be collected from superovulated non-human females the day after mating and injected with the expression construct. The injected oocytes are either cultured overnight or transferred directly into oviducts of <NUM>-day p. pseudopregnant females. Methods for superovulation, harvesting of oocytes, expression construct injection and embryo transfer are known in the art and described in <NPL>). Offspring can be evaluated for the presence of the introduced nucleic acid by DNA analysis (e.g., PCR, Southern blot, DNA sequencing, etc.) or by protein analysis (e.g., ELISA, Western blot, etc.).

In other examples, 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 can be evaluated for the presence of the introduced nucleic acid by DNA analysis (e.g., PCR, Southern blot, DNA sequencing, etc.) or by protein analysis (e.g., ELISA, Western blot, etc.). Cells determined to have incorporated the expression construct can then be microinjected into preimplantation embryos. For a detailed description of methods known in the art useful for the compositions and methods of the disclosure, see<NPL>), <NPL>), <CIT>, <CIT>, <CIT>, and <NPL>).

The genetically modified non-human animals of the disclosure can be crossed to immunodeficient animal to create an immunodeficient animal expressing at least one human nucleic acid. Various examples of the disclosure provide genetically modified animals that include a human nucleic acid in substantially all of their cells, as well as genetically modified animals that include a human nucleic acid in some, but not all their cells. One or multiple copies, adjacent or distant to one another, of the human nucleic acid may be integrated into the genome of the cells of the genetically modified animals.

The genetically modified mice used in the methods of the invention are engrafted with human hematopoietic cells. In other examples, the disclosure relates to a method of engrafting human hematopoietic cells in a genetically modified non-human animal. The engrafted human hematopoietic cells useful in the methods of the invention include any human hematopoietic cell. Non-limiting examples of human hematopoietic cells useful in the invention include, but are not limited to, HSC, HSPC, leukemia initiating cells (LIC), and hematopoietic cells of any lineage at any stage of differentiation, including terminally differentiated hematopoietic cells of any lineage. Such hematopoietic cells can be derived from any tissue or location of a human donor, including, but not limited to, bone marrow, peripheral blood, liver, fetal liver, or umbilical cord blood. Such hematopoietic cells can be isolated from any human donor, including healthy donors, as well as donors with disease, such as cancer, including leukemia.

In other examples, the disclosure relates to a method of engrafting human hematopoietic cells in a genetically modified non-human animal. In some examples, the genetically modified non-human animal into which human hematopoietic cells are engrafted is an immunodeficient animal. Engraftment of hematopoietic cells in the genetically modified animal of the disclosure is characterized by the presence of human hematopoietic cells in the engrafted animal. In particular examples, engraftment of hematopoietic cells in an immunodeficient animal is characterized by the presence of differentiated human hematopoietic cells in the engrafted animal in which hematopoietic cells are provided, as compared with appropriate control animals.

In methods according to the present invention, the mice are engrafted with human cancer cells (e.g., human solid tumors, etc.) in addition to human hematopoietic cells. In various embodiments, the human cancer cells can be a cancer cell line or primary human cancer cell isolated from a patient, from any of many different types of cancer (including, by way of non-limiting examples, melanoma, breast cancer, lung cancer, etc.) In some embodiments, the human cancer cell and the HSPC are isolated from the same patient and transplanted into the same non-human animal.

The genetically modified non-human animals provided in various examples of the present disclosure have various utilities such as, but not limited to, for use as models of growth and differentiation of hematopoietic cells, for the in vivo evaluation of human hematopoiesis, for the in vivo evaluation of cancer cells, for in vivo study of an immune response, for in vivo evaluation of vaccines and vaccination regimens, for the use in testing the effect of agents that modulate cancer cell growth or survival, for the in vivo evaluation of a treatment of cancer, for in vivo production and collection of immune mediators, such as an antibody, and for use in testing the effect of agents that affect hematopoietic and immune cell function.

Engraftment of human hematopoietic cells in genetically modified and/or immunodeficient non-human animals has traditionally required conditioning prior to administration of the hematopoietic cells, either sub-lethal irradiation of the recipient animal with high frequency electromagnetic radiation, generally using gamma or X-ray radiation, or treatment with a radiomimetic drug such as busulfan or nitrogen mustard. Conditioning is believed to reduce numbers of host hematopoietic cells, create appropriate microenvironmental factors for engraftment of human hematopoietic cells, and/or create microenvironmental niches for engraftment of human hematopoietic cells. Standard methods for conditioning are known in the art, such as described herein and in <NPL>. Methods for engraftment of human hematopoietic cells in immunodeficient animals are provided according to examples of the present disclosure which include providing human hematopoietic cells to the immunodeficient animals, with or without irradiating the animals prior to administration of the hematopoietic cells. Methods for engraftment of human hematopoietic cells in immunodeficient animals are provided according to examples of the present disclosure which include providing human hematopoietic cells to the genetically modified non-human animals of the disclosure, with or without, administering a radiomimetic drug, such as busulfan or nitrogen mustard, to the animals prior to administration of the hematopoietic cells.

In some examples, the methods of hematopoietic cell engraftment in a genetically modified non-human animal according to examples of the present disclosure include providing human hematopoietic cells to a genetically modified animal of the disclosure as elsewhere described here. In some examples, the genetically modified non-human animal of the disclosure is an immunodeficient animal that is deficient in non-human B cell number and/or function, non-human T cell number and/or function, and/or non-human NK cell number and/or function. In other examples, the immunodeficient animal has severe combined immune deficiency (SCID). SCID refers to a condition characterized by the absence of T cells and lack of B cell function. Examples 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(+). In some examples, the genetically modified non-human animal of the disclosure is RAG1-/-.

In some examples, the methods of hematopoietic cell engraftment in a genetically modified non-human animal according to embodiments of the present disclosure include providing human hematopoietic cell to in a genetically modified non-human animal 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 <NUM> as described in <NPL>). Mice homozygous for the scid mutation are characterized by an absence of functional T cells and B cells, lymphopenia, hypoglobulinemia and a normal hematopoietic microenvironment. The scid mutation can be detected, for example, by detection of markers of the scid mutation using well-known methods.

In other examples, the methods of hematopoietic cell engraftment in a genetically modified animal according to embodiments of the present disclsoure include providing human hematopoietic cells to genetically modified immunodeficient non-human animal having an IL2 receptor gamma chain deficiency, either alone, or 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.

In addition to the naturally occurring human nucleic acid and amino acid sequences, the term encompasses variants of human nucleic acid and amino acid sequences. As used herein, the term "variant" defines either an isolated naturally occurring genetic mutant of a human or a recombinantly prepared variation of a human, each of which contain one or more mutations compared with the corresponding wild-type human. For example, such mutations can be one or more amino acid substitutions, additions, and/or deletions. The term "variant" also includes non-human orthologues. In some embodiments, a variant polypeptide of the present disclsoure has at least <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or <NUM>% identity to a wild-type human polypeptide.

The percent identity between two sequences is determined using techniques as those described elsewhere herein. 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 proteins.

Conservative amino acid substitutions can be made in human proteins to produce human 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.

Human 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, <NUM>-phosphoserine, homoserine, <NUM>-hydroxytryptophan, <NUM>-methylhistidine, methylhistidine, and ornithine.

Human variants are encoded by nucleic acids having a high degree of identity with a nucleic acid encoding a wild-type human. The complement of a nucleic acid encoding a human variant specifically hybridizes with a nucleic acid encoding a wild-type human under high stringency conditions.

The term "nucleic acid" refers to RNA 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.

Nucleic acids encoding a human variant can be isolated or generated recombinantly or synthetically using well-known methodology.

Isolation of human hematopoietic cells, administration of the human hematopoietic cells to a host animal and methods for assessing engraftment thereof are well-known in the art. Hematopoietic cells for administration to host animal can be obtained from any tissue containing hematopoietic cells such as, but not limited to, umbilical cord blood, bone marrow, peripheral blood, cytokine or chemotherapy-mobilized peripheral blood and fetal liver. Hematopoietic cells can be administered into newborn or adult animals by administration via various routes, such as, but not limited to, intravenous, intrahepatic, intraperitoneal, intrafemoral and/or intratibial.

Engraftment of human hematopoietic cells in the genetically modified animal of the disclosure can be assessed by any of various methods, such as, but not limited to, flow cytometric analysis of cells in the animals to which the human hematopoietic cells are administered at one or more time points following the administration of hematopoietic cells.

Exemplary methods of isolating human hematopoietic cells, of administering human hematopoietic cells to a host animal, and of assessing engraftment of the human hematopoietic cells in the host animal are described herein and in <NPL>), <NPL>), <NPL>), <NPL>), <NPL>) and <NPL>).

In some embodiments of the invention, the human hematopoietic cells are isolated from an original source material to obtain a population of cells enriched for a particular hematopoietic cell population (e.g., HSCs, HSPCs, LICs, CD34+, CD34-, lineage specific marker, etc.). The isolated hematopoietic cells may or may not be a pure population. In one embodiment, hematopoietic cells useful in the methods of the invention are depleted of cells having a particular marker, such as CD34. In another embodiment, hematopoietic cells useful in the methods of the invention are enriched by selection for a marker, such as CD34. In some embodiments, hematopoietic cells useful in the methods of the invention are a population of cells in which CD34+ cells constitute about <NUM>-<NUM>% of the cells, although in certain embodiments, a population of cells in which CD34+ cells constitute fewer than <NUM>% of total cells can also be used. In certain embodiments, the hematopoietic cells useful in the methods of the invention are a T cell-depleted population of cells in which CD34+ cells make up about <NUM>-<NUM>% of total cells, a lineage-depleted population of cells in which CD34+ cells make up about <NUM>% of total cells, or a CD34+ positive selected population of cells in which CD34+ cells make up about <NUM>% of total cells.

The number of hematopoietic cells administered is not considered limiting with regard to the generation of a human hematopoietic and/or immune system in a genetically modified non-human animal expressing at least one human gene. Thus, by way of non-limiting example, the number of hematopoietic cells administered can range from about 1X10<NUM> to about 1X10<NUM>, although in various embodiments, more or fewer can also be used. By way of another non-limiting example, the number of HSPCs administered can range from about 3X10<NUM> to about 1X10<NUM> CD34+ cells when the recipient is a mouse, although in various embodiments, more or fewer can also be used. For other species of recipient, the number of cells that need to be administered can be determined using only routine experimentation.

Generally, engraftment can be considered successful when the number (or percentage) of human hematopoietic cells present in the genetically modified non-human animal is greater than the number (or percentage) of human cells that were administered to the non-human animal, at a point in time beyond the lifespan of the administered human hematopoietic cells. Detection of the progeny of the administered hematopoietic cells can be achieved by detection of human DNA in the recipient animal, for example, or by detection of intact human hematopoietic cells, such as by the detection of the human cell surface marker, such as CD45 for example. Serial transfer of human hematopoietic cells from a first recipient into a secondary recipient, and engraftment of human hematopoietic cells in the second recipient, is a further optional test of engraftment in the primary recipient. Engraftment can be detected by flow cytometry as <NUM>% or greater human CD45+ cells in the blood, spleen or bone marrow at <NUM>-<NUM> months after administration of the human hematopoietic cells. A cytokine (e.g., GM-CSF) can be used to mobilize stem cells, for example, as described in <NPL>).

These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present disclosure and practice the claimed methods. The following working examples therefore are not to be construed as limiting in any way the remainder of the disclosure.

As described herein, mice repopulated with a human hemato-lymphoid system (HHLS) represent a powerful tool for predictive human preclinical in vivo research. A major limitation of current HHLS mice is the defective development of human cells critical for innate immunity. Here, a novel mouse strain is reported in which multiple genes encoding cytokines are genetically humanized. These humanized cytokines act synergistically to efficiently support human hematopoiesis and the development and function of human monocytes/macrophages and NK cells. In a tumor microenvironment, human macrophages acquire an immunosuppressive phenotype and support the growth of a human cancer. With a more complete and functional human innate immune system, this novel model of HHLS mice has exceptional potential to facilitate the study of physiology and pathology of human innate immunity in vivo.

Monocytes and macrophages are major cellular components of the innate immune response (<NPL>). On the one hand, these cells are capable of sensing an infection and of mediating direct anti-microbial functions, by diverse mechanisms such as phagocytosis or the secretion of pro-inflammatory factors. On the other hand, monocytes/macrophages can acquire immunosuppressive functions, important for the resolution of inflammation and for tissue repair. Furthermore, these anti-inflammatory properties can be co-opted by tumor-infiltrating macrophages and provide a survival advantage to evolving tumors through a diversity of mechanisms (<NPL>; <NPL>).

Small animal models such as mice are frequently used to study in vivo mammalian immune responses. However, fundamental differences in immune function exist between species (<NPL>; <NPL>). In particular, major phenotypic and functional species-specific differences exist among monocytes/macrophages populations and generally, knowledge gained from mouse studies is only partly applicable to humans (<NPL>; <NPL>; <NPL>). One promising approach to study the specificities of human hematopoietic and immune function in vivo consists in using mice carrying a human hemato-lymphoid system (HHLS) (<NPL>; <NPL>). However, the development and function of several human immune cell types, such as monocytes/macrophages and NK cells, is largely defective in current HHLS mice (<NPL>). These defects are most likely due to reduced cross-reactivity of mouse cytokines on the corresponding human receptors (<NPL>). To circumvent this limitation, a strategy was developed to replace mouse genes encoding cytokines by their human counterpart (<NPL>) and this approach resulted in significant improvements in the development and function of individual human cell types (<FIG>) (<NPL>; <NPL>; <NPL>).

Hematopoiesis is a tightly regulated developmental process in which multipotent hematopoietic stem cells differentiate into more committed progenitors and then into mature blood cells (<NPL>; <NPL>). This process requires specific cytokines that support successive developmental steps (<FIG>). Perhaps synergy between multiple humanized cytokines would be required to fully recapitulate human myelopoiesis in the mouse. Thus, a novel mouse strain, named MISTRG, was generated in which the genes encoding M-CSF (<NPL>), IL-<NUM>/GM-CSF (<NPL>) and TPO (<NPL>) were replaced by their human counterparts (<NPL>) in the hSIRPAtg RAG2-/- IL-2Ry-/- background (<NPL>; <NPL>).

Newborn MISTRG mice and their littermates MITRG (lacking the hSIRPA transgene) were sublethally irradiated and transplanted with human fetal liver-derived CD34+ cells, following a standard protocol (<NPL>). RAG2-/- IL2-Rγ-/- (RG) mice that share the same genetic background but lack all the humanized alleles, and commercially available NOD-Scid IL2-Rγ-/- (NSG) mice served as controls. Blood engraftment levels (hCD45+ cell percentage; (<FIG> and <FIG>; and <FIG>) were lower in RG and higher in NSG recipients as previously reported (<NPL>; <NPL>). The percentage of blood hCD45+ cells was similar in MISTRG and in NSG. Blood engraftment was also significantly increased in MITRG compared to RG, suggesting that the combined humanization of genes overcomes the need to induce phagocytic tolerance through SIRPα/CD47 cross-reactivity (<NPL>; <NPL>; <NPL>), possibly by weakening the mouse innate response. Mice with at least <NUM>% human CD45+ cells in the blood were selected for further experimentation (<FIG>). In the bone marrow (BM), the percentages of hCD45+ cells exceeded <NUM>% and reached up to <NUM>% in the majority of both MISTRG recipients (<FIG> and <FIG>; and <FIG>), and the high efficiency of engraftment in the BM was independent of SIRPα/CD47 interaction. To test the capacity of humanized cytokines to support human hematopoiesis in more competitive conditions, human CD34+ cells were transplanted into non-irradiated MISTRG. This protocol resulted in human CD45+ cells in the blood and BM of all recipients (<FIG>) and remarkably, half of the mice showed chimerism as high as the highest levels measured in recipients engrafted after X-ray pre-conditioning (compare <FIG> and <FIG>). The data described herein show that the genetic replacement of multiple cytokines in MISTRG creates a microenvironment in which human hematopoiesis can almost completely displace mouse hematopoiesis in the bone marrow, and obviate the need for pathology-inducing irradiation.

Next, the capacity of MISTRG mice to support human myelopoiesis was assessed. Human myeloid cells (hCD33+) were present in significantly higher proportions in the blood and bone marrow of MISTRG compared to RG and NSG (<FIG>; and <FIG>). The increased proportion of myeloid cells in MISTRG resulted in a blood composition that resembles the physiological composition of human blood, which is rich in myeloid cells and radically different from that of lymphoid-rich mouse blood (<NPL>; <NPL>) (<FIG>; and <FIG>). While both monocytes (CD33hiSSCloCD66-) and granulocytes (CD33+SSChiCD66+) were present in the BM (<FIG>), human myeloid cell populations in peripheral blood were composed mostly of monocytes (<FIG>), suggesting that the terminal differentiation and egress from the BM or peripheral survival of human granulocytes is still suboptimal in this mouse environment. Importantly however, human myeloid cells were present in high numbers in non-lymphoid tissues such as lung, liver and colon of MISTRG as shown by immunohistochemistry (hCD68+ cells; (<FIG>) or by flow cytometry (hCD33+; (<FIG>), and significantly exceeded human myeloid cell numbers found in NSG mice by a factor of ~<NUM>.

In humans, three subsets of monocytes have been phenotypically and functionally described, based on the expression of the CD14 and CD16 markers (<NPL>; <NPL>). All three subpopulations of human monocytes (CD14+CD16-, CD14+CD16+and CD14dimCD16+) were present in the lymphoid and non-lymphoid tissues, such as lung and liver, of MISTRG (<FIG>; and <FIG> and <FIG>). In contrast in NSG, in addition to the lower frequency of myeloid cells, only CD14+CD16- and to some extent CD14+CD16+ monocytes could be consistently detected, while CD14dimCD16+ cells were only marginally represented. The extended immunophenotype (CD33, CD11b, CD115, CD62L and CX3CR1) of the monocyte subpopulations found in MISTRG compared closely to the equivalent subsets in human peripheral blood (<FIG>). Human CD14+CD16- and CD14+CD16+ monocytes isolated from the BM of MITRG produced high levels of inflammatory cytokines in response to TLR4 and TLR7/<NUM> ligands (LPS and R848, respectively) (<FIG> and sG). In an in vitro assay performed on WBCs of MITRG, both CD14+CD16- and CD14+CD16+ cells had a high capacity to phagocytose GFP-expressing E. coli, while CD14dimCD16+ monocytes had limited phagocytic ability (<FIG>), again reflecting the physiological properties of the corresponding subpopulations in human blood (<NPL>). When challenged in vivo with LPS or infected with the bacterial and viral human pathogens Listeria monocytogenes and influenza A, respectively, MISTRG mice responded with robust production of human inflammatory cytokines (TNFα, IL-<NUM> and IFNγ, respectively), while NSG mice showed significantly lower, about one log lower, responses (<FIG>). These results demonstrate that the human monocyte subsets that develop in MISTRG are functional in vitro and in vivo. However, a drawback of the presence of functional human phagocytic cells in the mouse is a breach of human-to-mouse phagocytic tolerance, to which mouse RBCs are particularly susceptible (<FIG>). This destruction of mouse RBCs resulted in anemia (<FIG>) and limited the lifespan of engrafted mice to <NUM>-<NUM> weeks (MISTRG) or <NUM>-<NUM> weeks (MITRG).

Myeloid cells can support the development and differentiation of other immune cells through the production of cytokines. Whether the myeloid compartment of MISTRG mice was a source of human cytokines, such as IL-<NUM>, was assessed. Consistent with this notion, it was found that mRNA expression of human IL-<NUM> and IL-15Rα was increased by a factor of greater than <NUM> in MISTRG when compared to NSG (<FIG>; and <FIG>). To define in more detail the cellular source of human IL-<NUM>/ IL-15Rα in MISTRG, the abundance of human IL-<NUM> and IL-15Rα transcripts in purified human cell populations was measured. Expression of human IL-15Rα mRNA was higher in human myeloid cells (hCD33+) than in non-myeloid cells (hCD33-) (<FIG>). In particular, CD14+CD16+ monocytes showed an enrichment of both IL-<NUM> and IL-15Rα transcripts (<FIG>). The expression of human IL-15Rα protein on the surface of human myeloid cells from MISTRG was confirmed by flow cytometry (<FIG>).

Based on these findings, whether MISTRG mice support the development of human immune cells dependent on IL-<NUM> trans-presentation, such as NK cells (<NPL>; <NPL>), was assessed. The efficient development of human NK cells in current HHLS mouse models requires the exogenous pharmacologic delivery of human IL-<NUM>/IL-15Rα (<NPL>; <NPL>; <NPL>) since mouse IL-<NUM> is not sufficient to support human NK cells in vivo. As previously reported (<NPL>; <NPL>; <NPL>), very few human NK cells (hNKp46+hCD3-) were observed in engrafted NSG (<FIG> and <FIG>; and <FIG> and <FIG>). In contrast, human NK cells were readily detected in multiple tissues of engrafted MISTRG and were increased by a factor of ~<NUM> compared to NSG (<FIG> and <FIG>; and <FIG> and <FIG>). Apart from the bone marrow, MITRG had less human NK cells than MISTRG, which is most likely due to the previously reported requirement for human SIRPα for the survival of human NK cells in the periphery (<NPL>). The hNKp46+hCD3- cells in MISTRG mice represented bona fide NK cells because they expressed the typical NK cell surface markers CD94, CD161, and killer inhibitory receptors (KIRs) closely mimicking human controls (<FIG> and <FIG>). In addition to its effect on development, IL-<NUM> also promotes the maturation of NK cells. Consistently, it was found that surface expression of the maturation marker CD16 and the amounts of the lytic granule protein perforin were higher on NK cells from MISTRG compared to NSG (<FIG>).

The cellular source of IL-<NUM> trans-presentation in vivo in humans is currently unknown, but human myeloid cells can support human NK cell proliferation in vitro (<NPL>). To test if trans-presentation of human IL-<NUM> by human monocytes/macrophages underlies the improved human NK cell development in MISTRG, the mice were treated with liposome-encapsulated clodronate to deplete phagocytic cells (<FIG>). The depletion of phagocytic cells also induced a significant reduction of human NK cells (<FIG>), suggesting that human monocytes/macrophages are indeed a critical cell type that trans-presents IL-<NUM> to support human NK cell homeostasis in vivo.

NK cells participate in the innate defense against pathogens by killing cells that lack the expression of MHC class I (missing-self) (<NPL>), and by producing the key cytokine IFNγ (<NPL>). Consistent with higher perforin expression (<FIG>), significantly enhanced NK cell cytotoxic activity against human cells lacking MHC class I was observed in vivo in MISTRG compared to NSG (<FIG>). NK cells are an early source of IFNγ after Listeria infection. Accordingly, it was found that expression of human IFNγ mRNA in the liver was more than <NUM>-fold higher in MISTRG than in NSG two days post-infection (<FIG>). At single-cell resolution, NK cells from Listeria-infected MISTRG showed production of human IFNγ without ex vivo restimulation (<FIG>), at frequencies significantly higher than in NSG (<FIG>). NK cells in MISTRG also had lytic activity (degranulation) after Listeria infection, as shown by plasma membrane exposure of CD107a (<FIG>). Overall, MISTRG via efficient production of human myeloid cells support the development, differentiation, and function of human NK cells, thereby overcoming one major limitation of current HHLS mouse models.

Next, the role of human myeloid cells in the context of a tumor microenvironment was assessed. Therefore, the human melanoma cell line Me290 was used as a tumor model (<NPL>). Clinical observations show that myeloid cells infiltrate tumors in several solid tumors, and high densities of infiltrating macrophages correlate with poor patient prognosis in most types of cancer (<NPL>; <NPL>; <NPL>; <NPL>; <NPL>). Accordingly, higher human myeloid cell infiltration was detected in tumors in MISTRG than in NSG, as shown by the expression of human PTPRC and ITGAM mRNA (encoding respectively CD45 and CD11b) (<FIG>). Closely resembling human tumors in patients, cells expressing the macrophage markers CD163 and CD14 were abundant in tumors in MISTRG, but were almost undetectable in the same tumors in NSG (<FIG>; and <FIG>). Most of the CD163+ cells also expressed low levels of HLA-DR and high levels of CD206 (<FIG> and <FIG>), an immunophenotype generally associated with "M2-like" macrophages (<NPL>; <NPL>).

The M2 subtype of macrophages promotes tumor progression via a variety of effector mechanisms, including proliferative signals to cancer cells, anti-apoptotic signals, pro-angiogenic activity, enabling cancer cell egress from primary tumors and formation of metastasis (<NPL>; <NPL>; <NPL>). Macrophage infiltration in tumors could promote tumor growth in MISTRG was assessed. Remarkably, it was observed that the size of the tumors in CD34+-engrafted MISTRG, which are heavily infiltrated by human CD163+ HLA-DRlow CD206+ macrophages, was significantly greater than tumors in NSG, which are not infiltrated by human macrophages and are the same small size seen in non-engrafted NSG or MISTRG mice (<FIG>). One of the mechanisms by which macrophages support tumor growth is through the production of cytokines or enzymes that promote vascularization and immune suppression. VEGF is an important polyfunctional tumor-supporting molecule (<NPL>; <NPL>), and to test whether this factor was involved in tumor growth in MISTRG, the mice were treated with the human-VEGF inhibitor Avastin™. This treatment completely reversed the tumor-growth phenotype (<FIG>), demonstrating that myeloid cells in MISTRG support melanoma growth through a VEGF-dependent mechanism. Overall, these results show that MISTRG mice recapitulate the role of human macrophages in tumor development and fulfill a critical need for models allowing studies of the interaction between human tumors and human macrophages in vivo, especially at onset of tumor development.

The data described here have demonstrated that the provision of multiple human cytokines in MISTRG mice resulted in synergistic effects (<FIG>) on human hematopoiesis and on direct or indirect support for human immune cell function. The MISTRG model of HHLS mice offers a unique opportunity to study human innate immune responses in vivo. The materials and methods are now described.

The generation of mice with knockin replacement of the genes encoding TPO, IL-<NUM>/GM-CSF and M-CSF or with BAC-transgenic expression of human SIRPα in the RAG2-/-γc-/- Balb/c x <NUM> genetic background was reported (<NPL>; <NPL>; <NPL>; <NPL>). These strains were crossbred to obtain MITRG (M-CSFh/hIL-<NUM>/GM-CSFh/hTPOh/hRAG2-/-γc-/-) and MISTRG (M-CSFh/hIL-<NUM>/GM-CSFh/hhSIRPAtgTPOh/hRAG2-/-γc-/-) mice. Those mice are viable, healthy and fertile. The mice were maintained under specific pathogen free conditions with continuous treatment with enrofloxacin in the drinking water (Baytril, <NUM>/ml). NOD Scid γc-/-(NSG) mice were obtained from Jackson Laboratory.

Recipient mice were engrafted with human hematopoietic stem and progenitor cells as described (<NPL>; <NPL>; <NPL>; <NPL>; <NPL>). Fetal liver samples were cut in small fragments, treated for <NUM> at <NUM> with Collagenase D (Roche, <NUM> ng/mL) and a cell suspension was prepared. Human CD34+ cells were purified by density gradient centrifugation (Lymphocyte Separation Medium, MP Biomedicals) followed by positive immunomagnetic selection with anti-human CD34 microbeads (Miltenyi Biotec). Cells were frozen in FBS containing <NUM>% DMSO and kept in liquid nitrogen.

For engraftment, newborn pups (within first <NUM> days of life) were sublethally irradiated (X-ray irradiation; RG, <NUM> x <NUM> cGy <NUM> apart; NSG, <NUM> x <NUM> cGy; MISTRG, <NUM> x <NUM> cGy) and <NUM>,<NUM> FL-CD34+ cells in <NUM>µL of PBS were injected into the liver with a <NUM>-gauge needle (Hamilton Company). In specific experiments (<FIG>), <NUM>,<NUM>-<NUM>,<NUM> cells were injected into non-irradiated MISTRG newborn recipients. The mice were bled <NUM>-<NUM> weeks later and the percentage of human CD45+ cells was measured by flow cytometry. Mice in which human CD45+ cells represented at least <NUM>% (RG) or <NUM>% (NSG, MITRG and MISTRG) of the total (mouse and human combined) CD45+ populations were selected for further experimentation. The mice were sacrificed or used for experiments <NUM>-<NUM> weeks after transplantation.

All experiments were performed in compliance with Yale University Human Investigation Committee and Yale Institutional Animal Care and Use Committee protocols.

To prepare WBCs, heparinized blood was treated twice with ACK lysis buffer to eliminate RBCs. Single cell suspension of the spleen and bone marrow (flushed from the femur and tibia) were treated with ACK lysis buffer. Liver and lung leukocytes were isolated by mechanically dissociating and digesting tissues with <NUM> U/ml collagenase IV and <NUM>/ml DNase I (Sigma) for <NUM> at <NUM>, followed by density gradient centrifugation.

For FACS analysis, antibodies against the following antigens were used:.

All antibodies were obtained from Biolegend, BD Biosciences or Miltenyi Biotec. Data were acquired with FACSDiva on a LSRII flow cytometer (BD Biosciences) and analyzed with FlowJo software.

For histological analysis, spleen, lung, liver and colon tissues were fixed overnight in IHC zinc fixative (BD Biosciences) or <NUM>% paraformaldehyde and embedded in paraffin. Sections were stained with hematoxylin and eosin, or with anti-human CD68 antibody (clone PGM1) followed by a HRP-conjugated secondary antibody and revealed with the peroxidase substrate <NUM>, <NUM>'-diaminobenzidine.

Coli expressing GFP were grown in LB medium overnight at <NUM> to an OD600 of <NUM>-<NUM>, at which point the bacteria were diluted and grown for <NUM>-<NUM> hours to an OD600 of approximately <NUM>. Coli were washed three times with PBS and incubated with WBCs from MITRG mice for <NUM> hours at <NUM> in a volume of 200µl with about 2x10<NUM> E. Coli per 1x10<NUM> WBCs. After the incubation, the cells were washed with PBS and analyzed by flow cytometry.

Human monocyte subsets were isolated from the BM of mice. Briefly, BM cells were recovered and pooled from the hind legs and the spine of six mice. Human CD33+ cells were enriched by magnetic isolation (EasySep CD33 selection kit, StemCell Technologies). CD14+CD16- and CD14+CD16+ subsets were purified on a FACSAria cell sorter (BD Biosciences). <NUM>,<NUM> cells in <NUM>µl media were cultivated overnight in the presence of the TLR4 ligand LPS (E. Coli <NUM>:B4, Sigma-Aldrich, <NUM> ng/ml) or the TLR7/<NUM> ligand R848 (Invivogen, <NUM>µg/ml).

For in vivo stimulation, <NUM>µg of LPS (E. Coli <NUM>:B4, Sigma-Aldrich) in <NUM>µl PBS were injected intra-peritoneally and the serum was collected <NUM> minutes later.

Mice were infected with 3x10<NUM> colony-forming units (CFU) of Listeria monocytogenes (strain <NUM>) by intravenous injection. Forty-eight hours after infection, sera and tissues were harvested for ELISA and qPCR, respectively. Liver lymphocytes from uninfected or infected mice were incubated at 37C°/<NUM>% CO<NUM> for <NUM> hours in medium containing monensin (GolgiStop, BD Biosciences) and anti-human CD107a antibody. Cells were then stained for surface antigens, permeabilized using Cytofix/Cytoperm kit (BD Biosciences), and stained for intracellular human IFNγ.

Mice were infected intranasally with <NUM> × <NUM><NUM> PFU of influenza A /PR8 (H1N1) virus, and lungs were harvested on day <NUM> postinfection for qPCR analysis.

Cytokine concentrations (human TNFα, IL-<NUM> and IL-1β) in mouse serum and in culture supernatants were measured using ELISA MAX Standard kits (Biolegend), following the manufacturer's instructions.

RBC counts were measured on a Hemavet <NUM> (Drew Scientific). Blood smears were stained with Wright-Giemsa. For mouse RBC transfer experiments, blood was obtained from RG mice, labeled with CFSE (<NUM>, <NUM> minutes at <NUM>), washed three times with PBS and <NUM>µl of labeled RBCs were injected by retro-orbital intravenous injection. The mice were bled <NUM> minutes later to determine the initial frequency (Day <NUM>, <NUM>%) of CFSE-positive cells among Ter119+ cells by flow cytometry. They were then bled at the indicated time points and the maintenance of CFSE-labeled Ter119+ cells was calculated as a percentage of Day <NUM> values.

Phagocytic cells were depleted by intravenous retro-orbital injection of <NUM>µl of clodronate-loaded liposomes (Van Rooijen and Sanders, <NUM>, Journal of immunological methods <NUM>, <NUM>). Clodronate-liposomes were injected <NUM> times daily and human NK cells in mouse liver were analyzed <NUM> after the last injection. For RBC phagocytosis assay, clodronate-liposomes were injected <NUM> days and <NUM> day prior to transfer of CFSE-labeled RBCs.

Total RNA was extracted from tissues or purified cells with TRIzol reagent (Invitrogen) according to the manufacturer's instructions and used for cDNA synthesis with the SuperScript First-Strand Synthesis System (Invitrogen). Quantitative RT-PCR was performed on a <NUM> Fast Real-Time PCR system with primer-probe sets purchased from ABI. Expression values were calculated using the comparative threshold cycle method and normalized to mouse Hprt or human HPRT, as indicated.

Human NK cell cytotoxicity in vivo was determined following a previously reported protocol (<NPL>). <NUM> (HLA class I negative) and LCL721. <NUM> (class I positive) cells were mixed in a <NUM>:<NUM> ratio, labeled with CellTrace Violet (Invitrogen) and injected intravenously (1x10<NUM> cells/mouse) into engrafted NSG or MISTRG mice. Mice were sacrificed <NUM> hours later and single cell suspension of the spleens were prepared and analyzed by flow cytometry. The proportions of HLA class I positive and negative among violet cells were measured and specific lysis was calculated as (MHC class I positive - MHC class I negative) x <NUM> / MHC class I positive.

The human melanoma cell line Me290 (<NPL>) was grown to ~<NUM>% confluency and the cells (~<NUM> million cells per mouse) were injected subcutaneously under anesthesia in the flank of the mouse. For some experiments, the mice were treated every other day, starting on the day of tumor implantation, with the anti-human VEGF antibody Avastin™ (Roche; <NUM>µg intravenously). The size of the tumors was measured <NUM> days later and the volume calculated using the following formula: Volume = <NUM> * Length2 * Width.

Patients and mouse tissues were frozen in Optimum Cutting Temperature (OCT, Sakura Finetek). Cryosections (<NUM>) were consecutively treated with Triton-100X <NUM>% for <NUM>, Hyaluronidase <NUM>% for <NUM>, Background Buster (Innovex bioscience) for <NUM>, Fc Receptor Block (Innovex bioscience) for <NUM> and Background Buster for an additional <NUM>. The sections were then stained with primary antibodies, diluted in PBS supplemented with <NUM>% BSA and <NUM>% Saponin for <NUM> at room temperature, washed and stained with the secondary antibodies at room temperature for <NUM> minutes. Nuclei were stained with <NUM>',<NUM>-diamidino-<NUM>-phenylindole (<NUM>µg/mL) for <NUM>.

Primary antibodies: human CD14 (<NUM>:<NUM>, UCHM1, AbD Serotec); human CD163 (<NUM>:<NUM>, EDHu-<NUM>, AbD Serotec); human CD206 (<NUM>:<NUM>, <NUM>-<NUM>, AbD Serotec); human HLA-DR (<NUM>:<NUM>, LN3, Biolegend). For CD163/CD206 combined staining, both antibodies were labeled with Alexa Fluor <NUM> or <NUM> Antibody Labeling Kit (Molecular Probes) prior tissue staining.

Secondary antibodies: goat anti-rat Alexa Fluor <NUM>; goat anti-mouse Alexa Fluor <NUM>; goat anti-mouse Alexa Fluor <NUM> or goat anti-mouse Alexa Fluor <NUM> (<NUM>:<NUM>, Molecular Probes).

Immunofluorescence imaging was performed on an Eclipse Ti inverted microscope system (Nikon Instruments Inc. ) operated via NIS-Element Ar software (Nikon Instruments Inc).

For quantification of the density of CD163+ cell infiltration, tumors from <NUM> different melanoma patients, <NUM> NSG and <NUM> MISTRG were selected. From each tumor, <NUM> cryosections were stained for human CD163. From each stained section <NUM> representative pictures were acquired, totaling <NUM> representative pictures from each group (Patients, MISTRG and NSG). For each picture, CD163+ cells were counted using the NIS-Element Ar software (Niko Instruments Inc. Each picture was analyzed using the "split channels + overlay" display and by zooming simultaneously on each separate channel and on the overlay.

Statistical analysis was performed with the GraphPad Prism <NUM> software, using one-way ANOVA followed by Tukey post hoc test, two-tailed unpaired Student's t-test or repeated measure ANOVA.

Myeloid leukemia is a form of cancer that affects cells of the myeloid lineage. Myeloid leukemias are classified in different types, including acute myeloid leukemia (AML), myeloproliferative disorder (MPD), chronic myelo-monocytic leukemia (CMML) and myelodysplastic syndrome (MDS). The risk of developing myeloid leukemias increases with age and the incidence of these diseases is likely to increase with ageing of the population. Although therapeutic and supportive care approaches are available in the clinic, a better understanding of this group of diseases and novel therapies are needed.

One of the methods used to study human leukemias relies on the xeno-transplantation of patient samples into immunodeficient mice. However, currently available recipient mice are not optimal for this purpose: only a subset of AML samples can be engrafted successfully; and robust engraftment of MPD, CMML or MDS (including RCUD, RAEB I and RAEB II) has not been reported so far. Thus, optimized strains of recipient mice are needed for better engraftment of human myeloid leukemia.

It is demonstrated herein that MISTRG supports better engraftment of human hematopoietic cells, leading to the almost complete replacement of mouse hematopoiesis by human hematopoiesis in the bone marrow. It is also shown herein that samples isolated from patients with AML, CMML and MDS can be engrafted in MISTRG (<FIG>).

Claim 1:
A method for testing the effect of an agent that modulates cancer cell growth or survival, or for the in vivo evaluation of a cancer treatment involving the agent, the method comprising:
administering the agent to a genetically modified mouse, wherein the genetically modified mouse comprises in its genome:
a replacement of a mouse M-CSF gene with a nucleic acid encoding a human M-CSF polypeptide operably linked to the mouse M-CSF promoter and regulatory elements at the mouse M-CSF gene locus,
a replacement of a mouse IL-<NUM> gene with a nucleic acid encoding a human IL-<NUM> polypeptide operably linked to the mouse IL-<NUM> promoter and regulatory elements at the mouse IL-<NUM> gene locus,
a replacement of a mouse GM-CSF gene with a nucleic acid encoding a human GM-CSF polypeptide operably linked to the mouse GM-CSF promoter and regulatory elements at the mouse GM-CSF gene locus,
a replacement of a mouse TPO gene with a nucleic acid encoding a human TPO polypeptide operably linked to the mouse TPO promoter and regulatory elements at the mouse TPO gene locus, and
an insertion of a nucleic acid encoding a human SIRPA polypeptide operably linked to a human SIRPA promoter and regulatory elements,
wherein the genetically modified mouse expresses the human M-CSF polypeptide, the human IL-<NUM> polypeptide, the human GM-CSF polypeptide, the human SIRPA polypeptide, and the human TPO polypeptide, and
wherein the genetically modified mouse is immunodeficient, engrafted with human hematopoietic cells, and engrafted with human cancer cells; and
assessing effect of the agent on cancer cell growth or survival in the genetically modified mouse.